Embryology - User contributions [en-gb]

Talk:2017 Group Project 3

2017-10-26T06:25:04Z

Z5178463:

{{ANAT2341Project2017discussion}}



==Suggested Starting Places==
[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 10:15, 14 August 2017 (AEST) OK Group 3 below are some starting places.

{{Heart Links}}

PubMed Searches: [http://www.ncbi.nlm.nih.gov/pubmed?term=Heart+Development ''Heart Development''] | [http://www.ncbi.nlm.nih.gov/pubmed?term=Cardiac+Development ''Cardiac Development'']

BMC Dev Biol Search: [http://www.biomedcentral.com/bmcdevbiol/search/results?terms=Heart+Development ''Heart Development'']

Recent papers

<pubmed limit=5>Heart+Development</pubmed>

==Group Topic Intro==

==Vidoes used from Mark's page==
I used the video for the heart tube folding and fusion under the development section (Z5178463)

=Peer Review=

'''Peer review project 3:'''

Some general comments to the project:
*The project contained both developmental origin, timeline, signalling processes, current research and findings, animals model and abnormal development sections. The project therefore has all the sections which were a requirement for the project.
* Overall, I think the project was good. It was well written, easy to understand as a student, the sections correlated well and the context was good. I especially liked the signalling section, even though some context is missing. I think the idea of adding a treatment part to project is a good but I could not find it in the project. As mentioned some context is missing, which is the notch pathway, sonic hedgehog and retinoic acid sections.
* The project has a good introduction. You have a clear idea of what you are about to read, which is nice.
* The layout could be a lot better, I think the picture location could be adjusted. In the developmental origin part, the pictures make the section look very confusing. Some of the subheadings, like the abnormal development is pushed to side by the pictures, so when you scroll down the project you miss it.
* In general, the pictures miss their caption, sources and number. Therefore, you do not know which picture there is referred to when you are reading the project. I miss some more pictures in the developmental sections. Some of the home made drawing is not very descriptive
* In the developmental origin section I think the last sentence is very long. You get so much information in one sentence that you sometimes forget what you just read.
* The timeline is easy to read and understand. Could contain some key discoveries.
* Thought the glossary of terms is a nice addition to the project.
* Overall the referencing is good, but in some sections like primary heart field and heart tube formation, the referencing is missing. In some sections the articles/or links are at the bottom of the section, which makes it a little confusing.
* I don’t feel like the primary heart field and heart tube formation correlates very well with the secondary heart field and cardiac looping section. When I start reading the latter I feel like I am starting on something completely new instead of continuing reading on the heart developing. I get the feeling I am reading two different persons work, and some work should be put into these sections to make it feel more fluent.
* An idea for your project could be that you add a short anatomy section after the introduction, so the reader gets a picture and an overview of how the heart is structured. Then it is easier to understand the developing of the heart when you know how the heart is going to end up looking like.

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Don’t forget to remove the hyperlinks that are under ‘Heart’ at the beginning of the page. Remove student numbers from the page. Add a brief description under images so that readers will understand what the image is showing. Remember to move references that are written in some sections to the ‘References’ subheading. The Notch Pathway is incomplete.

Subheadings and content that have been used show a good understanding of the topic area. The use of a table for the ‘Development Timeline’ shows the teams innovativeness. The use of simple sentences in the table allows readers to understand content simply. References have been done well, they are cited properly. The team have used their own images to show their understanding of the heart. The images that they have used have been properly cited: there are references, copyright statements and the Student Image template. Thorough description of abnormal development, animal models and current research which shows the comprehensive research that was done for the heart. The use of Glossary of Terms is helpful to readers who may not understand what some terms are.

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Very informative and well written wiki. The glossary of terms is particularly useful and presented beautifully. The frequent addition of images and hand-made drawings are really good as they provide a useful visual reference point. The inclusion of a brief overview about the different animals studied in regards to the heart is very interesting. There is a minor spelling error in the table referring to Developmental timeline (week 5). The use of references is great, however maybe just include the links at the bottom of the wiki, to assist with the flow of information. Also add a description of your images so that viewers can more easily identify how the image relates to the text and the relevance of it. Overall, a really good wiki and well done.

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The project is very good. There is a lot information on the page, there is a good description of each picture when you click on them, and there is a good brief introduction of each topic before going into depth on certain points. Most things are described clearly with pictures to support the information. Ending the project with a glossary of terms adds clarity to the project.

In particular, the “Developmental Signalling Processes” section is excellent. There is a description of where molecules are expressed, what cells they act on, the molecules’ roles in the cell signalling pathway on individual cells, and the molecules’ roles in overall heart development. The table describing different types of FGF signalling is excellent. In this section and throughout the project, there is a lot of description of research that has led to the discovery of the information presented on this page. The section “Animal Models” and “Current Research and Findings” add to this.

There are still a couple things to be fixed before the project is complete. There are some grammatical and spelling errors, particularly in “Proepicardium and Coronary Heart Development” that need to be edited. Some subheadings have nothing under them. Some references need to be fixed. Copyright information is needed when you click on some of the pictures. Instead of just using a paragraph style, emphasizing information by bolding specific words or using bulleted information may make the project easier to read and understand. Near the beginning of the project, a picture of the locations of the truncus arteriosus, bulbus cordis, primitive ventricle, and primitive atrium in the heart tube may add to the description of early development. In addition, a description of valve development could add to the project. Overall very good project.

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Use of headings and subheadings break this complex developmental process in to understandable, clear sections.
The images chosen to reinforce the material are appropriate and I particularly like that time has been taken to draw a number of these.
There does seem to be an awful lot of information, and I wonder if this can be cut down at all. For example there is the section on signaling during development, which is a more complex section to understand. This is greatly helped by the diagrams but I can see that there are additional headings that are yet to have information added. It might be an idea to pick a few signaling pathways that occur and really perfect those. I think it has the potential to become very confusing to the reader otherwise.
It is very useful to the reader that you have included a glossary of terms, however I wonder if it may be more effective if this table is placed at the beginning of the page, or as a link at the top that can be opened up, so as the reader can familirise themselves with the terms prior to reading the page.
The page appears to be referenced extensively throughout and appropriately. Good job

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*Overall, this page has a good structure and was enjoyable to read. The headings and subheadings were clear and made it easier to understand the developmental process of the Heart.

*Perhaps it would be better to include relevant background information of the heart (such as blood flow and structure) before delving into the developmental process straightaway.

*There is a good amount of images (and well-drawn images) accompanying the text which aided in understanding the content. However some were not labelled, such as in Developmental Origin where in the text you referred to the images “In figure 2…” and “See figure 4” but the images were not labelled so it was difficult to tell which images you were referring to.

*The table on the Development Timeline was short and concise which was good however it would be better to insert images in the different stages to make it easier to visualise.

*Most parts were cited correctly and properly, however some parts need to be fixed; the Pubmed article reference by “Antoon Moorman” appears in several sections of the page and needs to be deleted. Most sections had a good amount of references, however some sections weren't cited at all such as "Current Research and Findings and "Cardiac Stem Cells".

*Some sections were left blank (The Notch Pathway, Sonic Hedgehog, and Retinoic Acid) which gives the page an unfinished feel, however I assume they will be completed over time. Glossary of terms was clever and made the content easier to understand (the heart is quite complicated to understand). Well done overall.

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Overall, this page had a finished feel because the page is so heavily packed with information, there are some sections that were not completed. There is however, a lot of information that may leave the reader feeling a bit overwhelmed. Some sections are also hard to understand and comprehend especially due to the heavy use of biotechnological jargon (ie. SMAD-dependent, SMAD-independent pathways, β-catenin). A terminology/glossary section would be extremely helpful for this issue. I'd advise using more images in "Abnormal Development" (ie. x-rays or physical observations of sufferers) to help the reader visualise such abnormalities. Referencing under images should be moved to the references section and should be referenced using the '''''< ref >''' '''< / ref >''''' if in text. Overall, there is a lot of information, some of which is not necessarily important. I'd advise to cut down, make paragraphs more simple and straight to the point, and use images to help the reader visually understand and comprehend.

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Introduction is very good and explains a lot. Under the heading “Primary heart field and heart tube formation” – the reference at the bottom should be removed.
Secondary heart field and cardiac looping: First sentence doesn’t really make sense, maybe switch it up a bit.
Under current research and findings you have labelled a figure figure 1, when it is not the first figure in your wiki page, seems a little confusing. Images also don’t have appriopriate copyright info, description and referencing. Also figure 2 is placed right in the middle of the sentence, maybe put it to the right so it doesn’t interrupt reading.
Information could be formatted better under the heading atrial septal defect! Maybe some subheadings for the different defects? Same goes with the ventricular septal defect, its easier to read when things are broken up. Glossary is very good!

All the information written on your page is very well written and easily understood.
Images could be labelled better, add a figure to each of them as when you are referring to figure 1 and 2 in your writing, the images aren’t labelled so its hard to tell what image you are referring to.
With references, I don’t like how there is a bunch at the end of some headings? Could be because you still need to read them but looks messy.
There is an overwhelming amount of information, so good job on doing so much research but it was quite tedious to read, not sure if this much information is needed? But it is very hard to fault your wiki page so this might be a bit picky.

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The introduction is a brief and clear overview of the page. I liked how you acknowledged what your page will explore about the heart development. The “Developmental origin” subheading had good information and good diagrams in addition. However, I would adjust your layout a bit in this section so that the diagrams don’t look so awkward. You could do this by breaking down that second paragraph. The timeline provided a brief overview but I would also suggest adding another column for images. There is also a spelling error on week 5 – it says “srtats” where it should be “starts”.
As you go into “primary heart field and heart tube formation”, “secondary heart field and cardiac looping” and the next few sections, the references appear at the bottom of the sections. You should fix this so that they only appear in the reference list at the end. You could also probably bold “heart tube fusion”, “heart looping” and other terms in your developmental timeline since you explore them in depth.
The “developmental signalling process” subheading is very detailed. Since you also have a few more parts to complete into this section, it might be better to try to minimise some of the text. Your inclusion of current research is good and unique as you explore one paper in depth. However, I would suggest that you find another one or two. The “animal models” subheading should probably have a diagram or two of the referenced research papers if possible. Again with “abnormal development” subheading I would suggest some more images to see what these defects look like and possibly cutting down some text. In “future questions” you might also be able to provide a possible direction research might take to potentially answer this question. Also, you had a very good, long list of references.

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The page goes through everything required for the project page. It would be nice if the pictures on the page have a figure number and a short title on the figures, so it is easier for the reader to understand what figure belongs to what part of the section. A figure number on the picture makes it able for the writer to refer to a specific picture. There is a good use of tables and self-drawn figures/picture. This makes the page clearer and more readable. There are some references on the page that needs a different formatting, so it is not fully viewed in the sections. It is important that the references are given right after the specific section and not at the bottom of the section.

*'''The Introduction''' section gives a good excitement for the reader before reading the page. This gives an idea of what information to expect from the page. A little section about the anatomy of the heart and a picture could give a better preparation for the reader to understand the developmental part of the heart.
*'''The Development Origin''' section has a bit confusing layout.
*I like that the '''Cardiac Neural Crest and Outflow tract''' sections have a self-drawn picture, but maybe you can draw it a bit clearer, so it is easier to read the writing and understand the figure There is no figure text on the page of the figure.
*'''Current Research And Findings, Animal Models and Abnormal Development:''' These sections have a bit of a messy layout. The context is good, but there I a lot of text and pictures kind of mingling into each other. You could make these sections more separate in the layout.
*'''The Glossary of terms''' helps the reader a lot.

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The headings were all neat, concise and impressive. It successfully highlighted and sectioned the key topics in the development of the heart. The addition of the technical signalling pathways and the details of the development were well summarised with appropriate references in superscript format. There was a nice variety of visual resources, both hand drawn and externally sourced. Most images have their copyright approval and reference included perfectly, except "Figure 1 Morphological defects in CTCF mutant embryonic hearts" and "Figure 2 - defects of mitochondria in CTCF mutant hearts". There was a nice flow throughout the page through the use of effective paragraph sectioning. The table for the glossary of terms was really useful and neat.

Some of the images didn't have a box around it and these figures were not labelled, this should be easily changed in the edit mode. Some of the hand drawn images were somewhat unclear, due to the writing as well as the rough outline of the heart. Signatures should also be removed. The references were also retained in the bottom of the sections. It was a confusing because it wasn't next to any paragraphs that needed to be referenced. A reference was also repeated in this section.

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*Introduction
**Introduction is clear with explanation on why the group decided to focus on heart as well as a brief outline of the page.

*Developmental origin
**Good use of images that are relevant in explaining the developmental origin of the heart. References are also made to the figures. However, captions for the images are missing so it is unclear as to which is figure 2 that the author is making reference to.
**Clear explanation that is easy to understand.

*Developmental timeline
**Good use of a table in summarising the embryonic developmental timeline of heart.
**Elaboration for the development of heart during each week is also clear and extensive. A suggestion would be to include the week i.e. “Week 2: primary heart field and heart tube formation” for the subheadings as it can get confusing easily having to scroll back to the table.
**Some references are missing.
**Some images are well labeled but some are not.

*Developmental signaling processes
**Clear explanation that is coupled with images that are relevant to developmental signaling processes.

*Future questions
**The questions listed seem abrupt. Author may want to consider including the significance and need to further investigate these questions.

*Glossary of terms
**Author may want to consider arranging the glossaries in alphabetical order. Otherwise, good inclusion of a list of terms.

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''For example:''
"This image is based upon Robert H Anderson, Sandra Webb, Nigel A Brown, Wouter Lamers, Antoon Moorman Development of the heart: (3) formation of the ventricular outflow tracts, arterial valves, and intrapericardial arterial trunks. Heart: 2003, 89(9);1110-8 PubMed 12923046

Robert H Anderson, Sandra Webb, Nigel A Brown, Wouter Lamers, Antoon Moorman Development of the heart: (3) formation of the ventricular outflow tracts, arterial valves, and intrapericardial arterial trunks. Heart: 2003, 89(9);1110-8 PubMed 12923046

Marc Sylva, Maurice J B van den Hoff, Antoon F M Moorman Development of the human heart. Am. J. Med. Genet. A: 2014, 164A(6);1347-71 PubMed 23633400"

Finally, there is a great variety of reputable sources of information. The only thing that needs changing is that the reference list should be revised. Some were left as a link and the list were inconsistent with its reference format.

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This wikipage had all the sections required for this assignment and the team was very detailed in their content. There was also a good amount of referencing. However for certain areas, the entire reference was there instead of just the number. Also, the student numbers should be removed from the page. There was also a good number of photos used and it was good that they had a mix between self-drawn images and images obtained online. However, a way that could improve the images would be to add a description or a caption under each figure so it's easier to know what the picture is about. Some photos in this article lacked description, reference and copyright information, so that could be added as well. For most of the article, I can see that the team carried out a great amount of research for this topic, however it was a little difficult to understand some parts as they were very lengthy and slightly too content heavy. For the developmental timeline, the use of a table was good but the information could be presented in a more concise manner and the headings could be slightly more prominent to make it more readable. The signalling processes was also very well researched but quite lengthy, perhaps a few main signalling pathways could be chosen instead. For the abnormal developments, each abnormalities were well researched on. Perhaps images could be added to show the abnormalities and also maybe one or two more defects would be good. Overall, I think this group did a great job in researching and providing information on this wikipage. Maybe with a bit of tweaking here and there to make it more concise and readable, this wikipage would make a really good project.

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Well-structured page which seems to be quite detailed and long to read. The extensive use of subheadings make it a little harder to follow in some areas such as under “Developmental timeline”, where after the table the subheading “Primary Heart Field and…” appears to be a little misplaced or is lacking flow completely. Need to remove the student numbers from the page and also the two links under the initial “Heart” heading. In text referencing throughout the page seems to be consistent for the most part however, there are some areas where the correct format needs to be used (i.e., under “Wnt signaling” and “Cardiac Neural Crest and Outflow tract”). Some images do have a description of what is addressed however, many of them do not – this expansion would help with the overall reading experience as well as add further information for understanding. Overall, an extensive knowledge of the topic is well demonstrated through an attention to detail – but perhaps a more concise approach would add some clarity to the text.

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The introduction was very good! I like how it introduced why the heart is so critical in early development, explained what you were going to discuss and where there would be gaps due to a lack of medical knowledge. The information in developmental origin and the developmental timeline is really great, however, I think you need to consider joining these two headings and not splitting them into one. You also state in developmental origin "as seen in figure two", however, none of your images have figure titles so I am not sure which figure you're actually referring to. The timeline is a good basic reference point, so I think it would be nice for it to be before the origin outline as it gives the basics which you then go into more detail about. I like that you put in the developmental signalling processes and then outlined each one of these, obviously the rest of those processes that have subheadings but no information just need to be finished. The current research is really interesting, again images just need a figure of some sort. The future questions section is a little confusing as I'm not sure if that's an area you're going to go into more depth over or if that's a future question you think research should look in to? So a clarification would be good. The glossary of terms is super helpful and all referencing looks good!

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At first glance this project seems very detailed and lengthy. In my opinion, it could have too much text and maybe summarizing and condensing some sections could be beneficial. Collapsible windows or maybe more use of subheadings or dot points could be used to make the page clearer and less overwhelming. The diagrams and tables are very engaging and informative. However I do think the position and sizing of the diagrams could be improved to align it with the text better. The text and most of the diagrams seem to be well referenced. Another suggestion for this page would be to make the overall title of ‘Heart’ larger and clearer, perhaps include a diagram of the heart with the title to make it more attractive. The overall title should also be placed above the contents section.
Despite these suggestions, well done this page is very detailed and informative and you have clearly put a lot of work and effort into it.

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Great detailing of the development and signaling processes involved, it really showed me that the group had extensive knowledge on this topic. In addition, there is a good balance of personal images (e.g. wnt signaling diagram) and web images which showed me that the group dedicated time to make sure the reader fully understood each aspect of the topic. Most images also are properly cited with copyright statements, references, and description (some are missing, but overall are done well). It was very helpful to include a glossary of terms at the end for the reader to refer to. An image for the cardiac looping steps would help to visualize steps. Information needs to be added for the notch pathway, sonic hedgehog, retinoic acid. While the detail is very informative, there is a lot of information and can be a little overwhelming. It might help to add more bullet points (with only essential information) or to edit some of the superfluous information. In addition, captions for the images would help so that the reader knows which image you’re referring to when referring to them in the text.

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'Introduction' is clear and informative, well referenced and gives a good outline for the rest of the page. 'Developmental Origin' has a bit of a confusing set up.. pictures are seemingly scattered and need captions. The 'Morphology of the Heart tube formation' hand-drawn figure is almost an exact replica of the original.. not sure if this is allowed because they are so similar. 'Developmental Timeline' has a very brief table.. would benefit from a better description of each week of development. This section has a couple of random references that should be at the bottom. 'Developmental Signalling Processes' diagrams need captions. There is a lot of information here which is very detailed. 'Current Research and Findings' has good subheadings and picture use; pictures need to have copyright information and citations added. Very detailed with good references throughought. 'Future Questions' needs to be added to but has shown evidence of initial research into this area. 'Glossary of terms' is a very good idea that has not yet been shown in other group topics. Maybe look into researching how to link certain words in the article to redirect to the bottom of the page to the Glossary of terms for quick definitions? Some references also need to be properly cited in the 'References' section. Over all, really well researched with some sections needing a bit more work.

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Overall a very in-depth page with most of the required subjects covered. Some general notes; the figures would have benefited from appropriate captions but were helpful nonetheless. Referencing and overall visual formatting could be improved. The page is written well and enjoyable to read.

"Primary heart field and heart tube formation" could benefit from a diagram or figure since it is a lot of text that could be hard to conceptualise. The signalling processes are explained very well and in deep detail. Just a note though; the addressing of so many different variables in the signalling is a little confusing and hard to follow. Perhaps a more condensed response might be a little more straightforward. The diagram of the signalling pathways under "Wnt Signalling" was likewise hard to follow, and no key was given. Current Research and Animal Models were covered well and the explanation for their research and the key results highlighted were fascinating. Abnormal development was likewise addressed very well and the glossary of terms was very much appreciated.

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- Introduction was very well written and great spelling and grammar. Good referencing, simple way to start a page

- Developmental origin of the heart was explained well and I liked how they referenced to the figure in the text as well, showing that the picture is actually important in this section. Correct referencing was used in this section which is good. A moving video of developmental origin of the heart would have been really useful here

- Developmental timeline was a bit too brief, and would help to put some pictures to explain how some of the main steps looked like

- Further explanation of the development was separated and structured well and had good amount of information for each main step. Citation of the reference shouldn’t be here but in the references

- A nice picture was used to explain the difference between straight, looped and converged which I personally found very interesting and informative

- The drawings included were very well drawn, precise and different colours helped differentiate each part of the heart and also referenced.

- I really liked the developmental signalling processes and they had explained each important factor of the process in a lot of detail. Would have been good if they had finished this section. A good use of table to differentiate different FGF and their functions

- Current research and findings selected were definitely very new and also explained in a lot of depth

- Abnormalities section was slightly lacking, although there is good information, adding 3 or 4 more abnormalities would be even better. Some pictures in this section would have made it more interesting and explain the content better.

- Overall, a very nicely set out page with good information in each section and appropriate referencing in most parts. Inclusion of glossary and a large variety of subheadings made this page great. Some further work in some subheadings would make this page perfect.
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- I think your intro was really good because it was very straight to the point and gave a great summary of your project as a whole.
- Developmental origins was well done, not too complex. The diagrams definitely helped break down this complex model. I have seen some helpful videos online that summarise this process, so maybe you could add a video to it? Just because it is still a lot to grasp and if the person is anything like me, they'll find videos more helpful than the diagrams (but your choice of pictures were great and good referencing). The placement of the pictures looks a bit wonky, but I don't think it's something you have control of.
- Developmental timeline table was a bit brief, I think more detail needs to be added to it because the rest of the developmental section has a lot of content; the table should summarise it all so someone can have a quick idea of it all rather than having to read the whole section which is quite lengthy. Good job for having a table in the first place though. The diagrams in this section were relevant and well sized. Once again, a video would be nice but that's just me. Some referencing errors, but that can be easily fixed.
- Great work on the signalling section, it's really complex so I'm sure that would have been a mission to collate however I think it could be less wordy just because it is so hard to follow at times. You've done a lot of research which is great, but maybe shortening it a bit would be more beneficial to readers. Again, videos could help. Good use of the table to summarise it all. I can see that it's not finished, but it seems like you guys know where to go with it. Good referencing.
- I think your abnormal development section was well done, it wasn't too overwhelming and it is very detailed with proper referencing and suitable pictures.
- The stem cells section seemed a bit random to me because it wasn't mentioned in the intro. If it falls under "the possible treatments to be developed in the future", perhaps you could address that in the first sentence because right now it just seems like hey here's some info on stem cells... and I'm here wondering what the relevance of this is. Seems well researched though, just need to state its relevance.
- Future questions a bit empty.
- Glossary is great, maybe you could have a glossary for the signalling part too

Overall it is clear that you guys have put in a lot of time and effort, so well done on that. At times though, it just feels a bit overwhelming. There's good use of diagrams an tables, but I think the amount of content still needs to be a bit more concise. Your referencing for the most part is really good, however some parts in the development section are different, but it's nothing that can't be easily fixed. Overall, good job!

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* Introduction
** Justification of chosen topic was an interesting approach
* Developmental signalling processes
** Links between explored processes and heart development were made by could be more explicitly presented
* Current research and findings
** May have been too extensive at times
*** Felt like there was un-needed focus on methods used in the explored projects. Section could be streamlined to have less focus on methods and more highlighting of results of experiments presented
* Overall, well-structured and well written. However:
** Minor grammatical errors present throughout the page
** Some diagrams lacked descriptions and figure legends/abbreviation definitions – diagrams should be self-explanatory and be understandable in combination with their descriptions, when taken out of their contexts within the page
** Remember to remove zIDs before final submission

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GROUP 3

Introduction: Well written and concise - sets up a good expectation of whats to come Developmental Origin: I like how you have referenced the images in the text - though I would try to fix the image placement (it's hard - I am struggling also!) Developmental Timeline: This section is excellent - great amount of detail, well written, and great supporting images Developmental Signalling Processes: Also a very good section (just keep writing how you have for the rest of the subheadings!) - also need to fix up the referencing of that 2nd image Current Research And Findings: There is a commendable effort here to summarize current research. I might be nitpicking here but maybe try to make this section a little more concise as it is large blocks of text Glossary of Terms: Useful section to include

Overall: The level of research and depth of writing of this page is excellent. There isn't too much to change - and if the rest of the subheadings left to complete are in the same style as the rest of the page I think you guys should be pretty happy!

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'''Group 3- Heart'''

'''Regarding content:''' <br/>
Glossary is provided in which key terms are clearly defined for the reader. This is a commendable effort to make the project easy to understand. Overall, the project is also presented in simple terms making it easy to follow. Headings, subheadings and the use of paragraphs are also done appropriately. The topics chosen are all relevant to the topic.
However, for some topics, the information is a bit lengthy, such as that under the “Development Signalling Process”. It would also help to have the same structure throughout the project. There are variations under the headings. Perhaps highlighting key words by bolding or italics would make it easier to follow the text.

'''Referencing and Research:''' <br/>
Both referencing and research were done quite excellently in the project. The number of sources used were very extensive, and mostly being peer-reviewed articles. A variety of journals were also explored. The referencing list at the end had been properly listed and also throughout the project.
However, for some images, the references are not done correctly.

'''Other Comments:'''<br/>
A wide variety of useful images and diagrams have been used which enhances the understanding of the topic. There are good descriptions also provided when images are clicked on. However, it needs to be noted that on the page itself, descriptions of the images are missing. Adding a sentence or two would help. Also in some cases, the writing of the hand-written drawings was difficult to decipher.

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2017 Group Project 3

2017-10-26T03:29:05Z

Z5178463: /* Heart Tube Formation */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

The video above depicts the folding and fusion of Heart tubes taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Advanced_-_Heart_Tubes]

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

The video above depicts Cardiac Looping taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref name="PMID28007475><pubmed>28007475</pubmed></ref>At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref name="PMID28007475"/>

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise.
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T03:24:13Z

Z5178463: /* Heart Tube Formation */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>
The video above depicts the folding and fusion of Heart tubes taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Advanced_-_Heart_Tubes]

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

The video above depicts Cardiac Looping taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref name="PMID28007475><pubmed>28007475</pubmed></ref>At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref name="PMID28007475"/>

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise.
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T03:20:44Z

Z5178463: /* Heart Tube Formation */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>
The video above was taken from https://embryology.med.unsw.edu.au/embryology/index.php/Advanced_-_Heart_Tubes

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

The video above depicts Cardiac Looping taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref name="PMID28007475><pubmed>28007475</pubmed></ref>At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref name="PMID28007475"/>

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise.
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T03:15:45Z

Z5178463: /* Retinoic Acid */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

The video above depicts Cardiac Looping taken from [https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref name="PMID28007475><pubmed>28007475</pubmed></ref>At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref name="PMID28007475"/>

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise.
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T03:09:18Z

Z5178463: /* Retinoic Acid */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref name="PMID28007475><pubmed>28007475</pubmed></ref>At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise.
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T03:00:52Z

Z5178463: /* Transforming growth factor-β */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref name="PMID25813860><pubmed>25813860</pubmed></ref>
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise <ref><pubmed>3456489</pubmed></ref>
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-26T02:57:01Z

Z5178463: /* Retinoic Acid */

{{ANAT2341Project2017header}}


='''Heart'''=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the body. <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping - Student Image]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube.<ref name="PMID2794420><pubmed>2794420</pubmed></ref>

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart.<ref name="PMID2794420"/> The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy - Student Image]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 9) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors includes dickkopf-1(DKK1) which is an extracellular Wnt inhibitor that promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10: ''' Wnt signalling pathways - Student Image]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases.<ref name="PMID26793421><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions.<ref name="PMID26793421"/>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.11) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor ,called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11: ''' Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:''' Retinoic Acid activation pathway - Student Image]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. 12) <ref name="PMID25813860"/>

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart field which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===Sonic Hedgehog===

The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|400px|thumb|none|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 14) <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

From Figure 14, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 14A - 14D). Pericardial edema (Fig 14E) and non expanding cardiac chamber (Fig 14F) presented in E12.5 mutant embryo <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]

Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 14I and 14J). Fig 14G and 14H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 14K and 14L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 15A). Fig 15B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 15C and 15D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 15E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref name="PMID28846746><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders, such as heart block, long Q-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16: ''' Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline (Fig. 17). Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle. Therefore, Xenopus animal model has helped to have a better understanding or the role of CST in vertebrate cardiac development.

[[File:CST Is Required for Vertebrate Heart Development.PNG|300px|thumb|none|'''Figure 17: ''' CST Is Required for Vertebrate Heart Development]]

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 18: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 19: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 20:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>
[[File:Truncus Arteriosus.png|250px|thumb|left|'''Figure 21: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 22: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 23: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22.<ref name="PMID2651859"/> In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid.<ref name="PMID2651859"/> Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

===Role of bone morphogenetic protein (BMP) in cardiac neural crest development and migration===
The role of BMP has been implicated in many aspects of organogenesis including neural crest development and migration. However, it remains unclear to whether BMP impose a direct action on neural crest cells during cardiac morphogenesis or acts indirectly by affecting gene expression <ref><pubmed>15226263</pubmed></ref>. Also, studies of BMP receptor signaling have been hard to conduct as receptor deletion lead to death of the embryo at early stages of development. It was only recently that such studies of the receptor were performed by blocking the function of the receptor in neural crest cells. Thus, future experiments must be conducted to uncover the mechanism by which BMP act to regulate neural crest development and migration.

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|--bgcolor="FAF5FF"
|''' Anastomosis'''
| Term used to describe the connection between two tubes. Applied to describe the connection between peripheral blood vessels without an intervening capillary bed.
|-
| '''Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Axial mesoderm'''
| Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|--bgcolor="FAF5FF"
|'''Bulbar Ridge'''
| Term describing a spiral region in the developing heart bulbus cordis, there is a left and right bulbar ridge, that contribute to the septation of membranous portion of ventricular septum that is continuous with the outflow tract. These regions fuse to separate the single embryonic outflow tract into the aortic and pulmonary arteries.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|'''Cardiac Jelly'''
|Term used in early heart development to describe the initial gelatinous or sponge-like connective tissue separating the myocardium and the heart tube endothelium.
|-
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|--bgcolor="FAF5FF"
|'''Cyanotic Heart Disease'''
|Clinical term referring to a congenital heart abnormality (defect) resulting in lack of oxygen that causes cyanosis, a blue coloration of the skin and mucous membranes due to the presence of deoxygenated hemoglobin.
|-
|'''Ductus Arteriosus'''
|A prenatal vascular "shunt" which connects the left pulmonary artery and the descending aorta. Postnatal neonatal patency (patent ductus arteriosus, PDA) is a relatively common congenital cardiac anomaly occurring more frequently in premature infants.
|--bgcolor="FAF5FF"
|'''Endocardial Cushion'''
|Early heart development structures formed before atrial and ventricular septation occurs. These four heart wall in-folds (ventral, dorsal and two lateral) lie between the future atria and ventricles, later the posterior and anterior cushions fuse forming the primordial atrioventricular canals.
|-
|'''Endocardium'''
|The epithelial membrane lining the inside surface of heart, which along with the endothelial layer forms a continuous lining of the entire cardiovascular system.
|--bgcolor="FAF5FF"
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|-
|'''Heart Field'''
|The term used to describe the splanchnic mesoderm cardiogenic region in the trilaminar embryo that generates most of the heart. In humans, there are two fields the primary and secondary heart fields.
|--bgcolor="FAF5FF"
|'''Heart Valves'''
|The heart has a series of valves which regulate the directional flow of blood. The human heart has valves separating the atria from ventricles (atrioventricular, AV) and the ventricles from the outflow tract aortas. The left atrioventricular valve has two leaflets, anterior and posterior, and is the bicuspid valve or mitral valve. The right atrioventricular valve has a third leaflet (small, septal cusp) and is the tricuspid valve.
|-
|''' Inflow Tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|--bgcolor="FAF5FF"
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|-
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|--bgcolor="FAF5FF"
|'''Myocardium'''
|Layer that forms the muscular wall of the heart, the thickest layer formed by spirally arranged cardiac muscle cells.
|-
|''' Omphalomesenteric vein'''
| Vessels that provide the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|--bgcolor="FAF5FF"
|''' Outflow tract '''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|'''Truncus arteriosus'''
|An embryological heart outflow structure, that forms in early endocardial tube stage and will later divides into the pulmonary artery and aorta. Term is also used clinically to describe the malformation of the cardiac outflow pattern, where only one artery arises from the heart and forms the aorta and pulmonary artery (Persistent truncus arteriosus).
|-
|'''Vascular Endothelial Growth Factor (VEGF)
|A secreted protein growth factor family, which stimulates the proliferation of vascular endothelial cells and therefore blood vessel growth.
|--bgcolor="FAF5FF"
|'''Western Blotting'''
|An important technique used in cell and molecular biology. Researchers are able to identify specific proteins from a complex mixture of proteins extracted from cells with 3 techniques: (1) separation by size, (2) transfer to a solid support and (3) marking target protein using a proper primary and secondary antibody to visualise <ref><pubmed>3456489</pubmed></ref>
|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T13:23:19Z

Z5178463: /* Future Questions */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>. As a result, any defects occurring during the developmental periods can lead to congenital heart abnormalities.

Through researching the advances in technology, coupled with the biological use of suitable animal models our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development <ref name="PMID1767747"/>. This page will outline the embryonic development of the heart, the importance of how abnormalities arise, the treatments available and possible treatments that may be available in the future. Due to the certain knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 2: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 3:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 4:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 5:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 6:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 7:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development.<ref name="PMID3424040"/> Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 8:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions.<ref name="PMID3424040"/> The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF).<ref name="PMID24138816"/> The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue.<ref name="PMID24138816"/> The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis.<ref name="PMID3424040"/>

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart.<ref name="PMID3424040"/> The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves.<ref name="PMID5438177><pubmed>PMC5438177</pubmed></ref> The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve.<ref name="PMID5438177"/>

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet.<ref name="PMID3424040"/> The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve.<ref name="PMID3424040"/>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both of the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation]]

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===Sonic Hedgehog===

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|350px|thumb|left|'''Figure 13:''' Sonic Hedghehog (Shh) signalling pathway]]
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| '''Figure 14:''' Morphological defects in CTCF mutant embryonic hearts]]

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood.
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>.

From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|'''Figure 15:''' Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.

They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality <ref><pubmed>28846746</pubmed></ref>.

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

As the heart being the first organ to function during embryonic development (22 days in human, 8-8.5 days in mice) and is necessary for embryo survival. It depends on the sinoatrial node (SAN), the pacemaker of the heart’s electrical activity located in the right atrium of the heart and the conducting system, which transduces the electrical signal through the heart tissue for myocardial contraction. This system consists of atrioventricular node (AVN), atrioventricular bundle (AVB), bungle branches and Purkinje fibres, allows synchronized contraction of the atria and ventricles and a consistent heart rhythm. Thus, defects in the development of cardiac electrical function could lead to various heart disorders. Such as heart block, long T-T syndrome, atrial and ventricular fibrillation and tachycardia <ref name="PMID16643374><pubmed>16643374</pubmed></ref>

Epidermal Growth Factor Receptor 2 (Erbb2) is a transmembrane protein that is essential for normal embryonic development <ref name="PMID25269082><pubmed>25269082</pubmed></ref>.
It is known that at mid-gestation knockout mice lacking Erbb2 are lethal and die due to severe cardiac defects that show enlarged heart with thin ventricular myocardium, absent trabeculae and decreased atrioventricular cushions. Consequently, it results in poor circulation and irregular heartbeat <ref name="PMID7477377><pubmed>7477377</pubmed></ref>. Other studies suggested that Erbb2 participates in ventricular conduction system development and maturation. However, the role of Erbb2 in atrial conduction system development is still unknown.
In a study by Tenin et. al (2014), they showed that upon mutation in Erbb2 in I11Jus8 mice resulted in specific defect in atrial electrical propagation and thus displayed cardiac hemorrhage and failure in atrial function but did not affect ventricular conduction. The role of Erbb2 in I11Jus8 mouse line reported to have significant impact on the atrial function, which allows the embryo to survive. <ref name="PMID25269082><pubmed>25269082</pubmed></ref>. Although, other studies revealed that embryonic cardiac defects are not found in mice with deletion of Erbb2, it has yet to determine whether non-cardiomyocyte tissue requires Erbb2 function for cardiac conduction system development in Tenin and his colleagues’s experiment. Further more, it has been proposed that Erbb2 functions in adult cardiomyocytes where deletions of Erbb2 causes early cardiac dysfunction and dilated cardiomyopathy <ref name="PMID12072561><pubmed>12072561</pubmed></ref>.
It was also demonstrated that dilated cardiomyopathy in human and mice contributes to failure of the conduction system leading to arrhythmias and sudden death <ref name="PMID12072561><pubmed>12072561</pubmed></ref>,<ref name="PMID25269082><pubmed>25269082</pubmed></ref>

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors disrupt its production can cause a NAD deficiency resulting to cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in food such as meats and green vegetables. Research shows at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than it is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step would be to develop a diagnostic test to measure NAD levels. This would enable doctors to identify the women who are at greater risk of having a baby with a birth defect, and ensure they are getting sufficient amount of vitamin B3.

==Animal Models ==

===Mice Model===
Understanding the importance of different molecular pathways in heart development was made possible by studies conducted in different animal models including mice models. Such studies, in particular loss of function analysis studies, have allowed for the determination of specific roles of various signaling pathways in specific stages of cardiac development. For example, one of the studies conducted in mice embryo involved studying the effect of ALK2 receptor deletion <ref><pubmed>15226263</pubmed></ref>. This receptor is expressed on cardiac neural crest cells which contribute to the formation of cardiac outflow tract (OFT). Bone morphogenetic protein (BMP), an important protein in heart signaling processes, acts as a ligand for this receptor. Thus, by binding to this receptor, BMP play a role in neural crest development and migration. Therefore, since this receptor function to determine the downstream signaling specificity, understanding its function will allow the mechanisms by which BMP regulate heart development to be uncovered. However, the receptor was not deleted as this will lead to mice lethality, but instead its function was abolished in neural crest cells. Mice with abolished ALK2 receptor function revealed various cardiac defects such as aortic arch defect and cardiac OFT defect. These defects are very similar to the common human congenital heart disease. This occurrence of these cardiac defects is indicative of impaired neural crest cell migration and it provides evidence for the importance of BMP in heart development.

Since the origin of cells contributing to different parts of the valves remains controversial, another research on mice model <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labelled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different components of the valves. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin. Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves, the atrioventricular fibrous continuity and the leaflets of the aortic and pulmonary valves. However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Zebrafish Model===
[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px|thumb|'''Figure 16:'''Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos]]
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. 15). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

Other research into chick models reveals that cardiac progenitors of the splanchnic mesoderm, cardiac neural crest and the proepicardium are the major embryonic contributors to the development of the chick heart. The contribution of these components to cardiac development occurs with precise timing and regulation during such processes as primary heart tube fusion, cardiac looping and accretion, cardiac septation and the development of the coronary vasculature. In addition to these findings, chick models revealed that one role of the anterior endoderm is to create a “cardiac field” in the overlying anterior mesoderm, and that other events will restrict this cardiac field to the region of the embryo that will become the heart later in development.<ref><pubmed> 15986452</pubmed></ref>

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|300px|thumb|right|'''Figure 17: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure 18: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|300px|thumb|right|'''Figure 19:''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure 20: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies.<ref name="PMID21524457><pubmed>21524457</pubmed></ref> PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk.<ref name="PMID4021394><pubmed>PMC4021394</pubmed></ref> This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations.<ref name="PMID27126954><pubmed>27126954</pubmed></ref> Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation.<ref name="PMID4021394"/> PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year.<ref name="PMID21070356><pubmed>21070356</pubmed></ref>

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|250px|thumb|right|'''Figure 21: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births.<ref name="PMID2651859><pubmed>PMC2651859</pubmed></ref> It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations.<ref name="PMID2651859"/> The condition is characterised by four heart abnormalities:<ref name="PMID20091166><pubmed>20091166</pubmed></ref>

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn.<ref name="PMID20734579><pubmed>20734579</pubmed></ref> This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect.<ref name="PMID4460219><pubmed>PMC4460219</pubmed></ref> This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation.<ref name="PMID4460219"/>

Newborns with TOF typically undergo corrective surgery within the first month of life.<ref name="PMID2651859"/> Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta.<ref name="PMID4460219"/>

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|250px|thumb|right|'''Figure 22: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves.<ref name="PMID1877799><pubmed>PMC1877799</pubmed></ref> Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life.<ref name="PMID5313512><pubmed>PMC5313512</pubmed></ref> Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta.<ref name="PMID1877799"/>

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation.<ref name="PMID5313512"/> Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn.<ref name="PMID1877799"/> Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation.<ref name="PMID4460219"/>

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle.<ref name="PMID5313512"/> Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return.<ref name="PMID1877799"/> Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the now connected pulmonary artery and superior vena cava to the wall of the right atrium.<ref name="PMID1877799"/> These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system.<ref name="PMID5313512"/>

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===What is the Aetiology of Tetralogy of Fallot?===
The aetiology of Tetralogy of Fallot is complex and still poorly understood. Current research in genetics has linked the condition with various chromosomal abnormalities, including trisomy 13, 18 and 21, as well as microdeletions within chromosome 22. In addition, current research has identified risk factors such as maternal diabetes and exposure to retinoic acid. Further research in this area is required to achieve a greater understanding of the underlying causes of Tetralogy of Fallot.

===Regulation of forces involved in cardiac looping===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

===Specific identification of cardiac precursors===
Heart precursors were identified as occupying anterior mesoderm, proximal to the embryonic-extraembryonic boundary. This identification was achieved utilising cell transplantation, labelling of live embryos and conducting embryo culture. These embryonic manipulations highlighted the plasticity of populations that contribute to the heart. This means cells obtained from a different location or developmental time point can contribute to the heart once placed in the proper location. Subsequently, such experiments <ref><pubmed>23457256 </pubmed></ref> were able to locate the cardiac precursors but they failed to specifically identify these precursors and therefore cardiac precursors have not been identified and characterised yet. Thus, future experiments will aim to address this question.

===Cardiac Regeneration===
There are vast differences between the potential of cardiac regeneration in experimental models like zebrafish which is lost in adult mammals like humans. Researches speculate that it could be due to internal properties of cardiomyocytes or due to the failure of stem cell populations. They also hypothesize that it could be due to non-cardiomyocytes that could cause scarring which might hinder regeneration. In addition, more research has to be done on how regeneration begins and ends (signalling pathways involved etc).

==Glossary of Terms==

Definitions provided in this glossary are taken from lectures presented in the UNSW embryology course ANAT2341, as well as from the UNSW embryology wiki glossary provided online, which is linked below.

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"
|''' Cardiomyopathy'''
| Cardiomyopathy is a disease where the heart muscle becomes inflamed and enlarged and affects the way that the heart muscle pumps.

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

User:Z5178463

2017-10-25T08:08:28Z

Z5178463:

==Peer Reviews==

===Group 1(Cerebral Cortex)===

Overall, the page has a good structure and flow with good headings and subheadings. The information provided was concise and easy to comprehend. The introduction provides a brief overview and sufficient background knowledge about the cerebral cortex. I like how the team thought of mentioning about the early development of the brain before narrowing it down to the cerebral cortex. However these two sections do not seem to flow well. Maybe you could have 2-3 sentences that could help ease into the development of the cerebral cortex. I really love the timeline of corticogenesis. This part has been done really well. One minor improvement that could be made is to add images under each embryonic stage instead of just the last stage to better aid the reader into understanding the development. Also, a brief description of what corticogenesis is could be included before the table. For these two sections, there were a good amount of references.

For the anatomy of the cerebral cortex, it seems a little messy and hard to understand as its written in point forms. Perhaps, the dot points could be changed to proper sentences with histological images to tie it together. For the functions of the cerebral cortex, I think you could use a table to list down the areas and then provide a brief description of the functions of that particular part. The video is a good addition to the page. These two sections are lacking citations and references.The abnormalities section was well done. However, the citations should be added within the text instead of at the top of the page. Since there are a lot of abnormalities, maybe the team could list in a few sentences about all the abnormalities that they are going to discuss to have a better start to the section. For the images that are used on this page, the images should be labelled as “figure 1” or “table 1”. Maybe, sections on the “animal models” and “current research” could be added to wrap the page up.

===Group 2(Kidney)===

Overall, this project page is easy to read. Most of the information provided is very concise and specific. For the anatomical position and kidney structure, do remember to add in the references in the text. Before using the short form, do include the full name. For example Thoracic 12 (T12) instead of T12. I really appreciate the timeline of development table as it provides a brief overview before moving onto the details. The section of kidney development is well done with good subheadings to help with the flow of the content. However, more images or videos can be included for better understanding. Again, for the “nephrogenesis” and “ascension” and “genes expressed” section, its lacking references. For the developmental abnormalities, maybe a subheading could be used to categorise the first few paragraphs of information as it was hard to understand the flow of the content. Since it was mentioned that “there are defects in different stages of kidney development”, the team could use this as a basis in arranging the information. Perhaps, the team could assign one abnormality for each stage of the kidney development. I think that would help the section have a better flow. The team have also stated that the information for blood supply and current research is still ongoing. For the images, some images are lacking referencing, the copyright statement and also a brief description explaining the image. This team has kept their page simple and easy to understand. With a few more added information and slight tweaks, It would be a really good page.

===Group 4(Eye)===

Overall the page looks neat in the arrangement of the information. Before the anatomy of the eye, maybe a short paragraph on the general information of the eye could be included to have a good introduction to the project page. For the anatomy of the eye, there is a fair amount of information and good images to support the information. If the group wants to take this section a little further, they could include histological images. For the images that were drawn, perhaps a brief description could be included. For the overview of the eye development, I really like how there was a general table foe the different weeks of development and then another following table with the carniage stages. This helped the reader to have a broad overview before narrowing down to the specifics. However, I think images re needed to understand the stages better because its hard to picture the development without any pictorial aid. Also, I think the headings and subheadings for this part may need to be modified. Maybe you can start off with “Development of the Eye”. and instead of “short overview”, you can change it to “An overview of Eye Development”. Also, all the information was taken from only one source so maybe more articles could be sourced in order to have more credibility.

For the development of the eye components, the content is sufficient and concise but more images are necessary as some parts gets a little confusing. There is a good amount of references for this section. For the congenital abnormalities, the table is a good way to present the information. However, more information about the abnormalities is needed under the description column. As for the images, I think maybe you could create another column and add the image to that row for each abnormality. This would give the table a more complete look and the section will be really good. There is also good amount of references and the images are correctly referenced and the copyright statements are included so that’s well done.

===Group 5(Lung)===

Overall, this page has a good arrangement of information. For the lung anatomy, histology and cardiovasculature, the content is concise and good. The images were all self drawn and a lot of effort has been put to it. Good job to the person who did it. However, for the lung anatomy, histology and cardiovasculature, there are no references at all. Also, for the lung histology, perhaps adding in histological images and referencing it when writing the text would make the section better. The developmental timeline was also very well done. I love how all the information was presented in a table and was easy to follow through. The images had their copyright statements, brief overview and proper referencing. Again, there are no references for the structure of respiratory network and its sub sections and for the developmental signalling sections. Also, the images should be labelled as figure 1 or table 1 and could be mentioned in the text where appropriate. Perhaps a glossary could benefit this page. The abnormalities section was well referenced and there was a fair amount of abnormalities covered. Maybe more images could be added.

===Group 6(Cerebellum)===

Overall, I think this project page is really good and well done to the team. I think the headings and subheadings flow easily and there is a good arrangement of information. There is a good amount of referencing and the images have copyright statements and brief descriptions. For the “Neural Development” subsection, instead of placing it under the anatomy of the cerebellum, I think you should move it down to the development section as it has more relevance to that. I think the Cerebellum Developmental weeks should be shifted to before the description on cerebellum development. This way, the readers can have a general idea on the development and its stages before going through he description because the description is quite content heavy and if we were to read that first, its quite confusing and hard to understand. For the key historical discoveries, maybe you could use a table with two columns where one column can be the name of the discoverer and the other column could be a brief description. The abnormalities section was done well.

---

2017 Group Project 3

2017-10-25T07:39:30Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref><ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/><ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 13 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 14 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:36:56Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref>. <ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/> . <ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 13 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 14 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:33:58Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400><pubmed>23633400</pubmed></ref>. <ref name="PMID12923046><pubmed>12923046</pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery.

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 13 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 14 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:28:42Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref name="PMID23633400 ><pubmed>PMC23633400 </pubmed></ref>. <ref name="PMID12923046 ><pubmed>PMC12923046 </pubmed></ref>.

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery.

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 13 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 14 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:18:26Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 13) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 13 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 13, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 13A - 13D). Pericardial edema (Fig 13E) and non expanding cardiac chamber (Fig 13F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 13I and 13J). Fig 13G and 13H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 13K and 13L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 14 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 14A). Fig 14B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 14C and 14D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 14E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:12:24Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/>. <ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:08:42Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID23633400"/> <ref name="PMID12923046"/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:06:04Z

Z5178463: /* Cardiac Neural Crest and Outflow tract */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref name="PMID1767864"/> <ref name="PMID 23633400 "/>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T07:02:25Z

Z5178463: /* Introduction */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref name="PMID1767747"/> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T06:55:04Z

Z5178463: /* Secondary Heart Field */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation. <ref name="PMID1767747"/>

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

[[File:Development of the Semilunar Valves.jpg|400px|thumb|right|'''Figure 7:''' Development of the Semilunar Valves.]]

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T06:31:22Z

Z5178463: /* Primary Heart Field */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape. <ref name="PMID1767747><pubmed>PMC1767747</pubmed></ref>.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space. <ref name="PMID1767747"/>

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Heart Valve Development====

'''Gestational Week 8'''

Heart valve development commences in the endocardial cushions of the atrioventricular canal (AVC) and outflow tract (OFT) during the 7th week of embryological development [2]. Valve morphogenesis is comprised of four stages:

*Epithelial-to-mesenchymal transformation (EMT)
*Growth
*Remodelling
*Apoptosis

Development of the valves begins with the transformation of the endocardial cells into mesenchymal cells within the endocardial cushions [2]. The endocardial cushions then expand through cell proliferation and synthesis of the extracellular matrix, which is primarily regulated by vascular endothelial growth factor (VEGF) [1]. The elongating cushions then undergo remodeling, as mesenchymal cells differentiate into collagen and other fibrous tissue [1]. The valve leaflets achieve their thin, unique shape as cells near the base of the extending cushions undergo apoptosis. [2]

The semilunar valves differentiate from the conotruncal and intercalated endocardial cushions located at the outflow tract of the heart. The superior and inferior septal conotruncal cushions contribute to the left and right cusps of both the pulmonary and aortic valves. The right-posterior intercalated cushion contributes to the posterior leaflet of the aortic valve, while the left-anterior leaflet gives rise to the anterior leaflet of the pulmonary valve [3]

The atrioventricular valves arise from the atrioventricular (AV) endocardial cushions. As the atrioventricular canal divides during cardiac septation, fusion and proliferation of the superior and inferior AV endocardial cushions results in the formation of the anterior mitral leaflet and the septal tricuspid leaflet [2]. The right lateral AV cushion differentiates into the anterior and posterior leaflets of the tricuspid valve, while the left cushion gives rise to the posterior leaflet of the mitral valve. [2]

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T06:15:55Z

Z5178463: /* Heart Tube Formation */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction <ref name="PMID16567300><pubmed>16567300 </pubmed></ref>.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle <ref name="PMID17796013><pubmed>17796013 </pubmed></ref>
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|350px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space <ref><pubmed> PMC1767747 </pubmed></ref>.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk .<ref name="PMID1767747"/>

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|thumb|none|'''Figure 9:''' Signalling pathways in heart development]]

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px||thumb|none|'''Figure 10:'''Wnt signalling pathways]]

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png|500px||thumb|none|'''Figure 11:'''Regulation of Nodal-Activin signalling during heart formation<ref name="PMID25813860"/>]]

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|'''Figure 12:'''Retinoic Acid activation pathway ]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

[[File:The Sonic Hedghehog (Shh) signalling pathway.png|500px|thumb|none|'''Figure 12:''' Sonic Hedghehog (Shh) signalling pathway<ref name="PMID27507209"/>]]

==Current Research And Findings==

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

===Dietary supplementation of NAD in prevention of miscarriages and birth defects===
Sydney’s Victor Chang Cardiac Research Institute is the centre point of research to prevent recurrent miscarriages and multiple types of birth defects of the heart, spinel, kidney and cleft palate in newborn babies.

Scientists have demonstrated a potential cure, in the form of a common dietary supplement. The research study found that a deficiency in a vital molecule, known as NAD, can prevent a baby’s organs from developing correctly in the womb.
Nicotinamide adenine dinucleotide (NAD) is one of the most important molecules in all living cells. NAD synthesis is essential for energy production, DNA repair and cell communication. Environmental and genetic factors can disrupt its production, which can cause a NAD deficiency, which as a result can cripple an embryo when it forms.
The dietary supplement vitamin B3 is required to make NAD and is found in meats and green vegetables. Research show at least a third of women have low levels of vitamin B3 in their first trimester of pregnancy, which is the critical time in organ development. There is an indication that pregnant women may require more vitamin B3 than is currently available in most vitamin supplements.
Using a preclinical mouse model, vitamin B3 was introduced into the mother’s diet. The dietary change prevented both the miscarriages and birth defects completely. This discovery can be likened to the revolutionary breakthrough made last century that confirmed folic acid supplementation can prevent spina bifida and other neural tube defects in babies.

The next step will be to develop a diagnostic test to measure NAD levels. This will enable doctors to identify the women who are at greatest risk of having a baby with a birth defect, and ensure they are getting sufficient vitamin B3

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

===Regulation of forces involved in cardiac looping?===
Studies have identified some mechanical forces involved in looping, it remains to be established how these forces are regulated spatially and temporally to produce a looped heart tube. It needs to be further investigated to determine whether the proposed actin polymerization mechanism can produce the necessary changes in tissue shape on a global scale. In addition, the possible roles played by redundant mechanisms have not been elucidated. Finally, the mechanisms of initial looping of the heart tube and s-looping have received relatively little attention.
To gain a complete understanding of heart development requires finding the link between gene expression and morphomechanics, which will need to combine the expertise of developmental biologists and biomechanical engineers <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

===Signalling of the heart fields===
Multiple signalling pathways overlap to regulate SHF cell addition to the poles of the heart tube, as well as SHF proliferation and myocardial differentiation. The signalling molecules identified to highlight the continuance of SHF cells in a progenitor cell state and controls their progressive differentiation. However, the control of differentiation delay and SHF movement into the OFT remains obscure. Future research into gene regulatory networks and signalling pathways that control SHF development will identify mechanisms underlying these processes. This will provide a greater understanding of the extent to which these networks differ from those controlling differentiation of the linear heart tube.
Further exploration of the mechanisms underlying SHF progenitor cell development in the early embryo is required, as it will uncover the potential of progenitor and pluripotent stem cells for cardiac repair <ref name="PMID 19390062 ><pubmed> 19390062 </pubmed></ref>.

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T02:30:14Z

Z5178463: /* Cardiac Stem Cells */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|250px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk <ref><pubmed> PMC1767747 </pubmed></ref>.

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

[https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

z5059996

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|'''Figure 9:''' Signalling pathways in heart development]]

<ref><pubmed>12781678</pubmed></ref>

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px]] <pubmed>20830688</pubmed>

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png| '''Figure 10:''' Regulation of Nodal-Activin signalling during heart formation]] <ref><pubmed>25813860</pubmed></ref>.

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade <ref><pubmed>25206052</pubmed></ref>.

Shh signaling contributes to myocytes specification and a reduction in this signaling can cause a cardiomyocyte deficit whereas increased Shh signaling lead to a surplus. At early stages of cardiac development, Shh signaling induces a proper number of myocardial progenitor cells. These progenitor cells show a direct respond to SHH signals allowing it to control and determine the optimal number of cardiomyocytes <ref><pubmed>15936751</pubmed></ref>. At later stages of cardiac development, Shh plays an important role in survival and proliferation of neural crest cells (NCCs) and it maintains the proliferation of progenitor cells within the second heart field. These actions allow Shh to promote outflow tract morphogenesis. However, Shh does not act as a direct survival factor for NCCs, but rather, it acts indirectly by inducing a survival factor and inhibiting an apoptotic factor. NCCs of Shh null mice embryos show specification and migration but they are mislocalized and undergo apoptosis. Also, mice that lack Shh develop many cardiac abnormalities such as outflow tract shortening, ventricular hyperplasia and septation defects<ref><pubmed>18842815</pubmed></ref>.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|Figure Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

==Current Research And Findings==
https://www.victorchang.edu.au/heart-research/embryology

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

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===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

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===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

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===Persistent Truncus Arteriosus===

[[File:Truncus Arteriosus.png|250px|thumb|right|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]
Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

Hypoplastic Left Heart Syndrome (HLHS) is a congenital heart defect that involves the underdevelopment of cardiac structures of the left side of the heart; structures effected include the left ventricle, ascending aorta, aortic arch and the mitral and aortic valves [2]. Despite being one of the rarer heart defects, accounting for up to 3.8% of congenital cardiac malformations, HLHS is responsible for 23% of all cardiac-related deaths in the first week of life [1]. Severity of the disease depends on the extent of systemic outflow obstruction, the number of heart structures effected, and the degree of hypoplasia of the left ventricle and ascending aorta [2].

Initially, newborns with HLHS will appear healthy. This is because the presence of the foramen ovale and a patent ductus arteriosus allows blood to bypass the malformed left side of the heart and enter systemic circulation [1]. Once these structures close, inadequate blood flow in the systemic circulatory system results in hypoxemia and cardiogenic shock; rapid surgical intervention is required to prevent death of the newborn [2]. Corrective surgery for neonates with HLHS is performed in three stages, with the ultimate goal of increasing systemic circulation and bypassing the malformed side of the heart; the end result is a modified right ventricle that supports both systemic and pulmonary circulation. [3]

The first operation, known as the Norwood operation, is performed at birth. This involves the construction of a new ascending aorta and arch that extends from the right ventricle, as well as the establishment of a communication between the right ventricle and the pulmonary trunk, which allows for both pulmonary and systemic perfusion from the right ventricle [1]. Next, a Glenn shunt operation is performed at 6-8 months after birth, in which a surgical anastomosis is created between the right pulmonary artery and superior vena cava, which decreases the work on the right ventricle during venous return [2]. Finally, a Fontan procedure is performed 1.5-4 years after birth, which involves establishing a communication between the connected pulmonary artery and superior vena cava to the wall of the right atrium [2]. These surgeries collectively result in the development of univentricular circulation in which oxygenated and de-oxygenated blood are unable to mix, which increases the efficiency of the neonate’s cardiovascular system. [1]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity which did not form teratomas
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

What is the secreted protein that flows across the node, and what is the mechanism that ensures that the loop turns in rightward direction?

What are the developments of stem cell usage for developmental abnormalities?
http://circres.ahajournals.org/content/91/3/189.short

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T01:50:13Z

Z5178463: /* Cardiac Septation */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|250px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk <ref><pubmed> PMC1767747 </pubmed></ref>.

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

[https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
'''Gestational Week 6-7'''

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

z5059996

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|'''Figure 9:''' Signalling pathways in heart development]]

<ref><pubmed>12781678</pubmed></ref>

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px]] <pubmed>20830688</pubmed>

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png| '''Figure 10:''' Regulation of Nodal-Activin signalling during heart formation]] <ref><pubmed>25813860</pubmed></ref>.
===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|Figure Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

==Current Research And Findings==
https://www.victorchang.edu.au/heart-research/embryology

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

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===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

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===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

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===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

[[File:Truncus Arteriosus.png|300px|thumb|left|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

What is the secreted protein that flows across the node, and what is the mechanism that ensures that the loop turns in rightward direction?

What are the developments of stem cell usage for developmental abnormalities?
http://circres.ahajournals.org/content/91/3/189.short

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T01:38:53Z

Z5178463: /* Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|250px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk <ref><pubmed> PMC1767747 </pubmed></ref>.

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

[https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
=====Gestational week 6-7=====

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

z5059996

====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|'''Figure 9:''' Signalling pathways in heart development]]

<ref><pubmed>12781678</pubmed></ref>

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px]] <pubmed>20830688</pubmed>

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png| '''Figure 10:''' Regulation of Nodal-Activin signalling during heart formation]] <ref><pubmed>25813860</pubmed></ref>.
===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|Figure Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

==Current Research And Findings==
https://www.victorchang.edu.au/heart-research/embryology

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.<ref name="PMID28211263><pubmed>28211263</pubmed></ref>

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

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===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

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===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

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===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

[[File:Truncus Arteriosus.png|300px|thumb|left|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

What is the secreted protein that flows across the node, and what is the mechanism that ensures that the loop turns in rightward direction?

What are the developments of stem cell usage for developmental abnormalities?
http://circres.ahajournals.org/content/91/3/189.short

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T01:35:16Z

Z5178463: /* Retinoic Acid */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|250px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk <ref><pubmed> PMC1767747 </pubmed></ref>.

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

[https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
=====Gestational week 6-7=====

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

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====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|'''Figure 9:''' Signalling pathways in heart development]]

<ref><pubmed>12781678</pubmed></ref>

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px]] <pubmed>20830688</pubmed>

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png| '''Figure 10:''' Regulation of Nodal-Activin signalling during heart formation]] <ref><pubmed>25813860</pubmed></ref>.
===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|400px|thumb|right|Figure Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

==Current Research And Findings==
https://www.victorchang.edu.au/heart-research/embryology

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

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===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

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===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

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===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

[[File:Truncus Arteriosus.png|300px|thumb|left|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

What is the secreted protein that flows across the node, and what is the mechanism that ensures that the loop turns in rightward direction?

What are the developments of stem cell usage for developmental abnormalities?
http://circres.ahajournals.org/content/91/3/189.short

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>

2017 Group Project 3

2017-10-25T01:33:18Z

Z5178463: /* Retinoic Acid */

{{ANAT2341Project2017header}}


=Heart=

==Introduction==
The cardiovascular system is the first system to develop and function in the human embryo. Rapid cardiac development is essential as the growing embryo can no longer receive oxygen and essential nutrients via diffusion alone, hence a circulatory system and a contractile heart mechanism is required to supply the embryo.
We recognise the hearts normal development is vital for foetal life, and hence we have chosen to document the development of the heart from gastrulation to birth. Any defects occurring during the developmental processes can lead to congenital heart abnormalities. However, early cardiac development is a multifaceted procedure and is associated with other developmental processes such as: embryonic folding, coelom formation, and vascular development <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

Through researching the advances in technology, coupled with the biological use of suitable animal models <ref><pubmed> PMC1767747 </pubmed></ref> our understanding of embryological cardiac development has evolved, and we are piecing together the mechanism underlying this development. This page will outline the importance of how heart abnormalities arise, the treatments available and the possible treatments to be developed in the future. Due to the major knowledge gaps in current embryological heart research, we acknowledge that this will impact our assignment, and aim to address further research concepts that will improve our understanding.

==Anatomy of the Heart==

[[File:Basic anatomy of the heart.png|200px|thumb|right|'''Figure 1:''' Basic anatomy of the heart]]

The heart is the muscular organs that pumps blood around the body via the circulatory system. It is located within the thoracic cavity, in a compartment called the mediastinum. The heart is divided into four chambers including: left and right atria and ventricles which are compartmentalised by semilunar and atrioventricular valves. Blood moves via the systemic circuit to the organs of the body and back to the heart. The pulmonary circuit is responsible for the flow of blood between the lungs and the heart. Deoxygenated blood enters the right side of the heart, while Oxygenated blood returning from the lungs exits the left side. The heart’s electrical system uses electrical signals to cause the muscular walls to contract. The mechanical pumping of the heart is essential for movement of blood which exchanges gases and essential nutrients between organs of the bod <ref><pubmed> PMC1571338 </pubmed></ref>.

==Developmental Origin==

In the developing embryo the lateral plate mesoderm splits into somatic and splanchnic layers, the latter is comprised of cardiac progenitor cells. The somatic mesoderm lines the ectoderm, the splanchnic mesoderm lines the endoderm, and in between lies an embryonic coelom <ref><pubmed> PMC2691808 </pubmed></ref> . At the end of week three, the heart develops from splanchnic mesoderm of the cardiogenic region of the embryonic plate. At the cranial end of the embryo, anterior to the developing neural tube is the initial origin of heart formation.

[[File:Pericardial Development 4-6 Gestation weeks .jpg|250px|thumb|right|'''Figure 3: '''Pericardial Development 4-6 Gestation weeks ]]
Originally, the cardiogenic region forms laterally of the paraxial mesoderm and primitive streak of the embryonic disc. Due to the natural mesenchymal cell organisation of the mesoderm, cells ‘migrate’ and fuse at the midline of the cranial end of the embryo forming the cardiac crescent just prior cardiac tube formation and folding. Endoderm surrounding the primitive gut contracts bringing the cardiogenic precursor regions of the splanchnic region towards the midline. Early heart formation begins with angioblastic cords of the splanchnic mesoderm. The angioblastic cords develop into separate endochondral heart tubes moving closer together as the foregut pinches together and the yolk sac contracts into the embryo. Fusion of the two heart tubes is facilitated by apoptosis. The origin of the primitive heart is in the early pericardial coelom, which is later developed into the pericardial cavity, through fusion of the pleuropericardial folds to separate the pleural cavities at the later development of the lungs <ref><pubmed> PMC4374196 </pubmed></ref>.

==Developmental Timeline==

{| class="wikitable"
! style="background:#f4a941" | WEEK
! style="background:#f4a941" | DEVELOPMENT
! style="background:#f4a941" | HISTORIC CARDIOVASCULAR DISCOVERIES
|-
| 2 || Bilateral cardiogenic areas form
|| In 157 AD Galen (born on 9 September AD 129 in Pergamon, Greece) discovered the pulmonary circulation.
|-
| 3 || Mesoderm splits, Heart tubes are brought to the midline, Heart tube fusion, Heart beat
|| Realdo Columbo (1515–1559 discovered that the heart’s four valves permitted flow of blood in one direction only: from the right ventricle to the lungs, back to the left ventricle, and from there to the aorta.
|-
| 4 || Heart looping, Neural crest migration commences, Dorsal and ventral endocardial cushions fuse
|| William Harvey (born 1 April 1578) while studying in London estimated:
* the capacity of the heart was 43 g
* about 6 g of blood went through the heart every time it pumped
* the heart beats 1 000 times every half hour.
* therefore, the heart pumps about 5 kg of blood in a half hour, or about 245 kg in a day
|-
| 5 || Foramen primum closed, Septum secundum srtats developing, Muscular interventricular septum develops, Bulbar ridges and trabeculation become evident
|| Harvey proved blood flows in two separate loops: the pulmonary and systemic circulation <ref><pubmed>PMC3721262</pubmed></ref>.
|-
| 6 || Aortic and pulmonary trunks cleave
|| The earliest study of the partitioning of the heart was conducted by cardiologist and anatomist Wilhelm His Jr (1863-1934) in 1886. Later in 1893 he discovered the ''bundle of His'' -the specialized tissue in the heart that transmits the electrical impulses and helps synchronize contraction.
|-
| 7 || Valves develop
|| Franklin P. Mall (1862-1917) worked at the Department of Embryology at the Carnegie Institute of Washington where he studied the subdivisions of the primary heart tube, formation of the atrio-ventricular valves and bundle, and the musculature of the left ventricle. Additionally, in 1910 he demonstrated that the nascent atrium of the heart could be identified based on the close proximity of endothelium to the heart muscle
|}

====Primary Heart Field====

[[File:Morphology of Heart Tube Formation- Student Image .png|250px|thumb|right|'''Figure 4:''' Morphology of Heart Tube Formation- Student Image]]
'''Gestational Week 4-5'''

The first cells start to migrate through the primitive streak to the anterior and lateral sections of the cranial end of the embryonic disc, forming bilateral primary heart fields. These primary heart fields resemble a crescent shape.

The lateral plate mesoderm is split into two layers, namely the splanchnic mesoderm, facing the endoderm and the somatic mesoderm, facing the ectoderm. The former portion of the mesoderm gives rise to the heart. The portion between the splanchnic and somatic mesoderm is the presumptive pericardial space. Cells from the splanchnic mesoderm will merge to form 2 lateral endocardial tubes (also known as angioblastic cords) and as they form a lumen, are enveloped by myocardium. These endocardial tubes are as of now located inferior to the presumptive pericardial space.

====Heart Tube Formation====

'''Gestational Week 5'''

The embryonic disc starts to fold. This folding begins cranially and extends in a caudal direction. The endocardial tubes fuse and is now located between the pericardial space and newly formed foregut that becomes surrounded by pericardial space (also known as the pericardial coelom). At this stage, the myocardium does not completely engulf the endocardial tubes. Instead, it remains in a continuous attachment with the non-cardiac splanchnic mesoderm through a structure called the dorsal mesocardium.

At this point, the primitive heart tube is bilaterally symmetrical and resembles an inverted Y shape. Starting from the inflow tract, there is the right and left sinus venouses that receives blood from the embryo, followed by the primitive atrium, primitive ventricle, bulbus cordis and then the truncus asteriosus which gives rise to the aortic and pulmonary trunk <ref><pubmed> PMC1767747 </pubmed></ref>.

<html5media height="200" width="240">File:Heart_folding_001.mp4</html5media>

====Secondary Heart Field ====

'''Gestational Week 5-6'''

The secondary heart field (SHF) is a region of subpharyngeal mesodermal progenitor cells located medially and ventrally to the adjacent primary heart field (PHF) (that forms the initial heart tube). The PHF and SHF cells make up a region known as the cardiogenic field. Following heart tube fusion around 19-21 days, cells from the SHF migrate to the cranial and caudal ends of the tube and continue elongation <ref><pubmed> PMC1767747 </pubmed></ref>.

[[File:Schematic diagram of heart tube looping.png|250px|thumb|left|'''Figure 5:''' Schematic diagram of heart tube looping]]
It is understood that within the cardiac regions the progenitor cell population of the SHF is “pre-patterned”<ref>http://perspectivesinmedicine.cshlp.org/content/4/10/a015750.full</ref>, hence being termed “specified but undifferentiated”. It is the patterning of cells within the soon to become myocardium, that is responsible differentiation into chamber-specific myocytes (atrial and ventricular) and the conduction of cells. It is essential that the SHF cells remain undifferentiated and do not add prematurely to the heart tube <ref><pubmed> PMC2794420 </pubmed></ref>.

The SHF cells gives rise to endocardial, myocardial and smooth muscle cells through expression of transcription factor Islet -1 <ref>http://www.sciencedirect.com/science/article/pii/S1875213613002817 </ref>. It also contributes to the right ventricle, inflow and outflow tract (OFT), and the arteries and semilunar valves that meet to form the arterial pole of the heart <ref><pubmed> PMC2794420 </pubmed></ref>. The OFT is the point of exit from the heart via structures of early cardiac development that give rise the essential structures of the aorta and pulmonary artery. The SHF of the developing heart and its contribution the arterial pole involves complex and interconnected signalling pathways that will be covered later in further detail.
[[File:Cardiac Development Overview .jpg|500px|thumb|right|'''Figure 6:''' Cardiac Development Overview]]

====Cardiac Looping and Steps====

'''Gestational Week 6'''

In the early embryo, the same progenitor cells that constitute the SHF are responsible for the rapid growth occurring during cardiac looping morphogenesis. Looping is essential for cardiac development as it assist further growth of the originally straight heart tube, and ensures it fits within the pericardial celom. Additionally, looping of the heart tube is known to be the first visual evidence of embryonic asymmetry.

# Straight heart tube continues to elongate
#Rapid growth of the bulbus cordis and the primitive ventricle causes ventral bending and right rotation = C shaped loop (convex side on the right)
#The ventricular bend continues to move caudally, the inflow and outflow tracts are brought together at the atrial pole of the heart = S shape
#The truncus arteriosus if formed by adding myocardial cells at the top end of the heart. This portion will form roots and the proximal portion of the aorta and the pulmonary artery <ref name="PMID16479500><pubmed>16479500 </pubmed></ref>.

<html5media height="240" width="280">File:Heart looping 002.mp4</html5media>

[https://embryology.med.unsw.edu.au/embryology/index.php/Intermediate_-_Heart_Tube_Looping]

====Cardiac Septation====
=====Gestational week 6-7=====

This stage of heart morphogenesis refers to the development of the four main cardiac chambers from the primitive atrium and ventricle. Cardiac septation is comprised of three main events:

*Division of the atrioventricular canal
*Atrial septation
*Ventricular septation

'''Division of the atrioventricular canal'''

Division of the atrioventricular canal (AVC) begins with the formation of the superior and inferior endocardial cushions, which are located on the dorsal and ventral aspects of the AVC respectively.<ref name="PMID3424040><pubmed>PMC3424040</pubmed></ref> These cushions develop as mesenchymal cells invade and proliferate within swollen regions of cardiac jelly of the AVC; this mesenchyme is derived from endothelial cells that have transdifferentiated in the process of epithelial-mesenchymal transformation (EMT).<ref name="PMID1767797><pubmed>PMC1767797</pubmed></ref> Throughout the fifth week of development, the endocardial cushions project inwards and eventually fuse to partition the AVC into the left and right atrioventricular canals; these canals will serve as the orifices in which the tricuspid and mitral valves are situated.<ref name="PMID1767797"/>

'''Atrial septation'''

[[File:The Process of Atrial Septation.png|400px|thumb|right|'''Figure 7:''' The Process of Atrial Septation. Growth (A) and perforation (B) of septum primum. Growth of septum secundum (C). Formation of foramen ovale (D).]]
As the AVC is undergoing division, a muscular outgrowth, referred to as the septum primum, extends inferiorly from the roof of the primordial atrium.<ref name="PMID24138816><pubmed>24138816</pubmed></ref> This septum partially divides the atrial chamber into left and right halves, leaving a temporary communication located between the inferior border of septum primum and the endocardial cushions known as the foramen primum.<ref name="PMID3424040"/> As the size of the foramen primum diminishes, perforations in the superior portion of the septum primum develop as a result of apoptosis, forming a second communication between the atrial chambers called the foramen secundum.<ref name="PMID1767797"/> Concurrently, an additional muscular septum, known as the septum secundum, projects inferiorly to the right of the septum primum.<ref name="PMID3424040"/> Eventually, the septum secundum will extend beyond the length of the foramen secundum, generating a partial division of the atria that forms the upper boundary of the foramen ovale.<ref name="PMID24138816"/> The development of the foramen ovale is critical as it allows oxygen-rich blood from the placenta to bypass pulmonary circulation of the embryo and directly enter systemic circulation.<ref name="PMID27188965><pubmed>27188965</pubmed></ref> Following birth, the foramen ovale is obliterated as the septum primum and septum secundum fuse, resulting in complete formation of the interatrial septum.<ref name="PMID3424040"/>

'''Ventricular septation'''

As with atrial septation, differentiation of the ventricles is initiated within week 4 of development.<ref name="PMID3424040"/> A muscular ridge, referred to as the interventricular septum primordium, develops and extends superiorly from the caudal aspect of the primitive ventricular chamber.<ref name="PMID1767797"/> Growth of the septum is attributed to the expansion of the ventricles, which involves the development of muscular trabeculae as cardiomyocytes proliferate within the chamber walls. <ref name="PMID24138816"/> The end result is a partial division between the left and right ventricles, which forms the muscular portion of the interventricular septum.<ref name="PMID1767797"/> A communication between the ventricles, known as the interventricular foramen, remains until approximately week 7 of development.<ref name="PMID12739611><pubmed>12739611</pubmed></ref> The foramen is obliterated by the fusion of the septum intermedium and the bulbar ridges of the bulbus cordis; this constitutes the membranous portion of the interventricular septum.<ref name="PMID3424040"/>

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====Cardiac Neural Crest and Outflow tract====
'''Gestational Week 8'''

[[File:Outflow tract anatomy.png|250px|thumb|right|'''Figure 8:''' Outflow tract anatomy]]
The outflow tract is a tube that runs from the right ventricle to the aortic sac and presents with a distinctive dog-leg bend that separates the proximal (bulbus cordis) and distal (truncus arteriosus) ends of the tract. The endocardial jelly that lines the lumen of the outflow tract concentrates to form the endocardial cushion facing each other that spirals in a 180-degree twist through the length of the outflow tract. Like the outflow tract, these endocardial cushions can be divided into distal and proximal moieties. The distal endocardial cushions are also known as the truncal ridges and the proximal ones are also known as the bulbar ridges. Cells from the cardiac neural crest migrates out of the neural tube, through the pharyngeal arches and aortic sac and into the outflow tract, where it condenses in the ridges to support the septation of the outflow tract. <ref><pubmed> PMC1767864 </pubmed></ref> <ref><pubmed> 23633400 </pubmed></ref>

The fusion of the endocardial cushions starts from the distal end of the outflow tract and proceeds proximally. Fusion of the truncal endocardial cushions forms the aorticopulmonary septum that separates the truncus into an aortic and pulmonary trunk. The bulbar endocardial cushions fuse as they extend towards the interventricular septum, separating the proximal outflow tract into the prospective aortic and pulmonary trunks. As the outflow tract separates, the aortic trunk leads to the 3rd and 4th pharyngeal arch arteries and the pulmonary trunk leads to the 6th pharyngeal arch artery. <ref><pubmed> PMC1767864 </pubmed></ref><ref><pubmed> 23633400 </pubmed></ref>

====Proepicardium and Coronary Heart Development====

Coronary vasculature arises when the fourth layer of the primary heart tube is formed. This fourth layer is known as epicardium that surrounds the myocardium which allows the development of vascular structures over the heart surface.

The monolayered embryonic epicardium derives from mesothelial cells of the septum transversium and mainly from clustered of proepicardium cells. The process involves either of these two mechanisms, one is by detaching of these cells and attach to the pericardial cavity and spread over the heart surface or two is by directly attaching the proepicardium to the myocardium forming a permanent tissue bridge. The spreading of the monolayered embryonic epicardium over the bare myocardium initiates the vascularisation of coronary system. A new extracellular matrix (ECM) layer, the subepicardium forms between the epicardium and the myocardium actively promotes the development of coronary blood vessels. The coalescence of three lineages of the coronary vessel cells: smooth muscle, endothelial and connective tissue and fusion of vascular cell progenitors (angioblasts) form a vascular structure ''de novo'' via the process of vasculogenesis. They then migrate onto the primary heart tube between day 22 and day 28 of human development. <ref> Pérez-Pomares J, Pires-Gomes A, (2013) '''The Epicardium and Coronary Artery Formation''', Journal of Developmental Biology, Vol.1(3), pp.186-202, ISSN: 2221-3759, E-ISSN: 2221-3759, https://doaj.org/article/b3f5aa0ae8df409ab1664975469b95d1</ref>,<ref> Martinsen B, Lohr J, (2005) '''Cardiac Development''', Handbook of Cardiac Anatomy, Physiology and Devices Iaizzo, P.A. (Ed.), http://www.springer.com/cda/content/document/cda_downloaddocument/9781588294432-c2.pdf?SGWID=0-0-45-387660-p173728389</ref>

==Developmental Signalling Processes==

Heart development is a very complicated and dynamic process that requires a high degree of control and regulation. This control is achieved by several temporally regulated signalling cascade (Fig. 1) expressed at different stages of heart development.

[[File:Signalling pathways in heart development.png|400px|'''Figure 9:''' Signalling pathways in heart development]]

<ref><pubmed>12781678</pubmed></ref>

===Wnt signalling===

According to the primary mode of action, Wnt signalling pathways have been divided into two major classes, canonical and non-canonical Wnt signalling pathways. Both the pathways have a role in different stages of cardiac development which could be overlapping or independent of each other. The canonical Wnt signalling pathway involves β-catenin and is activated by a number of ligands such as Wnt-1, Wnt-2, Wnt-3A, Wnt-8A, Wnt-8B, Wnt-8C, Wnt-10A, and Wnt-10B. However, the non-canonical signalling pathway is associated with planar cell polarity and Wnt/Ca2+ pathways that are activated by different ligands such as Wnt4, Wnt5A, Wnt5B, Wnt6, Wnt7A, Wnt7B, and Wnt11 <ref><pubmed>25813860</pubmed></ref>.

Canonical/β -catenin signaling is essential for mesoderm formation. Upon binding of the Wnt ligands to the Frizzled receptor, a seven-transmembrane receptor, or the co-receptor LRP-5/6, the Canonical signaling pathway is activated. This leads to cytoplasmic accumulation of β -catenin in one side of the embryo and its translocation to the nucleus where it drives the activation of transcription factors required to determine the site at which mesoderm and endoderm formation will occur in the embryo.
Animal studies have shown that the lack of nuclear accumulation of β -catenin results in inability of axis formation and mesoderm development whereas overexpression of β -catenin leads to the formation of secondary axis and ectopic expression of mesoderm signals. Also, homozygous deletion of β -catenin in mouse models results in absence of primitive streak and mesoderm formation <ref><pubmed>16860783</pubmed></ref>.

Once mesoderm formation proceeds, the β -catenin signal must be shut down by inhibiting factors otherwise cardiac mesoderm formation will be stopped. This indicates that canonical signaling act as a switch that either induces or suppresses cardiac development. One of the inhibiting factors include dickkopf-1(DKK1) which is an extracellular Wnt inhibitor promotes the expression of cardiac-inducing factor in the endoderm such as Hex. The latter activates paracrine factors to direct adjoining cells towards cardiac fate <ref><pubmed>4533091</pubmed></ref>.

[[File:Wnt signalling pathways.png|500px]] <pubmed>20830688</pubmed>

===Transforming growth factor-β===
Transforming growth factor-β superfamily involves a large number of growth factors that are structurally related. These growth factors, including Nodal or its mimic Activin, bone morphogenic protein (BMP) and growth and differentiation factors, signal through SMAD-dependent and SMAD-independent pathways <ref><pubmed>18290874</pubmed></ref>.
Nodal and its mimic Activin act through SMAD-2 and -3 to activate transcription. During embryo development, epiblast cells produce high levels of Nodal growth factors leading to a gradient of nodal in these cells. This gradient is important for mesoderm patterning (left-right asymmetric heart development) and lineage specification. For this reason, this gradient is maintained by Nodal antagonist secreted from the anterior visceral endoderm or by BMP-4 and Wnt-3 feedback loop in the extraembryonic tissues (Fig.3) <ref><pubmed>25813860</pubmed></ref>.
Nodal/Activin’s important role in heart formation is demonstrated by animal models. Presence of Activin in amphibian embryo lead to activation of heart formation whereas the absence of Nodal co-receptor in, called Cripto, in mouse embryo resulted in failure of ES cells differentiation into cardiomyocytes <ref><pubmed>20830688</pubmed></ref>. Also, knocking out copies of SMAD-2 alone or both SMAD-2 ND -3 result in inappropriate specification of axial mesoderm <ref><pubmed>12842913</pubmed></ref>.

[[File:Regulation of Nodal-Activin signalling during heart formation.png| '''Figure 10:''' Regulation of Nodal-Activin signalling during heart formation]] <ref><pubmed>25813860</pubmed></ref>.
===FGF signalling===
Fibroblast growth factors (FGF) serve a variety of functions in development, the maintainence of health and disease. FGFs are signaling proteins mostly as paracrine growth factors or endocrine hormones. There are 22 types of human FGFs, whereby paracrine FGF8, FGF9, FGF10 and FGF16 serve a major role in embryonic heart development. Additionally, FGF2, FGF9, FGF10 and FGF16 are involved in postnatal heart pathophysiology<ref><pubmed>11493531</pubmed></ref>.

Communication between cardiac progenitor cells is required during embryonic heart development. During heart development, neural cress cells which migrate from the neuroectoderm of the dorsal neural tube will contribute to cushion formation and dictate the correct septation and alignment of the heart<ref><pubmed> 23799628</pubmed></ref>. In conjunction to this process FGF3, FGF8, FGF9, FGF10, FGF15, FGF19 and FGF16 function was paracrine signals in embryonic heart development.

A study conducted demonstrated how a combination of both FGF2 and Bone morphogenic protein 2 (BMP2) efficiently enhances the cariomyogenic differention of embryonic stem (ES) cells at an optimal concentration. When FGF2 was inhibited, this ultimately suppressed cardiomyogenic differentiation, thus indicating that FGF signaling play a crucial role in early cardiomyogenesis. The ability for FGF2 to induce cardiac differentiation may serve a key role in treatment for heart diseases. <ref><pubmed>26793421</pubmed></ref>
In addition, FGF10 also promotes cardiomyocytes differentiation from ES and induced pluripotent stem cells (iPS). In experiments, when FGF10 and ES cells were administered, this lead to the promotion of cardiomyocyte differentiation in the myocardium of the heart. This may thus serve as a treatment for those who have suffered from myocardial infarctions, where the myocardium is replaced by scar tissue. Providing new and fully functional cardiac muscle may help reduce complications. <ref><pubmed> 24307297 </pubmed></ref>

The table below outlines the different types of FGFs invovled in heart development and their functions. <ref><pubmed>26793421</pubmed></ref>
{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Fibroblast Growth Factor'''
|'''Function'''
|-bgcolor="FAF5FF"
| ''' FGF8'''
|FGF8 is expressed in the early embryonic stages. Required for cardiac looping and migratory cardiac neural crest cell survival. FGF8 is also required for anterior heart field development.
|-
|''' FGF9'''
|Activates FGFR1c with heparin sulfate as a co-factor in a paracrine manner. In doing so, this FGF plays a major role in stimulating the proliferation of cardiomyocytes.
|-bgcolor="FAF5FF"
|''' FGF10'''
| Preferentially activates FGFR2b with heparin sulfate as a co-factor. Serves to regulate regional-specific cardiomyocyte proliferation in the embryonic heart in an autocrine/paracrine manner. This FGF type is also essential for the movement of cardiac fibroblasts in the compact myocardium.
|-
|''' FGF15/19'''
| These FGF types are required for proper morphogenesis of the cardiac outflow tract. In addition these FGFs play a major role in paracrine signaling in heart development.
|-bgcolor="FAF5FF"
|''' FGF16'''
|FGF16 serves as a major growth factor involved in stimulating the growth of embryonic cardiomyocytes.
|-
|-bgcolor="FAF5FF"
|-
|}

===The Notch pathway===
The Notch gene is an essential gene, in that it codes for a signaling receptor that is required throughout development to regulate the processes known as spatial patterning, timing and outcomes of many different cell fate decisions. The receptor itself is a single spanning transmembrane protein which has a modular architecture including many repeats of a protein module which is reminiscent of the epidermal growth factor as well as 3 membrane-proximal Lin12/Notch/Glp-1 repeats. Notch also has an intracellular domain which is composed of four distinct regions known as the RAM domain, the Ankyrin repeats, a transcriptional activator domain as well as proline, glutamate, serine and threonine sequence. <ref><pubmed>12651094</pubmed></ref>. In the process of activation, a canonical Notch ligand binds the extracellular domain of the notch receptor. Ligand binding will thus induce the exposure of the cleavage site which will allow access by proteases responsible for cleavage under physiological conditions. This cleavage renders the remaining transmembrane-intracellular fragment a substrate for a complex known as the gamma-secretase complex. It is this complex which will catalyse the intramembrane proteolysis to release the Notch intracellular domain. Once this cleavage has occurred, the intracellular domain has the potential to enter into the nucleus and bind the DNA to induce gene transcription<ref><pubmed>27507209</pubmed></ref>.

With regard to heart development Notch signaling plays a key role in the pre-patterning of the cardiac mesoderm. In gastrula-stage embryos, Notch works with estrogen receptor 9 and GATA4 transcription factor to regulate the timing of heart field specification for early cardiogenesis which is necessary for normal cardiac development. Notch receptor types 1, 2 and delta-like 1 are required for the determination of the embryonic left-right axis as well as the proper looping of the heart tube. In addition to this, Notch signaling promotes myocardial trabecular proliferation, differentiation and maturation by inducing bone morphogenetic protein-10 and EphrinB2/EphrinB4 expression. Notch signaling also modulates coronary vessel morphogenesis, in which the embryonic epicardium actively participates. Notch signaling elements are differentially expressed throughout the proepicardial-epicardial-coronary transition phases and are required for vessel wall maturation during coronary vessel development. Notch also cooperates with transforming-growth factor-beta to regulate coronary smooth muscle differentiation from epicardium-derived cells to assist in the formation of a functional coronary system. Notch signaling will also regulate cardiac conduction system function, in particular the atrioventricular node <ref><pubmed>21252157</pubmed></ref>.

===Sonic Hedgehog===
The Sonic Hedgehog (Shh) signaling pathway is one of the major trafficking networks that is responsible for regulating the key events involved in the developmental processes. The regulatory action of Shh signaling pathway is linked to the secretion, uptake and translocation of ‘Shh protein’ an important Hedgehog ligand precursor. Activation of Shh signal requires the binding of Shh to the Ptc mediate Smoothened (Smo) (Ptc-smo) receptor complex as well as induction of downstream signaling cascade.

===Retinoic Acid===
[[File:Retinoic Acid activation pathway.png|500px|thumb|middle|Figure Retinoic Acid activation pathway]]
Retinoic acid (RA), a derivative of vitamin A, has been shown to play an important role in vertebrate embryogenesis especially heart formation. RA mediates its action by binding to two families of nuclear receptors, that is the RA and the retinoid receptors (RARs and RXRs). This binding allows RA to directly regulate gene transcription. The expression of at least one of the receptor types by almost all embryonic tissues gives these tissues responsiveness to RA. Signaling of RA is regulated by enzyme that either produce or inactivate retinoids (Fig. ?) <ref><pubmed>25813860</pubmed></ref>.

At early stages, RA signaling is suggested to regulate progenitor size in that increasing RA concentrations leads to reduced heart populations in chicken and zebrafish embryos, whereas micromolecular concentrations lead to no heart formation. This regulation is though to happen by RA’s direct action on Hox gene family members such as Hoxa1 transcription factor. These transcription factors have enhancer containing RA response element (RARE). Thus, a reduction or an increase in RA signaling affects the contribution of Hoxa1-; Hoxa3- and Hoxb1-expressing progenitor cells to the heart<ref><pubmed>28007475</pubmed></ref>. At later stages of heart development, RA is involved in establishing the second heart filed which forms atrial cells. Also, RA contributes to the proliferation of ventricular cells. In addition, RA has the potential to stimulate cellular differentiation of atrial and ventricular myocytes from human embryonic stem cells (hESCs). This capacity of RA has been exploited in regenerative medicine to promote atrial specification <ref><pubmed>25700171</pubmed></ref>.

Embryo obtains vitamin A from maternal delivery of retinol via transplacental transfer. This transfer of Vitamin A is essential for fetal growth and development and any deficiency can lead to severe congenital defects. In fact, the high prevalence of heart malformation in developing countries can be partly explained by vitamin A insufficiency. Conversely, elevated levels of vitamin A during pregnancy have a toxic effect on organogenesis including heart development. Treatment with RA in rodents was one of the earliest teratogenic models of heart defects <ref><pubmed>28007475</pubmed></ref>.

==Current Research And Findings==
https://www.victorchang.edu.au/heart-research/embryology

===Increased regurgitant flow causes endocardial cushion defects in an avian embryonic model of congenital heart disease===

it has been discovered that an elevated regurgitant flow during the early stages of embryonic heart development eventually causes abnormalities in the endocardial cushions. However, till today it remains unclear as to how defects in the endocardial cushions during the cardiac looping stages plays a part in the septation of the heart. Thus the aim of this research was to change the blood flow within the avian embryonic heart and monitor the effects it had on the morphology of the endocardial cushions and on Congenital Heart Diseases (CHD). Optical pacing was used to provide the regurgitant flow and optical coherence tomography (OCT) was used to monitor the regurgitation and morphology. The researchers used quail hearts for this research. The quail hearts were optically paced at around 180 beats per minute at stage 13 of their embryonic development (coincides with weeks 3-4 for human development) for 5 minutes. The elevated pacing of the heart tired the heart and this caused 1 hour of regurgitant flow. The morphological changes caused by the regurgitant flow was observed using OCT imaging at stage 19 of avian embryonic development (coincides with cardiac looping stages in human development) or stage 35 (coincides with week 8 of human development). The results showed that all the paced embryos that were observed at stage 19 showed morphological changes in their endocardial cushions. It was also noted that there was an inverse relationship between the regurgitant flow and cardiac cushion size 24 hours post pacing. Out of the embryos that survived till stage 35, 17/18 of them displayed CHDs such as valve defects, ventricular defects, hypo plastic ventricles and common AV valve.

===CTCF counter-regulates cardiomyocyte development and maturation programs in the embryonic heart===

CTCF is a DNA binding factor that is essential in the process of genome binding and cardiogenesis. It mediates genomic interaction and maturation in the developing heart, coordinates cardiomyocyte differentiation by facilitating enhancer-promoter interactions. Although the role of CTCF in genome organisation is not fully understood. <ref><pubmed>28846746</pubmed></ref>
In this study by ''Gomez-Velazquez et.al (2017)'', they studied the effect of genetically deleting CTCF in differentiating cardiomyocytes at early stages of mouse development. They deleted CTCF in a population of cardiac progenitor cells which results in malformation of the heart and death of embryo (Figure 12) <ref><pubmed>28846746</pubmed></ref>
[[File:Figure 1 Morphological defects in CTCF mutant embryonic hearts.PNG|300px|thumb|right| Figure 12 Morphological defects in CTCF mutant embryonic hearts]]
From Figure 12, they presented control and CTCF-absent mice in stage E9.5, E10.5, E11.5 and E12.5.
E10.5 and E11.5 mutant embryos appeared normal (Fig 12A - 12D). Pericardial edema (Fig 12E) and non expanding cardiac chamber (Fig 12F) presented in E12.5 mutant embryo.
Histological examination showed slightly disorganised interventricular septum and normal 4 chambers and atrioventricular canal in E10.5 mutant embryo (Fig 12I and 12J). Fig 12G and 12H showed no defects in E9.5 mutants hearts as compared to controls. Fig. 12K and 12L showed the thinning of myocardial wall in E11.5.
[[File:Figure 2 - defects of mitochondria in CTCF mutant hearts.PNG|300px|thumb|middle|Figure 13 Defects of mitochondria in CTCF mutant hearts]]<ref><pubmed>28846746</pubmed></ref>
They also carried out Western blot to analyse the component of oxidative phosphorylation pathway in mutant and control embryonic cardiomyocytes. They revealed that there was an increase in Complex IV subunit 1 (Cox 1, encoded in mitochondrial DNA) and Complex IV subunit IV (Cox IV) in CTCF mutants hearts at E10.5 and E11.5 (Figure 2A). Fig 2B showed an increased in Tom20 which is the major receptor of mitochondrial outer membrane translocase. Complex I and Complex V increased from E10.5 to E11.5 in control hearts, consistent with maturation of mitochondrial oxidative phosphorylation. However, they did not increase in CTCF mutant hearts and Complex V was similarly increased between E10.5 and E11.5 in both control and mutant hearts (Fig 2C and 2D). Thus, this suggests that despite increased transcription of subunit in complexes and supercomplexes at the mitochondrial inner membrane, maturation of the respiratory chain is blunted in the CTCF mutant heart.

Transmission electron microscopy (TEM) analysis (Fig 13E) presented immature but normal mitochondria in CTCF mutant cardiomyocytes. At E11.5, mutant cardiomyocyte's mitochondria are swollen, and larger than controls and are disorganised at E10.5

They concluded that when CTCF is removed, genes are misregulated which leads to faulty mitochondria and incorrect expression of cardiac patterning gene. These misregulated genes control opposing genetic program incharge of development and cardiomyocyte maturation. Subsequently, embryo lethality.<ref><pubmed>28846746</pubmed></ref>

===Erbb2 Is Required for Cardiac Atrial Electrical Activity during Development===

==Animal Models ==

===Mice Model===
Over the past few decades, intensive studies have been carried out in mice, Xenopu, zebrafish and chicken embryos to establish the role of Wnt signaling in cardiac development. Such studies, in particular, loss of function analysis studies, have allowed for the determination of specific roles of this signaling pathway in specific stages of cardiac development. For example, one of the studies conducted in 2000 on mice embryo established the role of β-catenin Wnt signaling in anterior-posterior axis formation. In this study, Huelsken et al. <ref><pubmed> 10662781 </pubmed></ref> produced β-catenin deficient mice and observed its impact on the anterior-posterior axis formation. At E6.0, the mice embryo showed block and inappropriate formation of anterior-poterior axis, mislocation of anterior visceral endoderm indicated by the Cerberus-like and Lim1 markers. Consequently, mesoderm and head structures were not developed and markers of posterior and anterior differentiation like Brachyury and Hex respectively were not expressed. However, adhesion was not affected by the lack of β-catenin due to the presence of substituents such as plakoglobin. In parallel to Xenopus and zebrafish <ref><pubmed>9914418 </pubmed></ref> <ref><pubmed> 7876319 </pubmed></ref> , the observed block in axis formation of β-catenin null mice demonstrates the signaling functions of β-catenin.

===Zebrafish Model===
Growth of the heart tube into a two-chambered heart in zebrafish is regulated by two separate phases of cardiomyocyte differentiation <ref><pubmed>19395641</pubmed></ref>. These two phases are detected using a developmental timing essay and a photoconvertible marker. In the first phase, a continuous wave of cardiomyocytes differentiation appears in the ventricle initially and extend through atrium allowing the addition of cardiomyocytes to the venous pole of the heart tube. This phase is regulated by the islet1 protein and islet mutants show reduced cardiomyocyte differentiation at the venous pole (Fig. ?). However, the second phase involves Fgf signaling and it is initiated by the addition of new cardiomyocytes to the arterial pole. Therefore, these two processes are independent of each other in space, time and regulation and they are essential for heart development in Zebrafish. Modified regulation of these two phases can result in a change of heart size and morphology.

[[File:Reduced cardiomyocyte differentiation at the venous pole in isl1 mutant embryos.png|300px]]

===Mice Model===
The developmental processes of cardiac valves formation have been well understood through animal models. Since the origin of cells contributing to different parts of the valves remains controversial, this study on mice models <ref><pubmed>15297379</pubmed></ref> provides further insight into the origin of these cells. In this study, myocardial, endocardial and neural crest cells were labeled irreversibly using a genetic system during embryonic development to determine their eventual contribution to different valvar components. At E17.5 majority of cells present in the cardiac valves were positive for β-galactosidase expression which is indicative of endocardial origin (Fig.?) Cells of the endocardial lineage were detected in leaflets and tendinous cords of the mitral and tricuspid valves (Fig. ?A - ?C), the atrioventricular fibrous continuity (Fig. ?G) and the leaflets of the aortic and pulmonary valves (Fig. ?I and ?J). However, these components showed minimal contribution from cells of the myocardial and neural crest lineages.

===Xenopus Model===
Along the molecular pathways mentioned in the heart signaling section, CASTOR (CST) protein previously identified in Drosophila for its role in maintaining stem cells competence at the dorsal midline, is now shown to play a role in cardiogenesis. A study using Xenopus as an animal model <ref><pubmed>18410736</pubmed></ref> identified CST expression in the myocardial layer of the heart in a gradient that extends from dorsal to ventral. The expression of CST in this region within a subset of cardiac progenitor cells is necessary for the initiation of cardiomyocyte differentiation at the ventral midline. Tracing the fate of these cardiac tissue, it is shown that these cardiac progenitor cells at the ventral midline region give rise to a population of cells in the outer curvature of the ventricle indicating their specific cell fate. However, progenitor cells that are deficient in CST protein overproliferate and do not end up integrating into cardiac muscle. This suggests that CST within the subset of cardiac progenitor cells in vertebrates is essential for proper timing of differentiation in order for these progenitor cells to give rise to the outer curvature of the ventricle.

===Chick Model===
Initial stages of cardiac looping involve the formation of a c-shaped tube that is curved towards the right side of the embryo. This c-shaped tube is formed as a result of ventral bending and rightward rotation. The genetic and molecular pathways involved in this process of cardiac looping have been well understand but the role mechanical forces play in this process remains poorly understood. This study on chick embryos <ref><pubmed>15282152</pubmed></ref> show that bending and rotation are mediated by different sets of forces. While bending is controlled by intrinsic forces to the heart, rotation into a c-shaped tube is driven by external forces exerted mainly by a membrane pushing against the heart tube called splanchnopleure (SPL). Also, some of these forces come from omphalomesenteric veins (OVs) which plays a role in the directionality of left-right looping. These results were obtained from analysis of tissue stress and strain using dissection and fluorescent labelling respectively after subjecting the embryos to mechanical perturbations.

==Abnormal Development==

===Atrial Septal Defect===

[[File:Atrial Septal Defect (ASD).png|400px|thumb|right|'''Figure: ''' Atrial Septal Defect (ASD), as demonstrated by the red arrow. ]]

Atrial septal defects (ASD) are one of the most prevalent congenital heart malformations, with an estimated 56 cases per 100,000 births.<ref name="PMID24725467><pubmed>24725467</pubmed></ref> This heart defect involves a communication between the left and right atria, and can be subdivided into five main types based on the nature of the defect.

The most common ASD is a patent foramen ovale. Throughout fetal development, the foramen ovale functions to shunt oxygenated blood from the right atrium into the left atrium.<ref name="PMID4711537><pubmed>PMC4711537</pubmed></ref> After birth, the now functioning respiratory system of the newborn results in elevated pressure in the left atrium relative to that of the right.<ref name="PMID5505397><pubmed>PMC5505397</pubmed></ref> This induces fusion of the septum primum and septum secundum which closes the foramen ovale, leaving a remnant known as the fossa ovalis.<ref name="PMID4711537"/> Incomplete closure results in a patent foramen ovale. This condition is present in nearly all newborns and typically closes within the first month, however complete closure is achieved in only 70-75% of adults.<ref name="PMID24725467"/> A patent foramen ovale is often asymptomatic and thus is not usually considered a clinically significant condition, however it can be problematic when other cardiac abnormalities are present.<ref name="PMID325204><pubmed>PMC325204</pubmed></ref>

A secundum ASD is the most common of the true (clinically significant) atrial septal defects. This malformation occurs within the region of the fossa ovalis and typically results from abnormal resorption of the septum primum during cardiac development.<ref name="PMID325204"/> Secundum ASDs enable left-to-right atrial shunting of blood, which increases the load on the right side of the heart and, consequently, the pulmonary circulation; this can result in the development of atrial arrhthymias and right ventricular failure.<ref name="PMID4711537"/>

Sinous venosus ASD refers to a communication between one of the right pulmonary veins (typically the upper vein) and the wall of the right atrium adjacent to the superior vena cava.<ref name="PMID325204"/> This defect can occur as a result of the resorption or partial absence of the septum secundum,<ref name="PMID325204"/> and accounts for 5-10% of all atrial septal defects.<ref name="PMID4373719><pubmed>PMC4373719</pubmed></ref>

A primum ASD is the result of an abnormality in the development of either the septum primum or septum secundum.<ref name="PMID325204"/> This condition is a type of atrioventricular septal defect, and will be discussed in more detail in the next section.

The most extreme ASD is a common atrium. This occurs when the septum primum and septum secundum fail to develop.<ref name="PMID24725467"/> This lack of an interatrial septum is often accompanied by other cardiac abnormalities, including clefts in the leaflets of the atrioventricular valves.<ref name="PMID325204"/>

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===Atrioventricular Septal Defect===

[[File:Stages of Atrial Septation.png|500px|thumb|right|'''Figure: ''' Comparison between a normal heart (A), a heart with a primum ASD (B) and a heart with an AVSD (C). ]]

An atrioventricular septal defect (AVSD) refers to a malformation involving the interatrial and/or interventricular septa, and has an estimated incidence of 1 in 2000 births.<ref name="PMID5267359><pubmed>PMC5267359</pubmed></ref> This defect typically occurs as a result of incomplete fusion of the endocardial cushions,<ref name="PMID4373719"/> although recent research has concluded that malformation of the dorsal mesenchymal protrusion (DMP) also contributes to the development of AVSDs.<ref name="PMID5267359"/> This can result in abnormal development of the interatrial septum, interventricular septum and/or the mitral and tricuspid valves.<ref name="PMID325204"/>

There are two main subdivisions of AVSDs, which differ according to whether one or both of the cardiac septa are affected. In a partial AVSD, there is a defect present in the inferoanterior region of the fossa ovalis, which allows the right-to-left shunting of blood across the atria.<ref name="PMID5267359"/> In this scenario, the lack of fusion of the endocardial cushions can also result in the presence of clefts in the septal leaflets of the atrioventricular valves.<ref name="PMID325204"/> However, in a complete AVSD, a defect in the membranous portion of the interventricular septum is also present.<ref name="PMID4373719"/> This condition results in direct communications between the atria and the ventricles, and pulmonary hypertension or congestive heart failure can occur if the anomaly is severe.<ref name="PMID5267359"/>

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===Ventricular Septal Defect===

[[File:Ventricular Septal Defect (VSD).jpeg|350px|thumb|right|'''Figure :''' Ventricular Septal Defect (as viewed from the left ventricle)]]
Ventricular septal defects are the most prevalent of all congenital heart defects (excluding patent foramen ovales), accounting for approximately 40% of all cardiac abnormalities among newborns.<ref name="PMID21349577><pubmed>21349577</pubmed></ref> The exact definition of a VSD is still a point of contention within the scientific community, but is generally referred to as a communication between the left and right ventricles; this anomaly can manifest as a lone defect, but is often present with other abnormalities such as Tetralogy of Fallot or a complete AVSD.<ref name="PMID4316658><pubmed>PMC4316658</pubmed></ref> occur as a result of abnormal development (or absence) of any of the components forming the interventricular septum.<ref name="PMID7437181><pubmed>7437181</pubmed></ref> Hence, a VSD can occur in either the muscular or the membranous portion of the septum, with the latter being more common.<ref name="PMID7437181"/>

A membranous VSD occurs when there is an incomplete occlusion of the interventricular foramen, which is due to the membranous portion of the septum failing to develop.<ref name="PMID325204"/> This is the result of a failure of fusion between the muscular portion of the interventricular septum and the endocardial cushions.<ref name="PMID325204"/> This defect is most commonly located in the outflow tract of the left ventricle.<ref name="PMID325204"/>

A muscular VSD results from abnormal development of the interventricular septum primordium during cardiac septation.<ref name="PMID4316658"/> This can refer to incomplete formation of the primitive septum, or to errors in the proliferation of trabeculae within the septum.<ref name="PMID21349577"/> VSDs can form anywhere within the muscular wall of the septum, and can extend into various regions of the right ventricle including the apex, inlet or outflow tract.<ref name="PMID21349577"/>

The most extreme form of VSD is a common ventricle. This very rare defect occurs when the interventricular septum primordium fails to develop.<ref name="PMID325204"/> Infants with common ventricle often die from congestive heart failure.<ref name="PMID325204"/>

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===Persistent Truncus Arteriosus===

Persistent truncus arteriosus (PTA) is a rare heart defect accounting for less than 1% of all congenital cardiac anomalies [4]. PTA occurs when the truncus arteriosus of the developing heart fails to partition into the ascending aorta and pulmonary trunk [2]. This results in the formation of a common arterial trunk that provides a mixture of oxygenated and de-oxygenated blood to systemic, pulmonary and coronary circulations [1]. Although the exact aetiology of PTA is unknown, cellular ablation studies have demonstrated that aberrant migration and/or development of neural crest cells can result in abnormal aorticopulmonary septation [2]. PTA has a poor prognosis without surgical intervention, with only 20% of patients surviving beyond the first year [5].

[[File:Truncus Arteriosus.png|300px|thumb|left|'''Figure: ''' Persistent Truncus Arteriosus (PTA). ]]

===Tetralogy of Fallot===

[[File:Tetralogy of Fallot (TOF).png|300px|thumb|right|'''Figure: ''' Characteristics of Tetralogy of Fallot (TOF). Ventricular septal defect (1) Pulmonary stenosis (2) Dextroposition of aorta (3) Right ventricular hypertrophy (4). ]]

Tetralogy of Fallot (TOF) is a complex congenital heart defect with an incidence of 3 cases per 10000 live births [1]. It is also the most common cyanotic cardiac abnormality, accounting for 7-10% of all congenital heart malformations [1]. The condition is characterized by four heart abnormalities: [2]

*''Pulmonary stenosis'' – narrowing of the pulmonary trunk and associated pulmonary valve.

*''Ventricular septal defect (VSD)'' – a communication between the left and right ventricles

*''Dextroposition of the aorta'' – rightward displacement of the aorta over the ventricular septal defect.

*''Right ventricular hypertrophy'' – enlargement of the myocardium of the right ventricle

Tetralogy of Fallot typically presents as cyanosis (bluish discolouration of the skin) in the newborn [3]. This is due obstruction of pulmonary outflow, as well as right-to-left shunting of blood across the ventricles via the ventricular septal defect [4]. This collectively results in poorly oxygenated blood being discharged from the left ventricle into systemic circulation [4].

Newborns with TOF typically undergo corrective surgery within the first month of life [1]. Pulmonary stenosis is relieved via widening of the pulmonary trunk and the ventricular septal defect is repaired, allowing the outflow of only well-oxygenated blood through the aorta [4].

===Hypoplastic Left Heart Syndrome===

[[File:Hypoplastic Left Heart Syndrome (HLHS).png|300px|thumb|right|'''Figure: ''' Hypoplastic Left Heart Syndrome (HLHS). An atrial shunt is represented by the red arrow.]]

==Cardiac Stem Cells==
Following a myocardial infarction (MI), a significant number of cardiomyocytes becomes damaged and the heart has very little regenerative functions. The loss of cardiomyocytes pose as a problem when an individual has had repeated MI or those suffering from end stage heart failure. For these individuals, heart transplantation remains as the main solution. However, there are not enough donors available. Therefore, stem cell therapy provides as an upcoming possibility in replacement for donor transplant. There are two types of cells that could be use which includes human embryonic Stem cells (hESCs) and Human Induced Pluripotent Stem Cells (hiPSCs). However, there are differences between these two cells and further testing and applications are still required to produce highly pure and mature cardiomyocytes.

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells
! style="background:#f4a941" | Human Induced Pluripotent Cells
|-
| Ethical Issues ||
*More ethical issues
*Need to use a large amount of embryos during derivation as the hESCs are obtained from the inner cell mass of the blastocysts<ref name="PMID9804556><pubmed>9804556</pubmed></ref>
*Associated with invasive procedures
||
*Lesser ethical issues
*Obtained from adult cells
*Need not be associated with invasive procedures as cells can be obtained from hair cells or blood cells<ref name="PMID20964482><pubmed>20964482</pubmed></ref>
|-
| Immune Reactions ||
*Cell genome not matching patient’ genome
*Possible immune rejection against allogenic hESCs
||
*Cell genome matching patient’s genome
*Less likely immune rejection for isogenic hiPSCs
|-
| Availability of Cells ||
*Only a small amount of hESCs are able to differentiate into cardiac myocytes
||
*Only a small amount of hiPSCs are able to differentiate into cardiac myocytes
*hiPSCs take a very long time for differentiation.
|-
|}

{| class="wikitable"
! style="background:#f4a941" | FACTOR
! style="background:#f4a941" | Human Embryonic Stem Cells and Human Induced Pluripotent Stem Cells
|-
| Teratoma formations || Both have tendency to form teratomas. Cardiomyocytes thus have to be highly purified. A few methods have been explored to obtain a highly purified culture of stem cells to prevent teratoma formation. The following methods include:
'''Mitochondrial Based Separation'''<ref name="PMID19946277><pubmed>19946277</pubmed></ref>
*Non-genetic approach
*Based on the fact that cardiac myocytes that are differentiated contains a high number of mitochondria
*Mitochondria specific fluorescent dye used to identify differentiated cardiomyocytes from non-differentiated ones. Cells then separated using flow cytometry.
*~ 99% purity
*Furthur research and successful applications in stem cell experiments are required. Technological advancements and improvements are also needed for large-scale use.

'''Biochemical Differences between Differentiated and Undifferentiated Cardiomyocytes'''<ref name="PMID23168164 ><pubmed>23168164 </pubmed></ref>
*Non-genetic approach
*Only differentiated cardiomyocytes could survive in a glucose depleted and lactate abundant culture.
*Simple applications compared to the mitochondrial based separation.
*~ 99% purity
However, because both methods do not have a 100% purity, teratomas could still be formed.
|-
| Cardiac Maturation ||
'''Ultrastructural Analysis'''
*The ultrastructural features of both hESCs and hiPSCs were both phenotypically immature, that is it had an abundant amount of lipid droplets and endoplasmic reticulum, elevated glycogen content and different degrees of myofibrillar organization <ref name="PMID21883888 ><pubmed>21883888</pubmed></ref>

'''Electrophysiological Properties'''
*Both hESCs and hiPSCs displayed all three action potential phenotypes: nodal, atrial and ventricular-like, generally exhibiting a phenotypically immature cardiomyocytes<ref name="PMID12791707 ><pubmed>12791707</pubmed></ref>

'''Contraction Properties'''
*The more mature the sarcoplasmic reticulum, the better the contractile properties of the cardiomyocytes.<ref name="PMID21614516 ><pubmed>21614516</pubmed></ref> hESCs had an immature sarcoplasmic reticulum (SR) whereas hiPSCs had a slightly more mature sarcoplasmic reticulum. <ref name="PMID16322641 ><pubmed>16322641 </pubmed></ref>

Since hESCs and hiPSCs are required to replace damaged cardiomyocytes, their obvious immaturity problem would have to be resolved before it can be utilized for transplantation or therapy.
|-
|}

==Future Questions==

What is the secreted protein that flows across the node, and what is the mechanism that ensures that the loop turns in rightward direction?

What are the developments of stem cell usage for developmental abnormalities?
http://circres.ahajournals.org/content/91/3/189.short

==Glossary of Terms==

{| class="pretty table"
|-bgcolor = "DDCEF2"
|'''Term'''
|'''Definition'''
|-bgcolor="FAF5FF"
| ''' Axial mesoderm'''
|Also referred to as notochord. It is an embryonic structure lying within the midline of the trilaminar embryo.
|-
|''' Angioblastic cords'''
| Groups or ‘columns’ of embryonic precursor cells which will form the walls of both arteries and veins.
|--bgcolor="FAF5FF"
|''' Blastocoel'''
|A fluid filled cavity formed during early embryonic development (Week 1-2) within the blastocyst. Once Morula is formed as a result of the initial cell division, further cell division followed by compaction leads to the formation of this cavity.
|-
|''' Bulbus cordis'''
| A region of the early developing heart tube forming the common outflow tract, will differentiate to form three regions of the heart.
|--bgcolor="FAF5FF"
|''' Cardiocyte'''
| Mature muscle cell of the heart. These cells are characterized by striation and separated by intercalated discs.
|-
|''' Epicardium'''
| The inner layer of the pericardium that is in contact with the surface of the heart
|--bgcolor="FAF5FF"
|''' Inflow tract'''
| Entrance of blood into the heart tube; the sinus venosus portion of the tube.
|-
|''' Mesothelial cells'''
| Epithelial cells of mesodermal origin
|--bgcolor="FAF5FF"
|''' Mitochondrial oxidative phosphorylation'''
| A synthesis of ATP powered by the free energy of reduced compounds that are produced by other metabolic pathways, including glycolysis and the TCA cycle, occurs in the mitochondria
|-
|''' Outflow tract'''
| Exit of blood from the heart tube formed by the truncus arteriosus.
|--bgcolor="FAF5FF"
|''' Omphalomesenteric vein'''
| Vessels that provid the venous pole input into the heart from the yolk sac connected at the umbilicus during embryogenesis
|-
|''' Pericardium'''
| The membranous sac filled with serous fluid that encloses the heart, aorta and large blood vessels
|--bgcolor="FAF5FF"
|''' Proepicardium'''
| Group of progenitor cells that forms near the venous pole of the heart gives rise to the epicardium
|-
|''' Sinus venosus'''
| An early developmental cardiovascular structure, thin walled cavity, forming the input to developing heart which has 3 venous inputs (vitelline vein, umbilical vein, common cardinal vein). Later in heart development this structure gets incorporated into the wall of the future right atrium.
|--bgcolor="FAF5FF"

|}

''Below are links to a more extensive glossary if additional definitions are needed''

[[A]] | [[B]] | [[C]] | [[D]] | [[E]] | [[F]] | [[G]] | [[H]] | [[I]] | [[J]] | [[K]] | [[L]] | [[M]] | [[N]] | [[O]] | [[P]] | [[Q]] | [[R]] | [[S]] | [[T]] | [[U]] | [[V]] | [[W]] | [[X]] | [[Y]] | [[Z]]

==References==
<references/>