Embryology - User contributions [en-gb]

User:Z3414482

2016-11-02T07:21:09Z

Z3414482: /* Lab Assessment 7 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?''' 
Dystrophin mutation is what occurs when there are abnormalities affecting the dystrophin gene, such as large deletions, small point mutations and duplications of one or more exons. Mutations within the dystrophin protein complex leads to many forms of autosomally inherited muscular dystrophy and the most well known disease is the X-linked muscle wasting disease of Duchenne muscular dystrophy. Both Duchenne (DMD) and Becker muscular dystrophy (BMD, a milder form of muscular dystrophy that appears at a later age than Duchenne) occur almost exclusively in males and characterized by progressive muscle weakness and atrophy. Whereas DMD is commonly characterised by complete absence of dystrophin, BMD patients display low levels of truncated dystrophin protein.
<pubmed>26721686</pubmed>
'''2. What is the function of dystrophin?'''
Dystrophin is a 427-kDa cytoskeletal protein produced by the largest known human gene. This protein is most commonly found in skeletal muscles and cardiac muscles although small amounts are also detected in nerve cells of the brain. Both in the skeletal and cardiac muscles, dystrophin play a role in strengthening and protecting the muscle fibres to prevent injury to the cells during its contraction and relaxation. The dystrophin complex allow for the muscle cell’s cytoskeleton to be connected with the network proteins and molecules outside the cell, thus acting as an anchor for the muscle cell. In doing so, the dystrophin complex also most likely participates in cell signalling between the cells and the proteins they interact with.
<pubmed>27458343</pubmed>
'''3. What other tissues/organs are affected by this disorder?''' 
Although little information is currently known about the role of dystrophin nerve cells, recent research have suggested that this protein is important for the normal structure and function of synapses. Furthermore, dystrophin is found to be an essential protein for tissues with a secretory function or those that form barriers between functional compartments such as the blood-brain barrier, choroid plexus or kidney. Thus, DMD or BMD would affect the above mentioned tissues and organs of the human body.
<pubmed>16710609</pubmed>
'''4. What therapies exist for DMD?''' 
There is currently no effective treatment available for this disease although much can be done for the management of the symptoms to improve the quality and length of life of patient. The only medication proven to slow the progression of DMD is steroid, which is not always chosen to be used due to the side effects that it may cause.
<pubmed>27621596</pubmed>
'''5. What animal models are available for muscular dystrophy?''' 
Murine (mdx mice) and dog models are currently available for the study of muscular dystrophy
<pubmed>9334342</pubmed>

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper''' 
<pubmed>26544927</pubmed>

'''2. Summary of main findings from research paper''' 
The aim of the research paper was to show how a 40% increase in ventricular cardiomyocyte number that takes place during preadolescence produces newly synthesized DNA in 57% of the cardiomyocyte nuclei. MHC-nLAC mice that expressed a nuclear-localised β-galactosidase were used to investigate further into this bust of preadolescent cell-cycle activity. Through co-localization of β-galactosidase and BrdU immune reactivity, cumulative ventricular cardiomyocyte DNA synthesis was able to be quantitated in the mice models. The results showed that only low levels of cardiomyocyte DNA synthesis were found in mice characterised by pumps from PN10 through PN19 or from PN12 through PN19. Only low levels of cardiomyocyte DNA synthesis were detected in C57BI/6J inbred mice. However BrdU on the otherhand, was not only detected in in small intestine crypt cells by 24 hr postimplantation of all the different mice model types but also at the end of the labelling period thus confirming its ability to continuously infuse. As the mice that received a single BrdU injeciton on PN14.5, PN15 or PN16 and PN19 also showeed little labeling from cardiomyocyte DNA synthesis, the study concluded that BrdU tyotoxicity and/or the presence of the osmotic mini-pump were not confounding factors.

'''3. Relationship between research paper & review article ''' 
Review article: <pubmed>26932668</pubmed>
The review article describes about the latent regenerative potential of cardiomyocytes that can be found in some animal models but not human heart. The review goes through the cellular and molecular mechanisms that facilitate the proliferation of cardiomyocytes. In doing so, it also shows how and why this capacity is lost in adult mammals normally, and therefore highlights some of the current research areas that might lead to restoration of this ability. The research article I chose was utilised within the review article to emphasise the ceasing of any proliferative activity within neonatal mammal cardiomyocytes. The research article was underlining the fact that despite certain reports that of a discovery in preadolescent proliferative burst of cardiomyocytes, that this in fact was not true as evidence of it could not be replicated by either cardiomyocyte assays or proliferation marker assays.

User:Z3414482

2016-11-02T07:11:18Z

Z3414482: /* Lab Assessment 11 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?''' 
Dystrophin mutation is what occurs when there are abnormalities affecting the dystrophin gene, such as large deletions, small point mutations and duplications of one or more exons. Mutations within the dystrophin protein complex leads to many forms of autosomally inherited muscular dystrophy and the most well known disease is the X-linked muscle wasting disease of Duchenne muscular dystrophy. Both Duchenne (DMD) and Becker muscular dystrophy (BMD, a milder form of muscular dystrophy that appears at a later age than Duchenne) occur almost exclusively in males and characterized by progressive muscle weakness and atrophy. Whereas DMD is commonly characterised by complete absence of dystrophin, BMD patients display low levels of truncated dystrophin protein.

'''2. What is the function of dystrophin?'''
Dystrophin is a 427-kDa cytoskeletal protein produced by the largest known human gene. This protein is most commonly found in skeletal muscles and cardiac muscles although small amounts are also detected in nerve cells of the brain. Both in the skeletal and cardiac muscles, dystrophin play a role in strengthening and protecting the muscle fibres to prevent injury to the cells during its contraction and relaxation. The dystrophin complex allow for the muscle cell’s cytoskeleton to be connected with the network proteins and molecules outside the cell, thus acting as an anchor for the muscle cell. In doing so, the dystrophin complex also most likely participates in cell signalling between the cells and the proteins they interact with.

'''3. What other tissues/organs are affected by this disorder?''' 
Although little information is currently known about the role of dystrophin nerve cells, recent research have suggested that this protein is important for the normal structure and function of synapses. Furthermore, dystrophin is found to be an essential protein for tissues with a secretory function or those that form barriers between functional compartments such as the blood-brain barrier, choroid plexus or kidney. Thus, DMD or BMD would affect the above mentioned tissues and organs of the human body.

'''4. What therapies exist for DMD?''' 
There is currently no effective treatment available for this disease although much can be done for the management of the symptoms to improve the quality and length of life of patient. The only medication proven to slow the progression of DMD is steroid, which is not always chosen to be used due to the side effects that it may cause.

'''5. What animal models are available for muscular dystrophy?''' 
Murine and dog models are currently available for the study of muscular dystrophy

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper''' 
<pubmed>26544927</pubmed>

'''2. Summary of main findings from research paper''' 
The aim of the research paper was to show how a 40% increase in ventricular cardiomyocyte number that takes place during preadolescence produces newly synthesized DNA in 57% of the cardiomyocyte nuclei. MHC-nLAC mice that expressed a nuclear-localised β-galactosidase were used to investigate further into this bust of preadolescent cell-cycle activity. Through co-localization of β-galactosidase and BrdU immune reactivity, cumulative ventricular cardiomyocyte DNA synthesis was able to be quantitated in the mice models. The results showed that only low levels of cardiomyocyte DNA synthesis were found in mice characterised by pumps from PN10 through PN19 or from PN12 through PN19. Only low levels of cardiomyocyte DNA synthesis were detected in C57BI/6J inbred mice. However BrdU on the otherhand, was not only detected in in small intestine crypt cells by 24 hr postimplantation of all the different mice model types but also at the end of the labelling period thus confirming its ability to continuously infuse. As the mice that received a single BrdU injeciton on PN14.5, PN15 or PN16 and PN19 also showeed little labeling from cardiomyocyte DNA synthesis, the study concluded that BrdU tyotoxicity and/or the presence of the osmotic mini-pump were not confounding factors.

'''3. Relationship between research paper & review article ''' 
Review article: <pubmed>26932668</pubmed>
The review article describes about the latent regenerative potential of cardiomyocytes that can be found in some animal models but not human heart. The review goes through the cellular and molecular mechanisms that facilitate the proliferation of cardiomyocytes. In doing so, it also shows how and why this capacity is lost in adult mammals normally, and therefore highlights some of the current research areas that might lead to restoration of this ability. The research article I chose was utilised within the review article to emphasise the ceasing of any proliferative activity within neonatal mammal cardiomyocytes. The research article was underlining the fact that despite certain reports that of a discovery in preadolescent proliferative burst of cardiomyocytes, that this in fact was not true as evidence of it could not be replicated by either cardiomyocyte assays or proliferation marker assays.

User:Z3414482

2016-11-02T07:10:41Z

Z3414482: /* Lab Assessment 7 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?''' 
Dystrophin mutation is what occurs when there are abnormalities affecting the dystrophin gene, such as large deletions, small point mutations and duplications of one or more exons. Mutations within the dystrophin protein complex leads to many forms of autosomally inherited muscular dystrophy and the most well known disease is the X-linked muscle wasting disease of Duchenne muscular dystrophy. Both Duchenne (DMD) and Becker muscular dystrophy (BMD, a milder form of muscular dystrophy that appears at a later age than Duchenne) occur almost exclusively in males and characterized by progressive muscle weakness and atrophy. Whereas DMD is commonly characterised by complete absence of dystrophin, BMD patients display low levels of truncated dystrophin protein.

'''2. What is the function of dystrophin?'''
Dystrophin is a 427-kDa cytoskeletal protein produced by the largest known human gene. This protein is most commonly found in skeletal muscles and cardiac muscles although small amounts are also detected in nerve cells of the brain. Both in the skeletal and cardiac muscles, dystrophin play a role in strengthening and protecting the muscle fibres to prevent injury to the cells during its contraction and relaxation. The dystrophin complex allow for the muscle cell’s cytoskeleton to be connected with the network proteins and molecules outside the cell, thus acting as an anchor for the muscle cell. In doing so, the dystrophin complex also most likely participates in cell signalling between the cells and the proteins they interact with.

'''3. What other tissues/organs are affected by this disorder?''' 
Although little information is currently known about the role of dystrophin nerve cells, recent research have suggested that this protein is important for the normal structure and function of synapses. Furthermore, dystrophin is found to be an essential protein for tissues with a secretory function or those that form barriers between functional compartments such as the blood-brain barrier, choroid plexus or kidney. Thus, DMD or BMD would affect the above mentioned tissues and organs of the human body.

'''4. What therapies exist for DMD?''' 
There is currently no effective treatment available for this disease although much can be done for the management of the symptoms to improve the quality and length of life of patient. The only medication proven to slow the progression of DMD is steroid, which is not always chosen to be used due to the side effects that it may cause.

'''5. What animal models are available for muscular dystrophy?''' 
Murine and dog models are currently available for the study of muscular dystrophy

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper''' 
<pubmed>26544927</pubmed>

'''2. Summary of main findings from research paper''' 
The aim of the research paper was to show how a 40% increase in ventricular cardiomyocyte number that takes place during preadolescence produces newly synthesized DNA in 57% of the cardiomyocyte nuclei. MHC-nLAC mice that expressed a nuclear-localised β-galactosidase were used to investigate further into this bust of preadolescent cell-cycle activity. Through co-localization of β-galactosidase and BrdU immune reactivity, cumulative ventricular cardiomyocyte DNA synthesis was able to be quantitated in the mice models. The results showed that only low levels of cardiomyocyte DNA synthesis were found in mice characterised by pumps from PN10 through PN19 or from PN12 through PN19. Only low levels of cardiomyocyte DNA synthesis were detected in C57BI/6J inbred mice. However BrdU on the otherhand, was not only detected in in small intestine crypt cells by 24 hr postimplantation of all the different mice model types but also at the end of the labelling period thus confirming its ability to continuously infuse. As the mice that received a single BrdU injeciton on PN14.5, PN15 or PN16 and PN19 also showeed little labeling from cardiomyocyte DNA synthesis, the study concluded that BrdU tyotoxicity and/or the presence of the osmotic mini-pump were not confounding factors.

'''3. Relationship between research paper & review article ''' 
Review article: <pubmed>26932668</pubmed> 
The review article describes about the latent regenerative potential of cardiomyocytes that can be found in some animal models but not human heart. The review goes through the cellular and molecular mechanisms that facilitate the proliferation of cardiomyocytes. In doing so, it also shows how and why this capacity is lost in adult mammals normally, and therefore highlights some of the current research areas that might lead to restoration of this ability. The research article I chose was utilised within the review article to emphasise the ceasing of any proliferative activity within neonatal mammal cardiomyocytes. The research article was underlining the fact that despite certain reports that of a discovery in preadolescent proliferative burst of cardiomyocytes, that this in fact was not true as evidence of it could not be replicated by either cardiomyocyte assays or proliferation marker assays.

User:Z3414482

2016-11-01T04:17:29Z

Z3414482: /* Lab Assessment 11 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper''' 
<pubmed>26544927</pubmed>

'''2. Summary of main findings from research paper''' 
The aim of the research paper was to show how a 40% increase in ventricular cardiomyocyte number that takes place during preadolescence produces newly synthesized DNA in 57% of the cardiomyocyte nuclei. MHC-nLAC mice that expressed a nuclear-localised β-galactosidase were used to investigate further into this bust of preadolescent cell-cycle activity. Through co-localization of β-galactosidase and BrdU immune reactivity, cumulative ventricular cardiomyocyte DNA synthesis was able to be quantitated in the mice models. The results showed that only low levels of cardiomyocyte DNA synthesis were found in mice characterised by pumps from PN10 through PN19 or from PN12 through PN19. Only low levels of cardiomyocyte DNA synthesis were detected in C57BI/6J inbred mice. However BrdU on the otherhand, was not only detected in in small intestine crypt cells by 24 hr postimplantation of all the different mice model types but also at the end of the labelling period thus confirming its ability to continuously infuse. As the mice that received a single BrdU injeciton on PN14.5, PN15 or PN16 and PN19 also showeed little labeling from cardiomyocyte DNA synthesis, the study concluded that BrdU tyotoxicity and/or the presence of the osmotic mini-pump were not confounding factors.

'''3. Relationship between research paper & review article ''' 
Review article: <pubmed>26932668</pubmed> 
The review article describes about the latent regenerative potential of cardiomyocytes that can be found in some animal models but not human heart. The review goes through the cellular and molecular mechanisms that facilitate the proliferation of cardiomyocytes. In doing so, it also shows how and why this capacity is lost in adult mammals normally, and therefore highlights some of the current research areas that might lead to restoration of this ability. The research article I chose was utilised within the review article to emphasise the ceasing of any proliferative activity within neonatal mammal cardiomyocytes. The research article was underlining the fact that despite certain reports that of a discovery in preadolescent proliferative burst of cardiomyocytes, that this in fact was not true as evidence of it could not be replicated by either cardiomyocyte assays or proliferation marker assays.

User:Z3414482

2016-10-28T05:15:31Z

Z3414482: /* Lab Assessment 11 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper'''
<pubmed>26544927</pubmed>

'''2. Summary of main findings from research paper'''
The research paper aimed to show a how a 40% increase in ventricular cardiomyocyte number during preadolescence should result in newly synthesized DNA in 57% of the cardiomyocyte nuclei.

To characterise this putative burst of peradolescent cell-cycle activity, MHC-nLAC mice, expressing a nuclear-localized B-galactosidase reporter in cardiomyocytes and maintained in a DBA/2J background were implanted with BrdU-containing osmotic mini pumps.

Through co-localizaiton of B-galactosidase and BrdU immune reactivity, cumulative ventricular cardiomyoctye DNA sysnthesis was quantitated.

'''3. Relationship between research paper & review article '''
Review article: <pubmed>26932668</pubmed>

User:Z3414482

2016-10-28T04:34:32Z

Z3414482: /* Lab Assessment 11 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
'''1. Research paper'''

'''2. Summary of main findings from research paper'''

'''3. Relationship between research paper & review article '''

User:Z3414482

2016-10-28T04:32:13Z

Z3414482:

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

==Lab Assessment 11==
Quiz on urogenital development completed

User:Z3414482

2016-10-21T02:19:29Z

Z3414482:

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:19, 21 October 2016 (AEDT)
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

{{Stem Cell Presentations 2016}}

2016 Group Project 1

2016-10-20T20:19:15Z

Z3414482: /* Quiz */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed></ref>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref> <ref name="PMID16685431"><pubmed>16685431</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear <ref name="PMID15930119"><pubmed>15930119</pubmed></ref>
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm. <ref name="PMID23307624"><pubmed>23307624</pubmed></ref>
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''. <ref name="PMID26583322"><pubmed>26583322</pubmed></ref>

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells

{Which of the following statement(s) are true? 
# Statement 1: Glycogen and CSK3 can only down regulating β-catenin in cellular cytoplasm.
# Statement 2: Through the various effect of β-catenin signalling in the nucleus, Wnt/β-catenin signalling pathway can not only activate DNA transcription but also repress DNA transcription.
# Statement 3: β-catenin enhances the function of DNA-bound transcription factor family known as 'TCF/LEF' that would normally transcript DNA genes.
|type="()"}

- Statement 1 only is true
+ Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true
</quiz>

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T20:08:04Z

Z3414482: /* Quiz */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed></ref>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref> <ref name="PMID16685431"><pubmed>16685431</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear <ref name="PMID15930119"><pubmed>15930119</pubmed></ref>
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm. <ref name="PMID23307624"><pubmed>23307624</pubmed></ref>
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''. <ref name="PMID26583322"><pubmed>26583322</pubmed></ref>

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells

{Which of the following statement(s) are true? 
# Statement 1:
# Statement 2:
# Statement 3:
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true
</quiz>

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T20:06:13Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed></ref>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref> <ref name="PMID16685431"><pubmed>16685431</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear <ref name="PMID15930119"><pubmed>15930119</pubmed></ref>
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm. <ref name="PMID23307624"><pubmed>23307624</pubmed></ref>
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''. <ref name="PMID26583322"><pubmed>26583322</pubmed></ref>

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T20:00:36Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed></ref>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref> <ref name="PMID16685431"><pubmed>16685431</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T19:59:17Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed></ref>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed><ref name="PMID16685431"><pubmed>16685431</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T19:56:22Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand. <ref name="PMID18358465"><pubmed>18358465</pubmed>

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T19:42:01Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation <ref name="PMID15473860"><pubmed>15473860</pubmed></ref>'''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation <ref name="PMID16019432"><pubmed>16019432</pubmed></ref>
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T19:29:32Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png|thumb|alt=Alt|Diagram of Wnt/β-catenin signalling pathway]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
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===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T19:26:38Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''[[File:Wnt signalling ONstate .png]]
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

File:Wnt signalling ONstate .png

2016-10-20T19:25:00Z

Z3414482: Diagram of Wnt/β-catenin signalling "Beginning six months after publication, I (student number) grant the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unpor...

Diagram of Wnt/β-catenin signalling

"Beginning six months after publication, I (student number) grant the public the non-exclusive right to copy, distribute, or display the Work under a Creative Commons Attribution-Noncommercial-Share Alike 3.0 Unported license, as described at http://creativecommons.org/licenses/by-nc-sa/3.0/ and http://creativecommons.org/licenses/by-nc-sa/3.0/legalcode."

2016 Group Project 1

2016-10-20T19:10:17Z

Z3414482: /* Quiz */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

{Which of the following statements are true? 
# False
# True
# True
|type="()"}
+ Statement 1 only is true
|| Statement 2 only is true.
- Statement 3 only is true
- None of the above statements are true.
- All of the above statements are true

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T18:50:24Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.<ref name="PMID15473860"><pubmed>15473860</pubmed></ref>

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T18:46:02Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation.<ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

15473860

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T18:40:46Z

Z3414482: /* Canonical Pathway - Gloria */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

===The signalling pathways:===
====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T18:36:42Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. <ref name="PMID19279717"><pubmed>19279717</pubmed></ref> Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. <ref><pubmed>23140633 </pubmed></ref>

[[File:PCP pathway leading to gastrulation.jpg|thumb|alt=Alt|upright=Factor]]

PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours. <ref><pubmed>19279717 </pubmed></ref>

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway, NRH1, Ryk, PTK7 or ROR2.
The main steps of the pathway are as follows:
*Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor.
*Dishevelled proteins are attracted by the receptor involved and thus form a linkage.
*This dishevelled protein utilises two sites on its structure, the PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1).
*The G-protein Rho is activated by DAAM1. It is activated by exchanging the base protein, guanine.
*Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator.
*At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin.
*The Rac1 initiates the role of JNL, causing actin polymerisation to occur.
*Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton. <ref><pubmed>1617723 </pubmed></ref> <ref><pubmed>16793760 </pubmed></ref>

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

[[File:Wnt Calcium ion pathway.JPG|thumb|alt=Alt|Interactions between main components of wnt/calcium ion pathway]]

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a specific set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is also narrowed. This has been identified in frogs (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. If CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. <ref><pubmed>23140633 </pubmed></ref> <ref><pubmed>1617723 </pubmed></ref>

The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close so that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close any part of the neural tube results in craniorachischisis. <ref><pubmed>15686623 </pubmed></ref> <ref><pubmed>15473860</pubmed></ref> <ref><pubmed>21181886</pubmed></ref>

Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, the hairs are faced in the same direction. If there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine their true role and effect. There are two major WnT proteins involved in gastrulation, WnT5b and Wnt11, they allow the formation of L-R asymmetry during gastrulation, regulates the direction of the nodal flow, which then results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals.

There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as deformations in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane.
===Human===

===Mouse===

===Xenopus and Zebrafish===

Research in the Xenopus and zebrafish shows that the PCP proteins regulates gastrulation during embryonic development. <ref><pubmed>26680417</pubmed></ref> <ref><pubmed>25266145</pubmed></ref>

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Apoptosis:''' the death of cells.
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesenchymal Cells:''' stem cells that can differentiate into many types of cells,
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Organogenesis:''' the formation of organs in animals or plants.
*'''Pleiotropic:''' when a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:''' the African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| WNT5A, WNT5B, WNT9B, WNT11
| Convergent extension during gastrulation, orientation of muscle fibers, regulation of muscle fiber elongation, normal sensory hair cell orientation, neural tube closure, polarised division of epithelial cells
| Spina bifida, anencephaly and craniorachischisis
|-
| '''Calcium Ion pathway'''
| WNT-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.

{What is the role of WnT11 in the PCP pathway.
|type="()"}
+ Convergent extension during gastrulation and orients muscle fibres and regulates their elongation
|| This is correct, as this gene has more than one role.
- Convergent extension during gastrulation
- Assists in sensory hair cell orientation in the inner ear and neural tube closure
- Convergent extension and polarised division of kidney epithelial cells
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T05:58:30Z

Z3414482: /* A table of all three pathways */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Pleiotropic:'''When a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:'''The African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Wnt ligands: WNT3, WNT4, WNT5B, WNT7A, WNT10A, WNT10B
| Normal function: Preserves β-catenin within cell cytoplasm so that it can get transported to cell nucleus to activate transcription of DNA
| Abnormalities with canonical pathway: Carcinogenisis (colon and breast), Bone conditions (Osteoporosis pseudoglioma syndrome, OPPG)
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T05:41:53Z

Z3414482: /* Glossary */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Haematopoietic:'''Pertaining to the formation of blood cells.
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Mitogenic:'''Has characteristics that induce mitosis.
*'''Pleiotropic:'''When a gene has effects on 2 or more seemingly unrelated phenotypic traits.
*'''Vertebrate:''' an animal with a backbone
*'''Xenopus:'''The African clawed frog that is used commonly in embryological research and previously for pregnancy testing too.

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T05:22:09Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous system development''' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T05:20:17Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
#'''CNS and Head formation (neural patterning)''' The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous sysem development'' In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter.
#'''Endodermal cells''' 
β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' 
An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T05:18:01Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer'''
A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
# '''CNS and Head formation (neural patterning)'''
The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear
#'''Nervous sysem development''
In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter. 
#'''Endodermal cells'''
β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoderm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification than neuroectoderm and mesoderm.
#'''Bone marrow''' 
An in vitro study of purified bone marrow haematopoietic stem cells (HSC's) have also uncovered the role of Wnt signaling in the maintenance and differentiation of multipotent stem cells. These cells when treated with purified Wnt3a protein or retroviral infection of a stabilized β-catenin consequently produced elevated Wnt signaling that led to a decrease in differentiation and contrastingly an increase in self-renewal. Not only that but these modified HSC cells showed capability to reconstitute the blood cells when transplanted into lethally irradiated mice.. Furthermore, blocking Wnt signaling through either overexpression of Axin or inhibiting the action of Fz receptor for Wnt binding, inhibited proliferation of HSC's ''in vitro'' and their ability to reconstitute blood cells ''in vivo''.

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T04:59:16Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' 
A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.
# '''CNS and Head formation (neural patterning)''' 
The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear.<
# '''Nervous sysem development''' 
In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter. Subsequently,
# '''Endodermal cells''' 
β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoerm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification thatn neuroectoderm and mesoderm.
'''Stem cell renewal ''' 
Finally, Wnt signalling also has recently been found to have a role in stem cell renewal

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T04:51:57Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation.

# '''Spermann-Mangold Organizer''' 
A lot of information about the role of Wnt canonical pathway in fetal development was found through study of the Xenopus system. Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of β-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, β-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development. 
# '''CNS and Head formation (neural patterning)''' 
The canonical Wnt pathway also regulates head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear β-catenin protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. '''R''' Furthermore, studies also performed with the brain of chicks have also found that increased Wnt protein levels along the anterior-posterior axis specifies neural cells with initial rostral character into gradually more caudal character. The canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well. The canonical Wnt signalling pathway involving β-catenin is necessary for the entire central nervous system also. It simultaneously promotes cell proliferation and bloks apoptosis and differentiation of the progenitor cell populations. Much of the mechanisms for how β-catenin exactly does so is still left unclear. 
# '''Nervous sysem development''' 
In pituiatary neurons and cardiac neural crest cells, β-catenin can increase transcriptio of PItx2 by binding to and displacing HDAC1 from Lef1 bound to PItx2 promoter. Subsequently,
# '''Endodermal cells''' 
β-catenin is an essential part of endoderm specification. Without this co-transcription factor, the cell fate of endodermal cells changes to become cardiac mesoderm instead. Although it is not yet fully understood how β-catenin mediates the fate choice between endoerm and mesoderm, it is understood that the role of β-catenin is much more highly regarded by endoderm specification thatn neuroectoderm and mesoderm.
'''Stem cell renewal ''' 
Finally, Wnt signalling also has recently been found to have a role in stem cell renewal

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T03:26:08Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
Wnt family of signalling proteins plays various roles in fetus development of embryogenesis. Wnt signals are pleiotropic and they have effects on mitogenic stimulation, cell fate specification and differentiation. A lot of information about the role of Wnt canonical pathway in fetus development was found through study of the Xenopus system.

'''Spermann-Mangold Organizer''' 
Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation. Dsh protein and other elements of the Wnt pathway that lead to stabilization of B-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation. Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation. In early development of Xenopus, B-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes. Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.

'''Anterior head formation and neural patterning''' 
The canonical Wnt pathway also regulates anterior head formation and neural patterning. A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation. The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear B-catenein protein in the anterior region and a higher level within posterior region of the gastrula embryo. This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering. However canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well as various organ systems such as the heart, lungs, kidney, skin and bone.

'''Stem cell renewal ''' 
Finally, Wnt signalling also has recently been found to have a role in stem cell renewal

Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-20T03:06:43Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

There are 19 wnt genes and 12 Frizzled receptors found in vertebrates which are responsible for regulating many cellular activities such as proliferation and cell migration. Failure in signalling transduction or overactivation may lead to serious birth defects or post-natal diseases. Although the wnt signalling pathways are relatively highly conserved across species providing many useful experimental models for research study, its involvement of massive cellular proteins and complicated crosstalk with wnt and other cell signalling pathways have made it difficult to reveal its mechanism and function clearly.<ref name="PMID18802045"><pubmed>18802045</pubmed></ref>

Based on the current research articles, our group has managed to briefly introduce the three main wnt signalling pathways, describing their molecular basis and functions in embryological development, and list a few animal models commonly used for research. Abnormalities caused by dysfunction of wnt signalling pathway will also be mentioned.

==History==

==Molecular Basis of Wnt signalling pathway==

Wnt ligands are glycoproteins with lipid modifications. They are secreted into the extracellular space and interact with the Frizzled (Fz) receptors on the surface of the effector cells.<ref name="PMID21903638"><pubmed>21903638</pubmed></ref> The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.<ref name="PMID17884187 "><pubmed>17884187 </pubmed></ref> After binding with a Fz receptor, the signal is transducted downstream either through G-proteins or through other proteins also coupled with the Fz receptor such as Dishevelled (Dsh/ Dvl).<ref name="PMID21903638"/>

===Canonical Pathway===
The Wnt family of secreted glycoproteins play an important role in the role of embryonic development and adult homeostasis. One method they do so is by regulating the level of transcriptional co-activator β-catenin. β-catenin can accumulate in the cytoplasm and eventually be translocated into the nucleus to form a complex with TCF (Transcription Factor) to activate transcription of Wnt target genes which are essential for development.The canonical Wnt pathway (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded as it is otherwise done through ubiquitination induced by destruction complexes. The conservation of canonical Wnt pathway is a key factor of stem cell pluripotency and cell fate decision during embryo genesis. '''(R)''' Consequently, this developmental cascade involves signals from other pathways according to the cell type and tissue it is affecting.

When the Wnt glycoprotein is secreted and binds to Frizzled receptors, a larger cell surface complex is formed in combination with LRP5/6. ZNRF3 and RNF43 are proteins that have the ability to ubiquitnate Frizzled receptors. However when R-spondins are bound to LGR5/6, Frizzled receptors are unable to be ubiquinated thus highlighting the function of R-spondins to increase sensitivity of cells to the Wnt ligand.

'''In the absence of Wnt'''
# Destruction complexes targeting for ubiquitination of β-catenin In the absence of Wnt proteins, the signalling pool of β-catenin remains at low levels as it is continuously degraded. β-catenin is targeted for ubiquitination by the Actin destruction complex which is made up of: scaffolding protein Axin, tumour suppressor adenomatosis polyposiscoli gene product (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1 (CK1). CK1 ad GSK3 have the specific roles of phosphorylating the amino terminal region of β-catenin which leads it be recognised by β-transducin repeat containing protein (βTrCP), an E3 ubiquitin ligase subunit. This pathway also known as the βTrCP/SKp pathway ultimately leads β-catenin to subsequent ubiquitination and proteasomal degradation. 
# Destruction complex The actual phosphorylation of β-catenin occurs in a multiprotein complex which is made up of axin, denomatous polyposis coli (APC) and diversin. <ref name="PMID15084453"><pubmed>15084453</pubmed></ref> 
#APC function adn APC-Axin cross regulation

'''In the presence of Wnt'''
# Activation of Wnt receptors Wnt signaling requires both Fz and LRP6 (or LRP5) and activation occurs likely through a Wnt-induced Fz-LRP6 complex. Wnt-induced LRP6 phosphorylation is a key event in Fz receptor activation. LRP6, LRP5 and Arrow each contain two of the five unit PPPSPxS (P, proline; S, serine or threonine, x, a variable residue), which are essential for LRP6 function and are each transferrable to another receptor to start β-catenin signaling. These PPPSPxS motifs when phosphorylated are docking sites for the Axin complex thus recruiting Axin to LRP6 (or LRP 5) upon Wnt stimulation. It is commonly thought as Wnt to induce PPSP phosphorylation which is surprisingly carried out by GSK3 and CK1 as is also the case for β-catenin phosphorylation '''R Wei et al 2007''' '''R Zeng et al 2005'''. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin 
# Inhibition of β-catenin phosphorylation The exact mechanism by which β-catenin phosphorylation is inhibited once the Fz receptor has been activated is still unknown although much data has been found to suggest possible ways. Some possible suggestions have been:
## Wnt-induced Axin dissociation Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation '''R LUo et al., 2007'''
## Inhibition of GSK3: Through in vitro experiments, phosphorylation of LRP6 cytoplasmic domain or individial phosphoPPPSPxS pepetides have been foudn to have a direct inhibitory effect on GSK3 phosphorylation of β-catenin
## Axin degradation: Overexpression of activated Wnt receptor may cause Axin degradation'''R Tolwinski et al 2003'''
## Transport and retention of β-catenin at nuclear level Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg). 
# Effect of Wnt signalling on nuclear functions & target genes Again the exact mechanisms for how β-catenin that has been retained is transported to the the nucleus and retained there is not yet fully understood. Through various studies though it has been suggested that the dynamic sum of multiple mechanisms of β-catenin shuttling and retention is what determines its distribution across the nucleus and cytoplasm of a cell. However, what is known though is that the family of DNA-bound transcription factors known as the 'TCF/LEF family' is the main partner for β-catenin in gene regulation. TCF's function is normally to repress gene expression in conjunction with another repressor known as 'Groucho' (TLE1 in humans) by promoting histone deacetylation and chromatin compaction. However through an increase in β-catenin level in the nucleus, TCF binds with β-catenin thus displacing Groucho and recruiting other coactivators for gene activation instead. TCF proteins are classified as high-mobility group (HMG) DNA-binding factors. Thus, TCF will bind to a DNA sequence known as the 'Wnt responsive element' can cause significant DNA bending that may later local chromatin structure. However the TCF/β-catenin complex produced by the Wnt signalling pathway, has also been found to have a repressive effect on some target genes.

Although still not clearly understood, β-catenin signalling clearly shows to have an affect of DNA-binding transcription factors by recruiting co-activators or co-repressors to either activate or repress DNA transcription respectively.

<pubmed>19279717 </pubmed>

===PCP Pathway===

The non-canonical, β-catenin independent pathway is also known as the Planar Cell Polarity pathway (PCP). A lot of research has been conducted on the behaviour of cells, as well as its interaction and apoptosis, however, there is a lot that remains unknown on the polarity of the cells. The polarity of some cells is knowns, for example, epithelial cells display apical-basal polarity, while neurons are classified as either receiving (dendrites) or transmitting (axons) cell signals. Mesenchymal cells also have a polarity that allows movement in different directions. PCP has several roles in the human body, many occurring during embryonic development. The roles of the pathway include the elongation of craniofacial processes, guidance of axons, organogenesis of the heart, lungs, kidneys, and eyes. The pathway also acts to regulate the polarity of cells as well as the movement of dorsal mesodermal cells at the time of convergent extension and throughout the neural tube closure process. There are two modules in place to explain the initiation and establishment of the PCP mechanism, these modules are known as the global and local modules, these are mainly based on studies performed on Drosophila.

*The local module explains the association between the PCP proteins across the cell membrane. It also explains how cells interact with each other, and thus create a positive feedback mechanism that regulates the PCP proteins and polarisation in cells.
*The global module explains the process by which the direction of PCP is associated with tissue axes. It is also thought that this module also regulates the Hippo pathway, whose role involves the regulation of organ size.

There is another model called the Frizzled gradient model, where a Frizzled activity gradient is formed due to a gradient of unknown signals. The signal is then utilised by the cells and compared to neighbouring cells, so that the cell can have a lower Frizzled activity in comparison to its neighbours.

The PCP pathway does not act alone, but is aided by a co-receptor, where it is though that one of the following plays a role in this pathway; use NRH1, Ryk, PTK7 or ROR2. Initially, the pathway initiates through the binding of Wnt to Fz and the co-receptor. Dishevelled proteins are attracted by the receptor involved. This protein utilises two sites on its structure, PDZ and DIX domains, which forms a linkage to the Dishevelled-associated activator of morphogenesis 1 (DAAM1). The G-protein Rho is activated by DAAM1, by exchanging the base protein, guanine. Rho, in turn, activates the Rho-associated kinase (ROCK), a cytoskeleton regulator. At the same time, the dishevelled protein binds with Rac1 and allows the binding of profilin to actin. The Rac1 initiates the role of JNL, causing actin polymerisation to occur. Also, the binding of profilin and actin causes gastrulation and a restructuring of the cytoskeleton.

===Calcium Ion Pathway===
The Wnt/calcium ion signalling pathway is activated by the wnt-5a family wnt ligands, including wnt-4, wnt-5a and wnt-11. The activation of this wnt pathway increases the activity of several calcium ion sensitive proteins such as CaMKII, PKC and calcineurin (CaCn/Cn).

As another pathway belongs to the wnt/non-canonical signalling pathway (the other one is the PCP pathway), beta-catenin is not involved in the signalling transduction either. Instead, the intracellular beta-catenin concentration is downregulated by the activity of wnt/calcium ion pathway. Therefore, the canonical pathway and the calcium ion pathway commonly antagonists each other and possesses opposite functions.

The calcium ion pathway is also different from the PCP pathway, however, not much. Both pathways share some common wnt ligands such as wnt-5a and wnt-11. It is note worthy that wnt-5a may also activate the canonical pathway under some conditions. Another difference between the PCP pathway and the calcium ion pathway is that different downstream effectors are involved. In PCP pathway, the main effectors are Rho GTPase and Jun-N-terminal kinase (JNK). However, cross talks between the two pathways are common especially when the common wnt ligand is used to induce the cells.<ref name="PMID14766423"><pubmed>14766423</pubmed></ref>

'''The general mechanism of wnt/calcium ion signalling is described below:'''
#Binding with Fz receptor Wnt ligands firstly interact and physically bind to the extracellular portion of the transmembrane Fz receptors (e.g. Fz-7), which are G-protein-coupled receptors. This interaction allows the formation of a functional complex assembled by Dishevelled (Dvl/Dsh), β and γ subunits of trimeric G-protein and other proteins such as beta-Arrastin 2 (Arrb2). <ref name="PMID24489854"><pubmed>24489854</pubmed></ref>
#G-protein activity The G-protein beta/gamma dimer formed then activates the enzyme phospholipase C (PLC) which cleaves phosphatidylinositol-4,5-bisphosphate (PIP2), which is a membrane protein, into inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 then migrates away from the cell membrane and binds to the IP3 receptor on the surface of endoplasmic reticulum (ER). The IP3 receptor functions as a calcium ion channel which activation causes release of calcium ion from ER into the cytoplasm and therefore, increases the intracellular calcium ion concentration.<ref><pubmed>9073455</pubmed></ref>
#Result of increased calcium ion concentration As mentioned before, increased calcium concentration leads to activation of several calcium sensitive proteins (CaMKII, CaCN and PKC). Their effects will then be discussed separately.
#*'''CaMKII''' CaMKII activity is modulated by calcium/calmodulin. Binding of calcium/calmodulin with the CaMKII induces a conformational change of CaMKII so that the catalytic site is now exposed towards the outside. <ref><pubmed>7778873</pubmed></ref>Further investigation has revealed TAK-1/NLK of being a downstream target of CaMKII following CaMKII activation. Both TAK-1 and NLK proteins were believed to belong to the mitogen-activated protein kinase (MAPK) pathway, which has been reported to be able to down regulate effect of canonical wnt pathway. As a result, it seems that wnt calcium ion pathway activates the TAK-1/NLK MAPK pathway through CaMKII and disrupts the binding of beta-catenin with TCF leading to downregulation of canonical pathway.<ref><pubmed>12482967</pubmed></ref> CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.<ref name="PMID14766423"/>
#*'''CaCN''' CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together<ref><pubmed>9593662</pubmed></ref>. Its activation may also lead to activation of ELK-1, indicating the potential cross talk between CaCN activity and CaMKII effects. More importantly, it may also activate nuclear factors of activated T-cells (NF-AT).<ref><pubmed>12015605</pubmed></ref> NF-AT may further activates AP1 or GATA transcription factors.<ref><pubmed>12975316</pubmed></ref> NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.<ref><pubmed>8684469</pubmed></ref> Taken together, NF-AT is a common node for many different pathways and may interpret distinct signals making it the more important downstream effector of CaCN signalling.
#*'''PKC''' Protein Kinase C (PKC) proteins are a family of catalytic proteins which are able to phosphorylate and activate downstream effector proteins. They can be classified into two types, the classical PKCs and the novel PKCs. Although both types are activated by DAG, the activation of classical PKCs requires the presence of calcium ions.<ref><pubmed>9792904</pubmed></ref> As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.<ref><pubmed>12842914</pubmed></ref> Recently, Arrb2, which forms the signalling complex with Dishevelled and β and γ subunits of trimeric G-protein, is also involved in activation and translocation of PKC alpha to the cellular membrane, indicating that location might also be important for the signalling downstream of PKC activation.<ref name="PMID24489854"/>

Overall, it seems that wnt/calcium ion pathway is mainly responsible for inhibition of TCF/beta-catenin signalling through CaMKII, determination of ventral fate through CaCN and tissue separation through PKC. However, the complicated cross talks between wnt/calcium ion pathway components, between wnt pathways and between wnt and other pathways make the in depth research on wnt signalling a rather difficult work.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* The canonical Wnt signalling pathway determines much of cell fate during embryogenesis.
* A lot of information about the role of Wnt canonical pathway in fetus development was found through study of the Xenopus system. (Xenopus is a type of highly aquatic frog that has been commonly studied as a model organism)

'''Spermann-Mangold Organizer'''
* Once fertilization has taken place, in order for the signalling centre known as the Spemann-Mangold Organizer to be formed at the dorsal side of the embryo, a dorsalizing factor is moved to the future dorsal side by the process of cortical rotation.
* Dsh protein and other elements of the Wnt pathway that lead to stabilization of B-catenin reach the future dorsal side of early Xenopus embryo via cortical rotation.
* Until recently it remained unclear whether a Wnt ligand caused the formation of this dorsal organizer or whether the Wnt signalling cascade was activated intracellularly without the need for a ligand. However recent studies have proved that Wnt11 in fact is the Wnt ligand responsible for this action in early dorsal axis formation.
* In early development of Xenopus, B-catenin/TCF complex has also been found to promote transcription of Twin and Siamosis which encode homeodomain transcription factors. Both Twin and Siamosis are crticial for the expression of organizer specific genes.
* Therefore from the data gathered, it can be concluded that the canonical pathway is required for dorsal axis formation during early development.

'''Anterior head formation and neural patterning'''
* The canonical Wnt pathway also regulates anterior head formation and neural patterning
* A number of Wnt inhibitors (such as Cerberus, WIF, Dickkopf and Frzb) are expressed in and secreted from the Spemann-Mangold Organizer to control the formation of the anterior of the embryo and also promote anterior head formation.
* The inhibitory proteins physically bind Wnt and prevent Wnt/Fx complez from being formed. This leads to to low levels of nuclear B-catenein protein in the anterior region and a higher level within posterior region of the gastrula embryo.
* This gradient of Wnt signal along anteroposterior axis has been found to be essential for the formation of anterior head structure and neuroectodermal pattering.
* However canonical Wnt signalling also plays a role in controlling of posterior patterning and tail formation as well as various organ systems such as the heart, lungs, kidney, skin and bone.
* Finally, Wnt signalling also has recently been found to have a role in stem cell renewal
* Thus is can be concluded that the canonical Wnt pathway impacts formation of all organ systems during embryogenesis, whether that be in direct or indirect ways.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

Observation of vertebrate embryos shows that a set of PCP genes have the roles of regulating convergent extension (CE). This involves the extension of the anterior–posterior (A–P) body axis, and during this time, the mediolateral (ML) axis is narrowed. This has been observed in frog (Xenopus laevis) and zebrafish (Danio rerio) embryos. The PCP pathway has many roles in mammalian development, including neural tube closure to determine the left–right (L–R) asymmetry, thus, it is vital for normal vertebrae development. Thus, if CE is affected or the process experiences any faults, neural tube closure will not occur, leading to the occurrence of spina bifida. The closure process of the neural tube occurs at different locations of the A-P axis, thus, there are two open ends of the neural tube, the head (cranial) and tail (caudal) ends, they are also called the anterior and posterior neuropores, respectively. These neuropores will eventually close that the neural tube is completely sealed. If the anterior neuropore fails to close, then anencephaly will occur. If the posterior neuropore fails to close, then spina bifida will occur. Failure to close of the entire neural tube results in craniorachischisis. Another major role of the PCP pathways is the formation of the sensory hair cells, located in the cochlea in the inner ear. There are three outer rows of hair cells, and one inner row. When the PCP pathway occurs normally, then the hairs are faced in the same direction, when there is an error in the PCP pathway, then the hairs are facing in random directions. Through technological development, some of the PCP proteins can be inactivated to determine what their true effect. There are two proteins involved in gastrulation, WnT5b and Wnt11, it allows the formation of L-R asymmetry during gastrulation, it regulates the direction of the nodal flow, which results in eliminating the bilateral symmetry. The absence of this protein results in random nodal flow, causing mutations. PCP is also required for the extension of limbs along the proximal-distal (P-D) axis. It is known that the PCP pathway is not regulated at the transcription level as none of the PCP proteins are transcription factors. But rather, it is regulated by asymmetrical protein localisation at the protein level. However, it is unknown whether the proteins in the PCP pathway act to initiate PCP, or whether they acts only as permissive signals. There are several genes associated with the PCP pathway, and each of the genes has a particular role. For example, the WnT5a gene is involved in sensory hair orientation, so that all hairs are facing the same direction. This gene also assists in neural tube closure, and if this process occurs efficiently, then birth defects will not occur. The WnT5b gene is involved in convergent extension during gastrulation. WnT9b is also involved in convergent extension, but also polarises the division of kidney epithelial cells. WnT11 is involved in convergent extension and regulates the extension of muscle fibres, as well as orienting the muscle cells.

PCP was originally identified in the insects Oncopeltus fasciatus, followed by extended research in the Drosophila melanogaster. A number of genes were discovered to plat a main role in the PCP pathway. It was found that any variation that occurred in this genes resulted in a deformation in the Drosophila, such as in the eyes or wings. The Drosophila allowed discovery of several parts of the PCP pathway, including seven-pass transmembrane proteins Frizzled (Fz), a four-pass transmembrane protein Van Gogh, and cytoplasmic proteins Dishevelled (Dsh). Once PCP has initiated, the genes associated with the pathway are evenly spread across the cell membrane, soon after, the Frizzled proteins and dishevelled proteins accumulate in the distal membrane, while the Van Gogh protein accumulates in the proximal side of the cell membrane. Research in the Xenopus and zebrafish shows that the proteins regulates gastrulation during embryonic development.

===What does Calcium ion pathway do?===

#'''Body Axis'''
#*'''Promoting involution during xenopus gastrulation''' Binding of wnt-11 with Fz-7 receptor activates canonical, PCP and calcium ion signalling pathways in the experimental xenopus embryos. Signalling of the calcium ion pathway is done through the G-protein coupled with Fz-7 and PKC-alpha. The cells making up the blastocoel roof (BCR) and the anterior mesoderm are possessing the same cadherin molecules. Moreover, there is no physical barrier between those two cell populations. Knocking down of Fz-7 results in failure in activating the wnt/calcium ion signalling pathway and fusion of two cell layers, which then leads to severe gastrulation defects. Moreover, mesodermal cells with Fz-7 activated remains separated with the endodermal BCR, while cells without Fz-7 activation eventually sink down to the BCR and fuse with the cells there. Therefore, it seems that Fz-7 activation is also related with mesodermal cell fate determination.<ref><pubmed>11677610 </pubmed></ref>
#*'''Promoting Convergent extension (CE)''' Research literatures have revealed that wnt-11 can also promote convergent extension in xenopus embryos. This signalling transduction is also conducted through a functional complex formed with Dishevelled (Dvl), beta-arrestin 2 (Arrb2) and beta and gamma-subunits of the G-protein coupled with Fz-7 receptor. This complex can then activate CaMKII and PKC-alpha. Arrb2 may also induce the translocation of PKC-alpha to the cellular membrane. Cdc42 protein, which can modify the actin skeleton of the cells, has also been shown to be a downstream target of PKC. Taken together, it seems that the result of the wnt-11 signalling is to promote the migration of cells which is critical for the CE movement in xenopus during gastrulation.<ref name="PMID24489854"/>
#'''Neural Crest Formation and ESC differentiation into osteogenic lineage''' ''In vitro'' experiments with mouse embryonic stem cells have revealed that wnt-5a signalling through the calcium ion signalling pathway might be related to the formation of cranial neural crest and determination of the cell fate. Although wnt-5a is able to activate the canonical and/or calcium ion signalling pathways, it is shown that nuclear beta-catenin stabilization was decreased following wnt-5a induction. Beta-catenin has generally been considered as an essential factor for stem cell differentiation to the osteogenic lineage. However, based on the data presented, early beta-catenin induction (day 5-7) actually down regulates the differentiation. While wnt-5a induction, which elevate the intracellular calcium ion concentration, upregulates the expression of several osteogenic markers and increases calcification at this early stage. The condition becomes opposite later. From day 7-9, further wnt-5a induction results in decreased degree of differentiation while wnt-3a (canonical pathway ligand) induction is able to antagonist the calcium ion pathway and promotes differentiation. It seems that wnt/calcium ion signalling pathway is more likely to be related with the embryonic stem cell fate decision (towards a osteogenic lineage) at the early stage rather than guiding the whole differentiation process. How wnt-5a signalling is related with neural crest formation was not mentioned in the literature, however, the mechanism can be expected to be similar to the mechanism taking place in xenopus gastrulation, since both CaMKII and PKC are activated in the experimental cells. <ref><pubmed>26956615 </pubmed></ref>
#'''Muscle development''' Experimental results have suggested that wnt-5a (stage 18) was expressed earlier than wnt-11 (late stage 22) during the development of limbs in the chicken model. It seems that wnt/canonical pathway, which can be activated by wnt-3a affects the number of the terminal muscle fibers, whereas wnt-5a and wnt-11 promotes the cell fate of those muscle fibers. Moreover, it is believed that wnt-3a and wnt-11 act in an opposite manner with wnt-3a decreasing the number of fast muscle fibers and increasing the number of slow muscle fibers and wnt-11 decreasing and increasing the number of slow and fast fibers respectively. Both ''in vivo'' and ''in vitro'' results have favoured this conclusion. However, the degree of influence in ''in vivo'' experiment is more moderate than that of the ''in vitro'' experiment, suggesting that epigenetic influences may also play a role in determining the muscle cell fate.<ref><pubmed>12810597 </pubmed></ref>
#'''Vascular development''' It is reported that wnt-5a is able to inhibit the proliferation of human hematopoietic stem cells (HSC) probably acting through BCL-2 and expression of ''Cdknib'' gene. Moreover, the canonical wnt signalling pathway is also inhibited following wnt-5a induction. It seems that on one hand, wnt-3a activates the canonical signalling pathway and promotes the proliferation of human HSCs, and on the other hand, wnt-5a may antagonist the effect of wnt-3a and keep HSCs at the quiescent phase. A hypothesis has also been proposed mentioning that wnt-5a not only silences the proliferation, but also induces self-renewal of the HSCs at the same time, leading to a larger proliferative capacity. <ref><pubmed>17881570 </pubmed></ref>

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,

===Drosophila===

===Human===

===Mouse===

===Xenopus===

==Diseases related with wnt dysfunction==

#Wnt calcium ion signalling pathway dysfunction may lead to cardiac hypertrophy. Experimental results have revealed that in experimental mouse model which possesses a phenotype of cardiac hypertrophy, wnt calcium ion signalling pathway is activated and further induces the activation of CaMKII. As a result, the cellular concentration of Histone deacetylase 4 (HDAC4) is signaficantly decreased. HDAC4 functions as a suppressor of myosin enhancer factor 2 (MEF2), and its down-regulation results in increased MEF2 activity which then leads to cardiac hypertrophy.<ref><pubmed>25489064</pubmed></ref>

==Glossary==
*'''Actin:''' a filamentous proteins (42 kD) involved in muscle contraction in both smooth and striated muscle and also serves as an important structural molecule for the cytoskeleton of many eukaryotic cells.
*'''Antagonistic effect:''' the effect produced by the contrasting actions of two (or more) chemical groups
*'''Blastocoel:''' the primordial, fluid-filled cavity inside the early forms of embryo, e.g. of blastula.
*'''Cadherin:''' any of a family of cell adhesion molecules that facilitate cell to cell adhesion in a homophilic manner and only when calcium ions are bound to it.
*'''Calcification:''' the process by which organic tissue becomes hardened by a deposit of calcium salts within its substance.
*'''Convergent extension:''' cell movement resulting in tissue elongation via intercalation of adjacent cells in an epithelial sheet to form a narrower, longer strip of tissue.
*'''Differentiation:''' the normal process by which a less specialized cell develops or matures to become more distinct in form and function.
*'''Endoderm:''' the single layer of cells surrounding the central stele (vascular tissue) in roots. The radial and transverse walls contain the hydrophobic Casparian band, that prevents water flow in or out of the stele through the apoplast.
*'''Epigenetic:''' the effect that regulates DNA expression without altering the DNA sequences.
*'''Gastrulation:''' the process in which the embryo develops into a gastrula following blastulation during the early embryonic development of animals
*'''Involution:''' the inward movement of an expanding outer layer of cells, thereby forming a dorsal lip in animal gastrulation.
*'''Lineage:''' a term used to describe cells with a common ancestry, that is developing from the same type of identifiable immature cell.
*'''Mesoderm:''' the middle of the three germ layers, gives rise to the musculoskeletal, blood, vascular and urinogenital systems, to connective tissue (including that of dermis) and contributes to some glands.
*'''Vertebrate:''' an animal with a backbone

==A table of all three pathways==
{|
|-
| '''Wnt Signalling Pathways'''
| '''Main Components'''
| '''Normal Functions'''
| '''Abnormailties'''
|-
| '''Canonical Pathway'''
| Main Components
| normal functions
| Abnormalities with canonical pathway
|-
| '''PCP Pathway'''
| Main Components
| Normal PCP
| Abnormalities with PCP
|-
| '''Calcium Ion pathway'''
| Main Wnt Ligands: Wnt-4, -5a, -11; Main mediators: CaCN, CaMKII, PKC
| Antagonist the canonical pathway(CaMKII); Gene expression(CaCN); Cell migration(PKC)
| Abnormalities: Decreased bone differentiation and slow muscle fibers; gastrulation defect
|}

==Quiz==
<quiz display=simple>
{Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.
|type="()"}
- Wnt-4 promotes the cardiac cell proliferation during embryological development.
|| wnt-4 has nothing to do with cell proliferation.
+ The calcium ion pathway may promote the migration of cells during gastrulation.
|| This statement is correct.
- Beta-catenin, as an important downstream target, is involved in all signalling transduction conducted by the calcium ion pathway.
|| beta-catenin is not a mediator of the calcium ion pathway.
- Wnt-11 upregulates the concentration of beta-catenin and promotes cell migration.
|| wnt-11 can promotes cell migration, however, it won't increase the concentration of beta-catenin. Cell migration is more likely to be completed through PKC activation.

{Wnt-5a may lead to increased beta-catenin stabilization and activation of mediators such as PKC, CaCN and CaMKII, therefore, wnt-5a activates both the canonical and the calcium ion pathway at the same time.
|type="()"}
+ The reason is correct, but the result is incorrect.
|| The reason is correct, however, it will not activate both pathways at the same time.
- The reason is incorrect, but the result is correct.
- Both the reason and the result are correct.
- Neither the reason nor the result are correct.
</quiz>

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====
PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>
<pubmed> 26035863</pubmed>
<pubmed> 25732825</pubmed>
<pubmed> 25428587</pubmed>
<pubmed> 25448697 </pubmed>
<pubmed> 25540130</pubmed>
<pubmed> 25410658</pubmed>
<pubmed> 25266145</pubmed>

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

==References==

2016 Group Project 1

2016-10-19T05:41:32Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-19T05:12:06Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-19T05:09:52Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-19T04:46:16Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-18T08:43:24Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-18T08:29:18Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-18T08:19:51Z

Z3414482: /* Canonical Pathway */

2016 Group Project 1

2016-10-17T22:44:31Z

Z3414482: /* What does canonical pathway do? */

2016 Group Project 1

2016-10-14T03:56:18Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:52:49Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:52:26Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:52:03Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:51:42Z

Z3414482: /* What does canonical pathway do? */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:51:03Z

Z3414482: /* Canonical Pathway: How it works - Gloria */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
* Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
* Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
* In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
* Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
* At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
* However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

2016 Group Project 1

2016-10-14T03:49:48Z

Z3414482: /* Canonical Pathway */

{{ANAT2341Project2016header}}{{Group Assessment Criteria table}}


==Introduction==

==History==

==Molecular Basis of Wnt signalling pathway==

Flow chart, molecules involved, function of each pathway
===Canonical Pathway===
* The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
* They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
* The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
* Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
* This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

===PCP Pathway===

===Calcium Ion Pathway===
Wnt calcium ion signalling pathway, same as wnt PCP pathway, also belongs to the non-canonical signalling pathway, which means that beta-catenin is not involved in signalling transduction. This pathway mainly regulates the canonical pathway for dorsal axis formation and the PCP pathway for cell movement in gastrulation. The key effector in this pathway is calcium ion.<ref><PMC>PMC2634250</PMC></ref> When wnt ligands (wnt-4, -5a and -11) interact with the Fz receptor protein across the membrane of the effector cell, the Dishevelled (Dvl/Dsh) protein with binds with the intracellular portion of Fz receptor forms a functional complex with beta-Arrastin 2 (Arrb2) and the β and γ subunits of trimeric G-proteins. <ref><pubmed>24489854</pubmed></ref>

PMID 24489854
beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction.
Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

==Wnt signalling in Embryonic Development==

===What does canonical pathway do?===

===What does PCP pathway do?===

===What does Calcium ion pathway do?===

PMCID: PMC2634250
====CAMKII====

====PKC====

==Experimental Models==
Mainly talking about advantages and disadvantages of each models, like can this model mimic the true condition in Human? cost, ethical issues,
===Human===

===Mouse===

===Xenopus===

==Glossary==

==Quiz/ summary table/ summary graph==

==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do/ Reference===
'''Friday 9/9/16:'''
1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways.
2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin)
3. Look for picture/diagrams/graphs etc.
''Let's get some more information up by next Thursday, 15/9/16''

'''Friday 16/9/16:'''
Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

===The signalling pathways:===
====Canonical Pathway - Gloria====
** The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
** They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
** The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
** Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
** This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

=====Canonical Pathway: How it works - Gloria=====
** Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
** Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
** In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
** Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
** At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
** However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

===== Canonical Pathway & Embryogenesis - Gloria =====

====Non-Canonical Pathway - Caroline====

=====Pathways=====

*The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
*The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
*The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
*The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

=====Role=====

*The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
*Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

======PCP Pathway======

*The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
*During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
*The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
*PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

=====Studies=====

*A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
*Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942
<pubmed>19279717</pubmed>
<pubmed>27101101</pubmed>
<pubmed>26680417</pubmed>
<pubmed>26555387 </pubmed>
<pubmed> 26499793</pubmed>
<pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

====WnT-Calcium Ion Pathway - Tony====
Wnt-Calcium Ion Pathway has generally been accepted as a non-cononical wnt signalling pathway using Calcium ions as secondary messengers. It main function is thought to antagonist the canonical wnt signalling pathway by decreasing the cellular beta-catenin concentration. Wnt-4, -5a & -11 were identified as ligands specifically activate this pathway.
Wnt (5a) interact with Fz receptor. Fz receptor functions as a G-protein coupled receptor which activates Gaq/11. Gaq/11 further cleaves PIP2 into DAG and IP3. IP3 then interacts with the IP3R on the surface of endoplasmic reticulum and causes increase in intracellular calcium ions. Those calcium ions will then react with CAMKII or calcineurin leading to further internal effects. DAG and calcium ions are also able to activate proteins belongs to PKC family which also posses various cellular effects.

*PMID 26956615
**In this article, several in vitro experiments using mouse ESCs, NC cells and mesenchymal stem cells were performed. Wnt5a was reported to posses an osteogenic effect when the induction was done before 7 days of incubation. Several osteoprogenitor markers such as ''Alp'', ''Opn'', ''Ocn'' and ''Bsp'' were found to have an elevated mRNA expression level, while expression of chondrocyte-specific markers such as ''Agg'' and ''Col2a1'' were down regulated. Moreover, EMT markers Snai 1&2 were also found to be increased in the induced cells. Downstream targets of wnt5a (CamkII-alpha, Jnk 1&2 and PKC) were reported to be upregulated at the mRNA level and their activities were also found to be increased. Furthermore, activation of wnt-calcium pathway has also been suggested to decrease the nuclease beta-catenin level and its inhibition may restore the beta-catenin level. However, when the wnt5a induction was performed after day 7, the expression of osteoprogenitor markers were not related with wnt5a. Instead, they were now upregulated by the induction of wnt3a, which activates the caniconical wnt-signalling pathway. Endogenous expression of wnt3a was also increased in the same cells. Interestingly, similar changes were not observed in mouse NC cells or mesenchymal stem cells, indicating this character might be ESC only. It is suggested that wnt-calcium pathway may antagonist the effect of caniconal wnt pathway. There is significant cross talk between those two pathways, and their effects may varies depending on the cell period and cell type.<ref><pubmed>26956615</pubmed></ref>

*PMID 21903638
**WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
**Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
**Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

*PMID 17881570
**It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of ''hes-1'' gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of ''c-myc'' whcih inhibits this action.

====How does it work in a foetus (skin formation) - Arsalan====

Wnt signalling is an important component of skin formation in the very early stages of foetus development. Once the embryo undergoes gastrulation, cells of the ectoderm will form differentiate to form the epidermis and the mesoderm will form the dermis.

WnT signalling strongly influences the decision of the ectoderm layer specifying as nervous system or skin epithelium. WnT signalling inhibits the ectoderm’s responsiveness to the fibroblast growth factors. This inhibition is essential for the ectodermal cells to be able to express bone morphogenetic proteins that result in blocking neural induction and directing the cells to differentiate into keratin expressing cells that form the epidermis.

In terms of the mesoderm WnT signals are important for the process of somitogenesis, in which the mesoderm segments into somites that eventually form the dermis. WnT signals will also instruct the lateral plate mesoderm and somite derived cells to create mesenchymal cells which are essential cells within the dermis.

<pubmed>PMC3552514</pubmed>

===What can go wrong?=== z341763

Cells need to communicate with each other so they can act in a coordinated manner in response to the environment. Communication occurs through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during the appropriate times. This can hence become a very pedantic and intricate process and little changes can interrupt the entire system. When things go wrong in signalling, cancer can happen.

In most normal cells the Wnt pathway is inactive and the beta catenin is destroyed and gene transcription is inhibited. In tumour cells, the Wnt pathway may be activated despite the absence of a Wnt signal. This is a result of a mutation of a gene that carries code for a protein complex and hence disintegrates the protein complex. The beta catenin is now no longer tagged for destruction and its cellular level continues to increase. This is very similar to a normal cell where the pathway was actually activated by the Wnt Signal. Beta catenin reaches the nucleus and activates the TCF and LEF transcription factors which activates an RNA polymerase and transcription of several genes begin. This however is inappropriate and uncontrolled and hence leads to cancer. One of the most common mutations is the mutation of the APC protein and is directly linked with the development of colon cancer. People who inherit defective APC alleles’ develop large numbers of polyps which become malignant adenocarcinoma(insertfootnote). People who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years.

Reference(im not sure how to reference things that arent from pubmed)
http://www.cell.com/cell/fulltext/S0092-8674(06)01344-4
Wnt/β-Catenin Signaling in Development and Disease
Clevers, Hans
Cell , Volume 127 , Issue 3 , 469 - 480

===References===
PMID 21903638

==Glossary==

User:Z3414482

2016-10-14T03:36:17Z

Z3414482: /* Lab Assessment 6 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

Mutations in T-box transcription factor 22 (TBX22) have been found to be one of the genetic factors causing cleft lip or palate with/without ankyloglossia. Ankyloglossia (“tongue tie”) is a birth defect of a short, tight, lingual frenulum that restricts movement of tongue inhibiting speech articulation. Recent studies have also determined that TBX22 mutation is not only associated with cleft palate and ankyloglossia but also cleft lip and palate and tooth agenesis.

'''2. Identify a recent research article on this gene.'''

PMID 21375406

'''3. How does this mutation affect developmental signalling in normal development.'''

During embryo development, TBX22 is expressed in the palatal shelves and reaches a peak prior to elevation to a horizontal position above the This gene’s mRNS has been detected also in the base of the tongue (in region of frenulum), interior portion of the nasal septum that fuses to the palatal shelves, the mesenchyme from which tooth buds develop and tooth buds themselves. TBX22 gene are essential for early development and particular mesoderm specification. Currently no gene deletions have been detected for TBX22 gene. However various points of mutation within the gene that cause defects have been identified. Some of these points are: splice site, frameshift and missense changes.

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

User:Z3414482

2016-10-13T05:44:16Z

Z3414482: /* Lab Assessment 9 */

{{ANAT2341Student2016}}

[[Student Page]]

== Lab Attendance==
[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 14:47, 12 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:15, 19 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:52, 26 August 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:43, 2 September 2016 (AEST)

[[User:Z3414482|Z3414482]] ([[User talk:Z3414482|talk]]) 13:24, 9 September 2016 (AEST)

[[User:Z8600021|Mark Hill]] ([[User talk:Z8600021|talk]]) 12:03, 13 October 2016 (AEDT) Where are your later lab attendance records?
==Lab 1 Assessment==
<pubmed>PMC4496430</pubmed>
'''Summary'''
This research article studies the effectiveness of artificial oocyte activation (AOA) with a calcium ionophore for fertilization under two circumstances. The first overall circumstance, under which the effectiveness of this procedure was tested for, was when there had been total fertilization failure in past IVF cycles. Secondly, the other more specific cases AOA was tested under, were those that showed severe male factor infertility with non-motile spermatozoa even after pentoxifylline (PF) treatement. AOA is a useful method in avoiding total fertilization failure in human in vitro fetilizaiton-embryo transfer (IVF-ET).
AOA attained through calcium ionophore can induce calcium oscillation in oocytes and initate the fertilization process.

For the purpose of this study, between January 2006 to June 2013, 29 intracytoplasmic sperm injection (ICSI) – AOA were performed. All cases were characterised by male factor infertility, however out of the 29 cases further division was made depending on sperm motility after PF treatment. From the data collected, it was concluded that regardless of whether sperm motility was restored after PF treatment or not, oocyte activation is a useful method to ensure fertilization in TESE-ICSI cycles.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - OK so this looks at mechanisms of how the oocyte can be artificially activated giving us insights into the normal in vivo mechanism. Good summary.
| Assessment 5/5
|}

==Lab 2 Assessment==

[[File:V polarity.jpg]]

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - You do not need to include the Reference, Copyright and Student Image template on your page. But you did need to have a link to the reference with the image legend, as shown below.
 

Position of echinoderms in phylogeny of bilateria and establishment of D/V polarity during early development of the sea urchin embryo.<ref><pubmed>19956794</pubmed></ref>
| Assessment 4/5
|}

===Reference===
<pubmed>19956794</pubmed>

===Copyright===
Copyright Lapraz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

{{Student Image}}

==Lab 4 Assessment==
<quiz display=simple>

{1. Which of the following options lists the order of events that happen at different stages in liver development of an embryo?
|type="()"}
- Cell differentiation, hepatic diverticulum development, enmeshing stromal capillaries
+ Hepatic diverticulum development, septum transversum forming liver stroma, epithelial cord profileration
- Hepatic diverticulum, development, epithelial cord proliferation, formation of hepatic trabeculae
- Cell differentiation, epithelial cord proliferation, formation of liver stroma
|| Option two is correct as the liver development occurs between Stages 11-13 in the order of: Stage 11 – hepatic diverticulum development/ Stage 12 – Cell differentiation, septum transversum forming liver stroma, hepatic diverticulum forming hepatic trabeculae/ Stage 13 – epithelial cord proliferation enmeshing stromal capillaries.

{2. Where does the mesoderm lie in relation to the notochord?
|type="()"}
- Ventrally
- Dorsally & Ventrally
+ Laterally, Rostrally & Ventrally
- Caudally
|| Option 2 is the correct answer. In relation to the notochord the mesoderm lies: laterally (on either side), rostrally (above the buccopharyngeal membrane) and ventrally (beneath).

{3. The process of ‘canalization’ that occurs at the endoderm of GIT wall from beginning of week 5 includes:
|type="()"}
+ Endoderm in the GIT wall proliferates, blockage of GIT endoderm lumen occurs, tissue degenerates to form a hollow tube once more
- The dorsal border of the tube where stomach will form in the embryo, begins to dilate and form an enlarged lumen
- Midgut grows in length as a loop extending ventrally, returning as hindgut
- At the level of stomach, dorsal mesogastrium extends as a fold forming the greater omentum
|| Option 1 is the correct answer. Canalization begins from the beginning of week 5 and is complete by end of week 8. Abnormalities in this process can lead to issues such as atresia, stenosis or duplications.

{4. In both the fetus and the adult, the celiac artery supplies:
|type="()"}
- Fetus: Foregut /Adult: Appendix
- Fetus: Midgut /Adult: Respiratory tract
+ Fetus: Foregut /Adult: Esophagus
- Fetus: Hindgut/Adult: Rectum
|| Option 3 is the correct answer. In the 4th week, the 3 distinct segments of the GIT (fore-, mid- and hind-gut) extend the length of embryo and contribute to different components. These 3 divisions are later identified their vascular (artery) supply. The Celiac artery supplies the: foregut (in fetus) & pharynx, esophagus, stomach, upper duodenum, respiratory tract, liver, gallbladder and pancreas (in adults)
</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem good quiz questions. Note that question 2 your answer says option 2 but it is actually 3 that you have shown as the correct answer.
| Assessment 5/5
|}

==Lab 5 Assessment==
Questionnaire done

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Questionnaire on course structure.
| Assessment 5/5
|}

==Lab Assessment 6==
'''1. Identify a known genetic mutation that is associated with cleft lip or palate.'''

'''2. Identify a recent research article on this gene.'''

'''3. How does this mutation affect developmental signalling in normal development.'''

==Lab Assessment 7==
'''1. What is/are the dystrophin mutation(s)?'''

'''2. What is the function of dystrophin?'''

'''3. What other tissues/organs are affected by this disorder?'''

'''4. What therapies exist for DMD?'''

'''5. What animal models are available for muscular dystrophy?'''

==Lab Assessment 8==
Quiz on urogenital development completed

==Lab Assessment 9==

===Group 2 Peer Review===
'''1. Strengths'''

Group 2’s wiki page is very clearly structured. The content has been structured and organised in a neat format making it easy for readers who have limited knowledge about the signalling pathway to have a comprehensive overview of the topic. I thought that the use of ‘history’ section and images made the information more entertaining to read and process. The information was not only written in clear sentences but cited well. This would be especially beneficial to readers who might be intrigued to read more about the studies done on animals or even certain diseases that are related to abnormalities in notch signalling.

'''2. Points for improvement'''

Although the overall wiki page was of high quality and looked as if it was almost complete, there were few headings/sub-headings such as “current areas of research” that were left blank. And as diagrams have been used in the content of the page, perhaps using pictures/graphs from recent research studies under sub headings such as “roles in embryonic development” would help the readers to understand better the large volume of information.

'''3. Overall'''

In conclusion, I thought that this wiki page was well put together and close to completion after minor adjustments to the existing content and further additions are made to it. From reading this page, I was able to gain a clear understanding for how the notch signalling pathway plays an important role in embryo development.

===Group 3 Peer Review===
'''1. Strengths'''

Group 3’s wiki page was entertaining to read and understand the information presented about FGFR pathway because of the variety of elements within the page. I appreciated looking at the various diagrams that were both drawn and provided through external resources. The referencing for the diagrams were well done, allowing readers to easily access research articles that can provide more detailed information about the diagrams. The table also was a great addition to the page that could present the information on subtypes of FGFR in a simple manner. Once the quiz at the end of page is actually completed, I think it would be also a great method for the readers to check their understanding of the information.

'''2. Points for improvement'''

Although there was a lot of information on the page, at times the content seemed a bit disorganised due to the incompletion of certain sections. Also there seemed to be not enough information regarding studies already done/currently being done on animal models to find out more about FGFR pathways. More information on the future of studies in regards to this signalling pathway would also be beneficial. I noticed that there were few typos and sentences that needed revision for correction to grammar/spelling and also information sections that were not adequately referenced e.g. “Bone Development”.

'''3. Overall'''

Group 3’s project has a lot of potential to become of even better quality than it is now. As the current wiki page that I have reviewed is a draft version, I believe that the final wiki page at the completion of this assignment could be a lot more engaging with completion of quiz/addition of more diagrams and provide a more complete information content for FGFR pathway once all the subheadings have been filled out. be a lot more engaging and complete in
===Group 4 Peer Review===
'''1. Strengths'''

From Group 4’s wiki page, I was particularly impressed by the content provided under the headings of “Mechanisms” and “Animal models”. The information was very detailed and referenced well, thus allowing the readers to easily access external resources should they want further information about a certain statement. A long list of reference could be found at the end of the page despite the wiki page being far from completion, indicating the group’s extensive research put in to create this page.

'''2. Points for improvement'''

I would have like to have seen an introduction for Group 4’s page rather than one single image of the Hedgehog signalling pathway being the only content found under the main heading. As there were no information for the subheadings of “History” nor “Function” provided yet, it was initially quite difficult to gage an idea of how and where this signalling pathway functions, especially in regards to embryogenesis. Some diagrams to assist the information under “mechanisms” would also be helpful for readers to understand better the content as well as a glossary as there are certain technical terms like “autocrine” and “paracrine” that readers might have difficulty understanding.

'''3. Overall'''
Overall, I think that Group 4 is off to a very promising start with their wiki page. Once more information has been added in, especially in terms of introducing the pathway, discussing more about the pathway’s role in embryo development, current and future researches on it with diagrams/tables included, I think that the wiki page would be of very high standard.

===Group 5 Peer Review===
'''1. Strengths'''

Inclusion of articles throughout the wikipage, Use of table/ Use of pargraphs
One of the positive points about Group 5 was their integration of relevant articles in certain sections of their wiki page (as is the case in “Introduction” and “Function of T-box in cardiac development”) rather than compiling a list of articles as a separate section. I found that this helped myself as a reader to know which resources I could read on to gain further understanding about specific aspects of T-box genes and their signalling. The use of the table as a “summary of the main T-box genes” organised the great volume of information in an easy way for the audience to read (although using dot points would have improved the organisation of it). It was also clear that a lot of research had been put into creating this wiki page. I particularly enjoyed reading about the “Functions of T-box in development” which was very thoroughly researched.

'''2. Points for improvement'''

Referencing sometimes seems a bit off (grouping of article references)/ Glossary/
There were some sections (such as “Other developmental events”) that were barely referenced or others (such as “TBX22/cleft palate”) where the referencing seemed inaccurate. It would have been to also have seen a glossary as there were a lot of technical words that readers with limited knowledge on this signalling pathway would not have understood as clearly.

'''3. Overall'''

Overall, I thought that Group 5’s wikipage was one of the best that I have seen in this course. The layout was near perfect and information about the signalling pathway was very comprehensive!
===Group 6 Peer Review===
'''1. Strengths'''

Group 6’s wiki page is off to a promising start. Although there is not much content yet on the page, there are subheadings which indicate that the authors of this page are aware of direction they should be researching towards.

'''2. Points for improvement'''

Firstly the wiki page could improve from a better organisation of the headings/subheadings it currently has. At present, all the subheadings are under the heading of “Introduction” which I believe is not true. Secondly, more content is needed as well as research on the TGF beta signalling pathway. At least compiling a list of useful resources to add to the “Reference list” might help the group get started in knowing which articles to start looking for to gain the information they need for each subheading. And more importantly, this wiki page needs to have a clear method of relating this signalling pathway to embryo development and demonstrate how progress has been made in understanding this signalling pathway through recent studies including those with animal models. Use of more images(with better citation), tables and diagrams would improve the quality of this wiki page also. Finally the current information uploaded on the wiki page needs to be correctly referenced (as it hardly is at this point!)

'''3. Overall'''

I think Group 6 has quite a bit of work to get done to reach completion of their wiki page however they are off to a promising start!

User:Z3414482

2016-10-13T02:04:10Z

Z3414482: /* Lab Assessment 9 */