Embryology - User contributions [en-gb]

User:Z3417363

2016-10-28T02:23:53Z

Z3417363:

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 2 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 16 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:27, 23 September 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - These seem reasonable quiz questions. Question 1 should better refer to the "intra-embryonic coelom". Question 2 really assesses what recanalization term means and options do not appropriately test. Question 3, is pretty easy. Question 4, vitelline duct may also contribute to malroation.

| Assessment 4.5/5
|}

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 11 October 2016 - Lab 6 - Cleft lip/Palate assessment missing.
|}

==Lab 7 Duchenne muscular dystrophy==

What is/are the dystrophin mutation(s)?

Dystrophin is encoded by the DMD gene which is a large muscle protein. Recessive mutations in the dystrophin gene on the x chromosome causes Duchenne muscular dystrophy which results in progressive deterioration of muscle tissue and weakness. The dystrophin gene is the largest known human gene and contains 79 exons, DMD occurs when there is a mutation deletion spanning on or multiple exons. These mutations disrupt's the protein's reading frame causing premature stop codons where the transcripts are now susceptible to nonsense decay.

PMC4767260

What is the function of dystrophin?

Dsytrophin is a an X-linked protein assembled by the dystrophin glycoprotein complex. Dystrophin plays an important role in striated muscle cells by linking the extracellular matrix to the actin cytoskeleton and also mediating structural stability of the plasma membrane, ion homoeostasis and transmembrane signalling.

<pubmed>16710609</pubmed>

What other tissues/organs are affected by this disorder?

Dystrophin also plays a significant role in the central and peripheral nervous system and in tissues with a secretory function that from barriers between two functional compartments. These include the blood-brain barrier, choroid plexus and the kidney. There mutations in dystrophin can have large impacts on these tissues/organs aswell.

<pubmed>16710609</pubmed>

What therapies exist for DMD?
The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects.

<pubmed>27621596</pubmed>

What animal models are available for muscular dystrophy?

The mdx mouse, a naturally occurring animal model for DMD, has been available for over a decade , others include hamsters and murines.

<pubmed>11005802</pubmed>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 13 October 2016 - Very good answers. There is also a dog model for DMD and "murine" includes mouse.
| Assessment 5/5
|}

==Lab 8 Assessment==
Completed the quiz during the lab.

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 27 October 2016 - You seem to have difficulties with Urogenital Development concepts, I suggest you look back through both the lectures and the lab associated with these concepts.
| Assessment 2.5/8
|}

===Lab 9 Assessment===

Group 2 Peer Assessment:

Positive Assessment:

So far this page looks great and very organised. I am really impressed by the set out of the information and the way the headings are arranged. It made it really easy for me to navigate around for particular information and not have to look for around aimlessly when I was looking for something in particular. Furthermore I think that the actual categories/sub headings used so far are very concise and effective. For example, I appreciate the brief introduction along with an overview of the molecular mechanisms involved in notch signalling before introducing its roles in embryonic development. This way I was able to have a understanding of what is really involved before understanding how it is important in embryonic development.

The references are also very neatly and correctly done and many times when I did not fully understand a concept I clicked on the citations which took me to the relevant articles and my understanding was clarified. I also really enjoyed the commentary on the specific research papers, for example cardiomyocyte specification and differentiation where you guys actually compared information from separate studies to make the information more whole and relevant.

In the abnormalities section, I think it was really awesome you guys included so many statistics and symptoms and not just a description of the abnormality.

Critical Assessment:

Although everything looks really amazing a couple of improvements that I personally think could be made would make this page really useful to students.

The introduction, although very informative can be simplified a bit more to address criteria 4 and make it a bit easier to understand. This can be done through including an interesting or very simplified diagram to engage the student from the beginning. I would also generally include more diagrams and drawings that are personally drawn as the pictures used although effective, can be difficult to understand when you are learning for the first time. It would also be nice if more words are included in the glossary because there was a quite few words I did not know the meaning of.
Lastly I think it would be a great addition to your page to include another subheading which outlines how the abnormalities are treated as this is something that I was intrigued to discover.

Overall I think your page is going great guys keep it going !

Group 3 Peer Assessment:

Positive Assessment

Wow this is a very professional looking page and one that I was immediately drawn to. The introduction is very clear and simple and I was able to understand the basic of FGFR straight away which made it so much easier for me to try to understand the rest of the information. I absolutely love the use of the tables to introduce the sub-types of FGFR as this is so much easier to read than blobs of information. The dot points are concise and to the point and introduce each sub-type along with its abnormality.

The signal transduction in any signalling pathway is probably the most confusing and hard to understand part. However this part of your project is my favourite and I was surprised as to how quickly I managed to understand the molecular mechanisms of FGFR. The hand drawn diagram is amazing and really clearly displays all the key elements in play for FGFR. What I really like about your page is that it is really user and student friendly. It really invites learning and encourages it. The use of a quiz is a great example of this and really does allow the student to reflect on their knowledge.

Critical Assessment:

The page is absolutely amazing but in my opinion there are a few ways that it could be made even more amazing.

Sometimes the information is a bit overwhelming, in that there is too much of it. For example in the sections Limb Bud formation and Bone development, for information that complicated it would probably be better to employ the use of dot points or tables just to make the information more digestible.

Although the hand drawing of the signal induction is extremely useful, I think it could be made even better by being accompanied with some specific step by step commentary which matches with the drawing. As a student this would make learning about FGFR a lot more engaging and easier.

The section on bone development although very informative could be more relevant to embryology and lastly a section outlining the treatments available for the abnormalities would be very interesting.

Overall great work guys !

Group 4 Peer Assessment:

Positive Assessment:

I am very impressed with the level and depth of information provided in this page so far. It is quite evident that you guys have gone to great effort and lengths to research and find relevant information regarding hedgehog signalling. The research conducted is also further solidified with the correct use of citations which link the information with their articles and allow the user to learn more if required. There is almost 34 references already provided which is a testament to the work that has been put in by the group. Well done!

I love the very detailed explanation of animal models used to investigate hedgehog signalling and there is an abundance of information provided for this where as I’ve noticed other groups tend to very lightly touch this topic.

Critical Assessment:

The page is looking very good so far but in my opinion there are a few ways in which it can be improved.

Although the information is in-depth and thorough it can be a little intense at times. I would recommend using more dot points or look into using tables to categorise information into a more user friendly structure. This can also be achieved by using more subheadings to further dissect the information and make it less imposing when reading as this content can be difficult to understand at first. I would also have a nice and clear introduction at the beginning of your page as it essential for the students entering your page to be able to familiarise themselves with Hedgehog signalling before diving into the more complicated information.

I would also make better use of the subheadings, so that they reflect more of the marking criteria in particular hedgehog signalling role in embryology. I didn’t see too much content outlining and explaining this and this is a major part of the project. It would also be a good idea to draw a picture rather than using one to explain the mechanism as simplified visual aids always help. Lastly, try including a glossary as there were many terms that I was very unfamiliar with, such as organogenesis.

Group 5 Peer Assessment:

Positive Assessment:

This is a very well, put together and organised page. Everything is very simple and straight-forward make it extremely student friendly and something I would definitely use to learn about T-Box genes. The introduction along with explanation of the actual meaning of T-box is both informative and also interesting and gives students a good chance to take a break from the heavy load of information and actually indulge in some interesting facts.

Following this, the table is one of the most useful things on the entire page and is extremely concise and structurally pleasing. It provides the key and relevant information and allowed me to make connections with T-box genes and their functions straight away. There is also the use of many pictures throughout the page which definitely aids in visual learning and the more the used the better.

The chronological structuring of the ¬page is also very impressive. As I was reading the page I felt like the information that I was gathering was carrying on and helping me understand what was talked about in the next sections.

Critical Assessment:

The chronological structuring although very impressive did fall a little out of place when the sub-headings “Ancient origins and evolution of the T-box gene family” appeared at the end of the page when it seems like this is something that should be included in the start. It would be thoroughly recommended to utilized hand drawings to explain some of the concepts, especially when introducing the signalling because this would really compliment your already easy to understand introductions and really enhance learning/understanding.

Lastly I think it would be a good addition to your project to include some information on the current research that is being conducted on t-box genes and also if there are treatments for the abnormalities. This page is looking amazing so far so keep up the good work!

Group 6:

Positive Feedback:

The introduction is very simple and clear making it a perfect way to familiarise with the TGF-beta signalling before diving into any more information.
The process is also described very well as it is aided with two pictures which were excellent choices. Together the information and the pictures collate to create a stable understanding of how TGF-beta signals.

Critical Feedback:

You guys can definitely focus on explaining its role specifically in embryo development which is a key criteria for this project. This can be done in many was such as tables or pictures if there is too much information. It would also be a good idea to talk about the things that could go wrong with TGF-beta signalling pathway and the current research being done to rectify this as this is something that is very interesting and also relevant.

You guys should also start referencing early as it can become very problematic later on to keep track and doing very quick citations would go a long way later when editing the project. Lastly it would possibly help if everyone brainstormed some subheadings and categories that you further want to talk about e.g. animal models and that way you know what information you are looking for.
This page has a very strong start and if that quality is carried through to the rest of the content it will be a very successful page.

{{Stem Cell Presentations 2016}}

===Lab 11 Assessment===

Summary of Article "Extensive scar formation and regression during heart regeneration after cryoinjury in zebrafish"

This research article presents and discusses the results obtained using zebrafish models to investigate the cellular response and regenerative capacity of the heart after cyroinjury. The process was performed by exposing the zebrafish to cyrocauterization which is a procedure that uses very cold temperatures to treat abnormalities. This induced damage in the ventricles and cell death was observed. The investigation then observed proliferative responses in the endocardium, pericardium and myocardium and within the first three weeks a massive scar formed which eventually degraded and was replaced by cardiac tissue. These results show that the zebrafish heart undergoes massive fibrosis after cardiac damage and continue to lose this scaring and regenerate the losses and heal. This article is used in the review article to highlight the usefulness of the zebra model and its importance as a tool to investigate the regeneration of heart tissue in humans. The review article says “zebrafish hearts are possibly the most developed model for adult heart regeneration” and this is backed up by the research article.
The research article directly states the similarity between the zebrafish heart and mammalian heart whereby the response of the zebrafish to injury yielded nonhomogeneous ventricular contraction and thickened ventricular wall which was mentioned to be parallel to “mammalian ventricular remodelling after acute MI”. Therefore the review article used this research article as means to justify the continual use of zebrafish in investigating the regeneration of the mammalian heart.

<pubmed>21429987</pubmed>

2016 Group Project 1

2016-10-21T02:21:18Z

Z3417363: /* 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==

{| class="pretty table"
| 1982
| Roel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors.

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus ===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex?
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

{Which of the following models has the most similarity to the human Wnt pathway system?
|type="()"}
- Xenopus
- Zebrafish
+ Mice
|| This is correct, both humans and mice have 19 Wnt glands and 10 frizzled(Fz) receptors.
- Drosophila

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:17:06Z

Z3417363: /* Mouse */

{{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==

{| class="pretty table"
| 1982
| Roel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors.

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus ===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:15:32Z

Z3417363: /* =Xenopus */

{{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==

{| class="pretty table"
| 1982
| Roel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus ===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:14:20Z

Z3417363: /* Xenopus and Zebrafish */

{{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==

{| class="pretty table"
| 1982
| Roel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus ==

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:12:18Z

Z3417363: /* History */

{{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==

{| class="pretty table"
| 1982
| Roel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:10:13Z

Z3417363: /* Mouse */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T02:04:34Z

Z3417363: /* 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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

{Which of the following cancers is most linked to the mutation of the APC complex
|type="()"}
+ Colon Cancer
|| This is correct, studies have shown that people who have mutant APC have nearly a 100% chance of developing colon cancer by the age of 40 years
- Liver Cancer
- Skin Cancer
- Prostate Cancer

</quiz>

==References==

2016 Group Project 1

2016-10-21T01:58:10Z

Z3417363: /* Diseases related with wnt dysfunction */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signalling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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>

==References==

2016 Group Project 1

2016-10-21T01:55:43Z

Z3417363: /* Diseases related with wnt dysfunction */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signaling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==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

2016-10-21T01:53:33Z

Z3417363: /* Experimental Models */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

Wnt signaling generates pattern in all animal embryos, from flies and worms to humans, and promotes the undifferentiated, proliferative state critical for stem cells in adult tissues. Here some of the models and their use is discussed.

===Drosophila===

Over the last 30 years, the use of genetic engineering in Dorsophilia has revealed majority of what is known about the pathway in terms of function, developmental roles and molecular mechanisms. <ref><pubmed>PMC3367557</pubmed></ref> In general, a single Wnt molecule in dorsophilia is encoded by Wg(wingless) and results in chain of cell decisions and networking that his very similar to the combined activities taking place in the Wnt family members in vertebrates.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). It is a simple and accessible tissue that has a system that is highly sensitive and hence is a very powerful tool to observe small changes in the pathway activity.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==Diseases related with wnt dysfunction==

==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

2016-10-21T01:32:48Z

Z3417363: /* Experimental Models */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </pubmed></ref>

==Experimental Models==

===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.

===Mouse===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development and the canonical pathway. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</pubmed></ref>

===Human===

It is clear that majority of the work done to investigate Wnt signalling performed using animal models, is done to apply it on the human model. However there has been many studies using actual adult stem cells that have given great insight and proliferated our understanding of this process. These studies include responses to Wnt signal activation in human embryonic stem cells and human and adult stem cells of mesenchymal, hematopetic, intestinal, gastric, epidermal, mammary and neural lineages<ref><pubmed>24323281 </pubmed></ref>. Due to the vital action of Wnt Signalling in developing and maintaining all these processes, these studies really allow a broad spectrum of information to be collected that helps our understanding of how cancer begins to form. There are of course many ethical issues regarding the use of human stem cells, mainly in the way that they are acquired but manipulation of Wnt signals in both in vivo and in vitro stem cells carries potential to create therapeutic approaches such as tissue engineering, regenerative medicine and anti-cancer treatment and this would change the course of history.

==Diseases related with wnt dysfunction==

==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

2016-10-21T00:47:09Z

Z3417363: /* Xenopus and Zebrafish */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </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===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===Xenopus and Zebrafish===

Xenopus is also another very commonly used model and can give great insight into the study of Wnt signaling in vertebrates, especially in embroyonic development. Xenopus has a relatively large sized embryo and this comes with many advantages as it makes microinjection experiments possible. These studies have allowed for key discoveries about the functional role of Wnt signalling in vertebrates and also the molecular mechanisms involved in vertebrate cells.

Another big advantage is that the xenopause model can use a large number of embryo's which develop rapidly and which can be studied in separation, allowing careful study of embroyonic development. As more technology emerges such as morpholino technology which eliminate the degradation of target RNA molecules and the development of the Xenopus tropicalis, a diploid genetic model system that boasts a shorter generation time and also possesses a genome similar to higher vertebrates, the Xenopus model's value to the study of Wnt pathway only increases.<ref><pubmed>19109718</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

2016-10-21T00:05:24Z

Z3417363: /* Experimental Models */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </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===

One of the widest used models in Wnt signalling is the use of the mouse. Engineering advancements in the field of genetics have allowed researchers to be able to work with mouse stem cells and manipulate them with great precision and detail. This has lead to the creation of very thorough activation profiles of the Beta-Catenin canonical Wnt pathway that takes place during embryonic mouse development. The mouse model is often regarded as the most reliable as amongst all the other models the mouse shares the greatest genetic, structural and physiological similarities with humans. Wnt signalling in particular is very effective with both mice and humans having 19 Wnt glands and 10 frizzled(Fz) receptors).

In general, in mice, canonical Wnt signalling takes place by binding of Wnt molecules to Fz receptors, inducing the dishevelled protein to restrict the destruction complex from phosphorylating/dissociating and hence stabilising Beta catenin which is trans-located to the nucleus where it activates gene transcription. This process can be easily observed through mice models by creating very useful mouse stem cell lines and then scrutinised.

Many studies have allowed researches to learn about critical and significant functions within this process and within early mouse development. However due to the many parallels, these studies have also revealed how the Wnt Pathway interacts with other pathways, forming networks that are apparent and unique in mammalian embryogenisis.<ref><pubmed>22550229 </pubmed></ref>

===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

2016-10-20T23:12:42Z

Z3417363: /* History */

{{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==

{| class="pretty table"
| 1982
| IRoel Nusse and Harold Varmus infected mice with mouse mammary tumor and discovered a a new proto-oncogene that they named int1 (integration 1)
|-
| 1987
| Int1 is highly present in multiple species including Drosophilia and humans. However Its presence in Drosophilia melanogaster revealed to researchers that Int1 is identical to Wingless(Wg)
|-
| 1989
| Mutations in the gene APC which is a part of the protein complex regulated by the Wnt pathway was shown to be correlated with colon cancer.
|-
| 1990
| Beta-catenin, which is the the protein that regulates gene transcription within the Wnt pathway was molecularly cloned.
|-
| 1993
| APC was found to directly interact with Beta-catenin
|-
| 1994
| Dishevelled was found to be an essential part of the Wnt Pathway where ts activation induces the dissociation of the protein destruction complex.
|-
|1995
|Nobel prize awarded to Christiane Nüsslein-Volhard and Eric Wieschaus for their discoveries of the genetic control of Wg in early embryonic developme
|-
|1996
|Beta-Catenin asymmetry and nuclear location was found to regulate embryonic axis formation in Xenopus.
|-
|1996
| The homeotic gene Ubx was identified as the first target Wnt gene
|-
|1998
|Axin 1 and 2 was discovered to interact with Beta-catenin, GSK3B and APC and to promote GSK3B dependent phosphorylation and degradation of Beta-catenin
|-
|1999
|WIF, a secreted protein, inhibits WnT molecules.
|-
|2001
|Wnt over expression was linked with carcino-genesis
|-
|2003
| Dishevelled is over expressed in carcino-genesis
|-
|2006
|Wntless(eveness interrupted) was found to promote secretion of wnt in early embroyonic development in Drosophilia
|-
|2007-Present
| LGR5 was discovered as a Wnt target gene and stem cell marker in the intestine. LRP5 mutations were found to be linked with thyroid tumours. Dishevelled was found to polymerise at the actual plasma membrane and after being stimulated by Wnt gather Axin
|}

==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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </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

2016-10-20T22:13:15Z

Z3417363:

{{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>

==Abnormalities==
Cells communicate with each other through signalling pathways which, stimulate, inhibit and coordinate the behaviour of cells to grow or divide during appropriate times. This is a very pedantic, 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.

===Colon Cancer===

Wnt Signalling factors involved in the canonical Wnt pathway initiate a myriad of intracellular events which results in the creation of a genetic program that controls and co-ordinates the expansion and sorting of the epithelial cells within the colon. <ref name="PMC1856802"><pubmed>PMC1856802</pubmed></ref> In colon cancer, these epithelial cells initially proliferate in an appropriate manner because they contain mutations in the components of the pathway. This is usually the mutation of the adenomatous polyposis coli gene (APC) and is a critical event in the development of the colon cancer. <ref name="PMC3072659"><pubmed>PMC3072659</pubmed></ref> Once this mutation has occurred, a situation where it is as if there is permanent/chronic Wnt stimulation is generated resulting in the recapitulation of the mutated cell and the formation of adenomas that ultimately progress to adeno-carcinomas.

Studies conducted on mutations in the murine homolog of the APC gene revealed the role of mutation of APC leading to the proliferation of tumours in the intestine. The mice were initially heterozygous for the APC gene but loss of this heterozygousity left only mutant APC, which was not sufficient to participate in the important cytoplasmic degradation complex.<ref><pubmed>1350108</pubmed></ref> Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.<ref><pubmed>9065402</pubmed></ref> Lastly, sporadic studies have shown that abnormalities in Wnt signalling have been linked with 90% of patients with colon cancer, with 60 percent containing a mutated APC gene.<ref><pubmed>1528264</pubmed></ref>

===Skin cancer===

Wnt signalling is one of the significant pathways involved in controlling the morphogenesis and development of the skin and hair and having an impact on the way stem cells proliferate along a lineage of the skin and its appendages. <ref><pubmed>PMC3552514</pubmed></ref> It is therefore safe to conclude that given the significance of the pathway any abnormalities or disturbances in the signalling can lead to problems.

One of the observed activities that lead to tumour formation is the stabilization/truncation of B-catenin which is a direct result of prolonged activation of the Wnt pathway. This leads to the formation of pilomatricomas which is a skin tumour that is densely packed within the matrix cells of hair follicles. More study conducted in the Huelsken laboratory yielded results that further support the conclusion that cancer stem cells within the epidermis are as a result of enriched Wnt signalling. <ref><pubmed>3331705</pubmed></ref> These observations were both observed in the studies taken with mice cells <ref><pubmed>9845363</pubmed></ref> and also human cells <ref><pubmed>10192393 </pubmed></ref>. On the contrary if Wnt Signalling is inhibited rather than prolonged, studies show that sebaceous gland tumours with the epidermis begin to proliferate at an alarming rate where in human studies 33% of the tumours studied contained mutations directly linked to the Wnt pathway. <ref><pubmed>16565724 </pubmed></ref>

All these studies taken together allow for the conclusion that skin homoeostasis is highly dependant on the Wnt signalling/beta canin function and that usually a optimal level is required. If there is deviation from this level, hyperactivation or inhibition of the signalling can lead to tumour formation.

===Liver Cancer===

Wnt signalling is once again an imperative pathway that is pivotal in liver development in both governing cell proliferation and also essential functions of the liver.<ref><pubmed>15057752</pubmed></ref>. And once again, abnormalities and aberration Wnt Signalling lead to many tumours found in the liver. These tumours also known as hepatocellular carcinoma have been linked to mutations in the beta-catenin, where b-catenin is activated for prolonged periods. Furthermore the main receptor (frizzled-7) in the Wnt pathway is often seen to be over expressed cells containing in liver cancer as reviewed in the paper <ref><pubmed>10936068 </pubmed></ref> . In terms of statistical analysis studies, hepatocellular adenoma was shown initially shown to be linked with only 12% of beta-catenin mutations but from this 46% of adenomas progressed to carcinomas. <ref><pubmed>16496320 </pubmed></ref>This result demonstrates that beta catenin/Wnt signalling mutations do not play a direct role in the creation of the carcinoma but more so the progression of it.

===Prostate cancer===

The prostate gland in males, is dependant on androgen, which through adrogen receptors regulates the initial growth of prostatic tumour. Therefore Androgen is one of the key factors in the growth of tumour and is often removed in surgery as a treatment measure for prostatic tumour. Androgen is related to Wnt signalling as the androgen receptors bind to the hormones of androgen and hence interact with Beta-catenin to stimulate transcriptional activity. When abnormalities occur, beta catenin binds the receptors and activates target gene expression. Studies on mice show that these mutations of beta-catenin can now induce hyperplasia, cell differentiation and most dangerously, neoplasia which is the uncontrolled growth of cells that is not under physiological control.<ref><pubmed>12037666 </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==

User:Z3417363

2016-10-06T18:06:46Z

Z3417363:

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 2 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 16 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:27, 23 September 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

==Lab 7 Duchenne muscular dystrophy==

What is/are the dystrophin mutation(s)?

Dystrophin is encoded by the DMD gene which is a large muscle protein. Recessive mutations in the dystrophin gene on the x chromosome causes Duchenne muscular dystrophy which results in progressive deterioration of muscle tissue and weakness. The dystrophin gene is the largest known human gene and contains 79 exons, DMD occurs when there is a mutation deletion spanning on or multiple exons. These mutations disrupt's the protein's reading frame causing premature stop codons where the transcripts are now susceptible to nonsense decay.

PMC4767260

What is the function of dystrophin?

Dsytrophin is a an X-linked protein assembled by the dystrophin glycoprotein complex. Dystrophin plays an important role in striated muscle cells by linking the extracellular matrix to the actin cytoskeleton and also mediating structural stability of the plasma membrane, ion homoeostasis and transmembrane signalling.

<pubmed>16710609</pubmed>

What other tissues/organs are affected by this disorder?

Dystrophin also plays a significant role in the central and peripheral nervous system and in tissues with a secretory function that from barriers between two functional compartments. These include the blood-brain barrier, choroid plexus and the kidney. There mutations in dystrophin can have large impacts on these tissues/organs aswell.

<pubmed>16710609</pubmed>

What therapies exist for DMD?
The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects.

<pubmed>27621596</pubmed>

What animal models are available for muscular dystrophy?

The mdx mouse, a naturally occurring animal model for DMD, has been available for over a decade , others include hamsters and murines.

<pubmed>11005802</pubmed>

===Lab 9 Assessment===

Group 2 Peer Assessment:

Positive Assessment:

So far this page looks great and very organised. I am really impressed by the set out of the information and the way the headings are arranged. It made it really easy for me to navigate around for particular information and not have to look for around aimlessly when I was looking for something in particular. Furthermore I think that the actual categories/sub headings used so far are very concise and effective. For example, I appreciate the brief introduction along with an overview of the molecular mechanisms involved in notch signalling before introducing its roles in embryonic development. This way I was able to have a understanding of what is really involved before understanding how it is important in embryonic development.

The references are also very neatly and correctly done and many times when I did not fully understand a concept I clicked on the citations which took me to the relevant articles and my understanding was clarified. I also really enjoyed the commentary on the specific research papers, for example cardiomyocyte specification and differentiation where you guys actually compared information from separate studies to make the information more whole and relevant.

In the abnormalities section, I think it was really awesome you guys included so many statistics and symptoms and not just a description of the abnormality.

Critical Assessment:

Although everything looks really amazing a couple of improvements that I personally think could be made would make this page really useful to students.

The introduction, although very informative can be simplified a bit more to address criteria 4 and make it a bit easier to understand. This can be done through including an interesting or very simplified diagram to engage the student from the beginning. I would also generally include more diagrams and drawings that are personally drawn as the pictures used although effective, can be difficult to understand when you are learning for the first time. It would also be nice if more words are included in the glossary because there was a quite few words I did not know the meaning of.
Lastly I think it would be a great addition to your page to include another subheading which outlines how the abnormalities are treated as this is something that I was intrigued to discover.

Overall I think your page is going great guys keep it going !

Group 3 Peer Assessment:

Positive Assessment

Wow this is a very professional looking page and one that I was immediately drawn to. The introduction is very clear and simple and I was able to understand the basic of FGFR straight away which made it so much easier for me to try to understand the rest of the information. I absolutely love the use of the tables to introduce the sub-types of FGFR as this is so much easier to read than blobs of information. The dot points are concise and to the point and introduce each sub-type along with its abnormality.

The signal transduction in any signalling pathway is probably the most confusing and hard to understand part. However this part of your project is my favourite and I was surprised as to how quickly I managed to understand the molecular mechanisms of FGFR. The hand drawn diagram is amazing and really clearly displays all the key elements in play for FGFR. What I really like about your page is that it is really user and student friendly. It really invites learning and encourages it. The use of a quiz is a great example of this and really does allow the student to reflect on their knowledge.

Critical Assessment:

The page is absolutely amazing but in my opinion there are a few ways that it could be made even more amazing.

Sometimes the information is a bit overwhelming, in that there is too much of it. For example in the sections Limb Bud formation and Bone development, for information that complicated it would probably be better to employ the use of dot points or tables just to make the information more digestible.

Although the hand drawing of the signal induction is extremely useful, I think it could be made even better by being accompanied with some specific step by step commentary which matches with the drawing. As a student this would make learning about FGFR a lot more engaging and easier.

The section on bone development although very informative could be more relevant to embryology and lastly a section outlining the treatments available for the abnormalities would be very interesting.

Overall great work guys !

Group 4 Peer Assessment:

Positive Assessment:

I am very impressed with the level and depth of information provided in this page so far. It is quite evident that you guys have gone to great effort and lengths to research and find relevant information regarding hedgehog signalling. The research conducted is also further solidified with the correct use of citations which link the information with their articles and allow the user to learn more if required. There is almost 34 references already provided which is a testament to the work that has been put in by the group. Well done!

I love the very detailed explanation of animal models used to investigate hedgehog signalling and there is an abundance of information provided for this where as I’ve noticed other groups tend to very lightly touch this topic.

Critical Assessment:

The page is looking very good so far but in my opinion there are a few ways in which it can be improved.

Although the information is in-depth and thorough it can be a little intense at times. I would recommend using more dot points or look into using tables to categorise information into a more user friendly structure. This can also be achieved by using more subheadings to further dissect the information and make it less imposing when reading as this content can be difficult to understand at first. I would also have a nice and clear introduction at the beginning of your page as it essential for the students entering your page to be able to familiarise themselves with Hedgehog signalling before diving into the more complicated information.

I would also make better use of the subheadings, so that they reflect more of the marking criteria in particular hedgehog signalling role in embryology. I didn’t see too much content outlining and explaining this and this is a major part of the project. It would also be a good idea to draw a picture rather than using one to explain the mechanism as simplified visual aids always help. Lastly, try including a glossary as there were many terms that I was very unfamiliar with, such as organogenesis.

Group 5 Peer Assessment:

Positive Assessment:

This is a very well, put together and organised page. Everything is very simple and straight-forward make it extremely student friendly and something I would definitely use to learn about T-Box genes. The introduction along with explanation of the actual meaning of T-box is both informative and also interesting and gives students a good chance to take a break from the heavy load of information and actually indulge in some interesting facts.

Following this, the table is one of the most useful things on the entire page and is extremely concise and structurally pleasing. It provides the key and relevant information and allowed me to make connections with T-box genes and their functions straight away. There is also the use of many pictures throughout the page which definitely aids in visual learning and the more the used the better.

The chronological structuring of the ¬page is also very impressive. As I was reading the page I felt like the information that I was gathering was carrying on and helping me understand what was talked about in the next sections.

Critical Assessment:

The chronological structuring although very impressive did fall a little out of place when the sub-headings “Ancient origins and evolution of the T-box gene family” appeared at the end of the page when it seems like this is something that should be included in the start. It would be thoroughly recommended to utilized hand drawings to explain some of the concepts, especially when introducing the signalling because this would really compliment your already easy to understand introductions and really enhance learning/understanding.

Lastly I think it would be a good addition to your project to include some information on the current research that is being conducted on t-box genes and also if there are treatments for the abnormalities. This page is looking amazing so far so keep up the good work!

Group 6:

Positive Feedback:

The introduction is very simple and clear making it a perfect way to familiarise with the TGF-beta signalling before diving into any more information.
The process is also described very well as it is aided with two pictures which were excellent choices. Together the information and the pictures collate to create a stable understanding of how TGF-beta signals.

Critical Feedback:

You guys can definitely focus on explaining its role specifically in embryo development which is a key criteria for this project. This can be done in many was such as tables or pictures if there is too much information. It would also be a good idea to talk about the things that could go wrong with TGF-beta signalling pathway and the current research being done to rectify this as this is something that is very interesting and also relevant.

You guys should also start referencing early as it can become very problematic later on to keep track and doing very quick citations would go a long way later when editing the project. Lastly it would possibly help if everyone brainstormed some subheadings and categories that you further want to talk about e.g. animal models and that way you know what information you are looking for.
This page has a very strong start and if that quality is carried through to the rest of the content it will be a very successful page.

User:Z3417363

2016-10-06T18:06:09Z

Z3417363:

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 2 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 16 September 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:27, 23 September 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

==Lab 7 Duchenne muscular dystrophy==

What is/are the dystrophin mutation(s)?

Dystrophin is encoded by the DMD gene which is a large muscle protein. Recessive mutations in the dystrophin gene on the x chromosome causes Duchenne muscular dystrophy which results in progressive deterioration of muscle tissue and weakness. The dystrophin gene is the largest known human gene and contains 79 exons, DMD occurs when there is a mutation deletion spanning on or multiple exons. These mutations disrupt's the protein's reading frame causing premature stop codons where the transcripts are now susceptible to nonsense decay.

PMC4767260

What is the function of dystrophin?

Dsytrophin is a an X-linked protein assembled by the dystrophin glycoprotein complex. Dystrophin plays an important role in striated muscle cells by linking the extracellular matrix to the actin cytoskeleton and also mediating structural stability of the plasma membrane, ion homoeostasis and transmembrane signalling.

<pubmed>16710609</pubmed>

What other tissues/organs are affected by this disorder?

Dystrophin also plays a significant role in the central and peripheral nervous system and in tissues with a secretory function that from barriers between two functional compartments. These include the blood-brain barrier, choroid plexus and the kidney. There mutations in dystrophin can have large impacts on these tissues/organs aswell.

<pubmed>16710609</pubmed>

What therapies exist for DMD?
The only established pharmacological treatment for DMD is corticosteroids to suppress muscle inflammation, however this treatment is limited by its insufficient therapeutic efficacy and considerable side effects.

<pubmed>27621596</pubmed>

What animal models are available for muscular dystrophy?

The mdx mouse, a naturally occurring animal model for DMD, has been available for over a decade , others include hamsters and murines.

<pubmed>11005802</pubmed>

===Lab 9 Assessment===

Group 2 Peer Assessment:

Positive Assessment:

So far this page looks great and very organised. I am really impressed by the set out of the information and the way the headings are arranged. It made it really easy for me to navigate around for particular information and not have to look for around aimlessly when I was looking for something in particular. Furthermore I think that the actual categories/sub headings used so far are very concise and effective. For example, I appreciate the brief introduction along with an overview of the molecular mechanisms involved in notch signalling before introducing its roles in embryonic development. This way I was able to have a understanding of what is really involved before understanding how it is important in embryonic development.

The references are also very neatly and correctly done and many times when I did not fully understand a concept I clicked on the citations which took me to the relevant articles and my understanding was clarified. I also really enjoyed the commentary on the specific research papers, for example cardiomyocyte specification and differentiation where you guys actually compared information from separate studies to make the information more whole and relevant.

In the abnormalities section, I think it was really awesome you guys included so many statistics and symptoms and not just a description of the abnormality.

Critical Assessment:

Although everything looks really amazing a couple of improvements that I personally think could be made would make this page really useful to students.

The introduction, although very informative can be simplified a bit more to address criteria 4 and make it a bit easier to understand. This can be done through including an interesting or very simplified diagram to engage the student from the beginning. I would also generally include more diagrams and drawings that are personally drawn as the pictures used although effective, can be difficult to understand when you are learning for the first time. It would also be nice if more words are included in the glossary because there was a quite few words I did not know the meaning of.
Lastly I think it would be a great addition to your page to include another subheading which outlines how the abnormalities are treated as this is something that I was intrigued to discover.

Overall I think your page is going great guys keep it going !

Group 3 Peer Assessment:

Positive Assessment

Wow this is a very professional looking page and one that I was immediately drawn to. The introduction is very clear and simple and I was able to understand the basic of FGFR straight away which made it so much easier for me to try to understand the rest of the information. I absolutely love the use of the tables to introduce the sub-types of FGFR as this is so much easier to read than blobs of information. The dot points are concise and to the point and introduce each sub-type along with its abnormality.

The signal transduction in any signalling pathway is probably the most confusing and hard to understand part. However this part of your project is my favourite and I was surprised as to how quickly I managed to understand the molecular mechanisms of FGFR. The hand drawn diagram is amazing and really clearly displays all the key elements in play for FGFR. What I really like about your page is that it is really user and student friendly. It really invites learning and encourages it. The use of a quiz is a great example of this and really does allow the student to reflect on their knowledge.

Critical Assessment:

The page is absolutely amazing but in my opinion there are a few ways that it could be made even more amazing.

Sometimes the information is a bit overwhelming, in that there is too much of it. For example in the sections Limb Bud formation and Bone development, for information that complicated it would probably be better to employ the use of dot points or tables just to make the information more digestible.

Although the hand drawing of the signal induction is extremely useful, I think it could be made even better by being accompanied with some specific step by step commentary which matches with the drawing. As a student this would make learning about FGFR a lot more engaging and easier.

The section on bone development although very informative could be more relevant to embryology and lastly a section outlining the treatments available for the abnormalities would be very interesting.

Overall great work guys !

Group 4 Peer Assessment:

Positive Assessment:

I am very impressed with the level and depth of information provided in this page so far. It is quite evident that you guys have gone to great effort and lengths to research and find relevant information regarding hedgehog signalling. The research conducted is also further solidified with the correct use of citations which link the information with their articles and allow the user to learn more if required. There is almost 34 references already provided which is a testament to the work that has been put in by the group. Well done!

I love the very detailed explanation of animal models used to investigate hedgehog signalling and there is an abundance of information provided for this where as I’ve noticed other groups tend to very lightly touch this topic.

Critical Assessment:

The page is looking very good so far but in my opinion there are a few ways in which it can be improved.

Although the information is in-depth and thorough it can be a little intense at times. I would recommend using more dot points or look into using tables to categorise information into a more user friendly structure. This can also be achieved by using more subheadings to further dissect the information and make it less imposing when reading as this content can be difficult to understand at first. I would also have a nice and clear introduction at the beginning of your page as it essential for the students entering your page to be able to familiarise themselves with Hedgehog signalling before diving into the more complicated information.

I would also make better use of the subheadings, so that they reflect more of the marking criteria in particular hedgehog signalling role in embryology. I didn’t see too much content outlining and explaining this and this is a major part of the project. It would also be a good idea to draw a picture rather than using one to explain the mechanism as simplified visual aids always help. Lastly, try including a glossary as there were many terms that I was very unfamiliar with, such as organogenesis.

Group 5 Peer Assessment:

Positive Assessment:

This is a very well, put together and organised page. Everything is very simple and straight-forward make it extremely student friendly and something I would definitely use to learn about T-Box genes. The introduction along with explanation of the actual meaning of T-box is both informative and also interesting and gives students a good chance to take a break from the heavy load of information and actually indulge in some interesting facts.

Following this, the table is one of the most useful things on the entire page and is extremely concise and structurally pleasing. It provides the key and relevant information and allowed me to make connections with T-box genes and their functions straight away. There is also the use of many pictures throughout the page which definitely aids in visual learning and the more the used the better.

The chronological structuring of the ¬page is also very impressive. As I was reading the page I felt like the information that I was gathering was carrying on and helping me understand what was talked about in the next sections.

Critical Assessment:

The chronological structuring although very impressive did fall a little out of place when the sub-headings “Ancient origins and evolution of the T-box gene family” appeared at the end of the page when it seems like this is something that should be included in the start. It would be thoroughly recommended to utilized hand drawings to explain some of the concepts, especially when introducing the signalling because this would really compliment your already easy to understand introductions and really enhance learning/understanding.

Lastly I think it would be a good addition to your project to include some information on the current research that is being conducted on t-box genes and also if there are treatments for the abnormalities. This page is looking amazing so far so keep up the good work!

Group 6:

Positive Feedback:

The introduction is very simple and clear making it a perfect way to familiarise with the TGF-beta signalling before diving into any more information.
The process is also described very well as it is aided with two pictures which were excellent choices. Together the information and the pictures collate to create a stable understanding of how TGF-beta signals.

Critical Feedback:

You guys can definitely focus on explaining its role specifically in embryo development which is a key criteria for this project. This can be done in many was such as tables or pictures if there is too much information. It would also be a good idea to talk about the things that could go wrong with TGF-beta signalling pathway and the current research being done to rectify this as this is something that is very interesting and also relevant.

You guys should also start referencing early as it can become very problematic later on to keep track and doing very quick citations would go a long way later when editing the project. Lastly it would possibly help if everyone brainstormed some subheadings and categories that you further want to talk about e.g. animal models and that way you know what information you are looking for.
This page has a very strong start and if that quality is carried through to the rest of the content it will be a very successful page.

Talk:2016 Group Project 2

2016-10-06T18:02:31Z

Z3417363: /* Peer Review */

{{ANAT2341Project2016discussionheader}}
=Peer Review=
 
 
Group 2:
 
 
Positive aspects of the project and suggested improvements: 
Group 2 has provided a variety of different topics related to the Notch receptor, such as its molecular pathway, its role in embryonic developing both in humans and animals as well as abnormalities caused by disruption in the receptor’s normal function (criteria 6). This variety is excellent, as it informs the audience of various aspects of the Notch receptor ranging from normal to abnormal development as well as newly emerging research (criteria 1.). Group 2 has also utilised both tables and diagrams to represent Notch receptor’s history and signalling pathway respectively (criteria 2). The use of diagrams is a great idea as it allows peers to understand the complexity of the signalling pathway in a much simpler manner (criteria 4).
 
 
In addition, the authors have correctly utilised in text citations when referencing all sources and have created a list of references at the conclusion of the page (criteria 3). Group 2 also investigated specific components of organ development which was another magnificent feature of their page, such that they divided cardiovascular development into different stages including “heart valve development” and “trabeculation” for example. This allows for an in-depth understanding of organ development with respect to the Notch receptor, rather than a general overview of the receptor’s involvement (criteria 5 and 6). The authors also have extended beyond Notch’s involvement in human embryonic development by exploring its role in animal embryonic development (criteria 5).
 
 
Although there are many positives, a possible improvement to this outstanding wiki would be to include a table of the different types of Notch receptors that exist and their different roles in embryonic development. This will allow the audience to understand that there is not just a single receptor playing a role in embryonic development but multiple. Another suggestion would be to add more subheadings under the “Central nervous system” development, as this subheading appears to have a lot less information compared to others. Also, it is obvious that there are different pathways for this receptor such as “Canonical” and “Non-canonical”, therefore it would be a great idea to include a youtube video to summarise these pathways and reinforce the in-depth description already provided on the page.
 
 

Negative aspects of the project and suggested improvements:
 
 

It was also noticed that a variety of terms were utilised which were not defined in the glossary such as “cyclins”, “pluripotent stem cells” and “ligands” for example. It is important to consider that the wiki should be able to teach at a peer level (criteria 4), as some students may not understand these terms. Therefore it is important to define them so audiences can develop a coherent understanding of the information. Another negative feature of the page was that it lacked interactivity. Indeed the page is very informative, however to further engage the audience, a suggestion would be to include a set of multiple choice questions at the end of the page which tests peers about the content covered.
 
 
It was also noticed that the page had a very limited number of subheadings regarding Notch’s involvement in embryonic development. A possible improvement would be to investigate Notch’s involvement in organ systems other than Cardiovascular and central nervous system. This will add a greater variety to the page and provide a greater depth of understanding regarding the role of the Notch signalling pathway in embryonic development.

 
 

===Group 2===

Well done on the progress you have made thus far! You guys have chosen appropriate headings and subheadings that effectively break down the Notch signalling pathway. A coherent introduction has been provided, giving a taste of what is to be expected in this project. The use of a table to explore the history of this signalling pathway was particularly useful in making the information understandable and relevant. Though you have done an excellent job, was there any reason you stopped at 1989? It may even be useful to create a brief timeline of events, thus allowing you to better explore current areas of research by considering past studies that have been performed.

You’ve provided a good overview of the canonical pathway with the appropriate use of a diagram which aids reader’s understanding of the information provided. In saying this, I think it would be useful to expand on how this pathway is tightly controlled, is it through transcriptional regulation or through other means? In addition, it may be useful to explain the differences in the non-canonical and canonical pathways in terms of their significance and role in embryonic development. I’ve noticed that you have provided a general overview of the role of Notch signalling pathway in embryonic development, do these roles differ between the canonical and non-canonical pathways?

In addition, it’s good that you have included the role of the Notch signalling pathway in animal development as it explores the scope of this pathway beyond human embryology but it may also be useful to explore animal models in research, especially considering that the ‘first description of a “notch” defect’ was discovered in Drosophila. By combining the role of animal models in expanding our knowledge of the Notch signalling pathway with the effect of this pathway in animals, it provides a more rounded approach to explaining and discussing this signalling pathway.

I particularly like how you have included statistics in the ‘Abnormalities of Notch signalling’ section as it provides insight into the importance of this pathway in embryological development. You have successfully described the type of mutation that results in the particularly disease in most cases except for Alagille syndrome. More detail in how the mutation causes the syndrome would be useful with an explanation of how the mutation is brought about.

Overall, you guys have done a fantastic job! You have appropriately referenced and cited all the information provided and have included useful flowcharts, tables and diagrams that aid understanding of the text provided. Providing more detail to each of the sections and communicating with all your team members in the discussion page will ensure that you produce an excellent project! Good luck!

===Group 2 Critical Assessment===

A job well done with the introduction! The introduction is brief however manages to link the topic to embryonic development, different medical conditions, whilst also outlining the function and elements of the pathway. Being brief and succinct, it allows the viewer to continue exploring the page without experiencing confusion at the first lot of information. Further to this, the history of the pathway is formatted well and is not too overwhelming or boring. It is evident you have decided ‘Current Areas of Research’ will also be included in your page which is a great idea as you have included a section on History. This would ensure your Wiki flows well, and covers the pathway from start to finish.

Images have been included to visually represent the elements of the general pathway, as well as the elements specific to the pathway in cardiac development, which forms a great aid for viewers in understanding the content. Videos explaining the different canonical and non- canonical pathways could also be included for viewers with a video learning preference.

Use of in text citations neatens the layout of information and enables viewers to access the article should they find the point interesting. Numerous subheadings have been included which further break down the page into small sections of information. This is a fantastic positive as viewers can locate information in which they are interested in easily instead of having to read through long paragraphs of text. I feel as if linking the topic clinically is extremely important which you have done a great job in! Along with the text explaining the disease, you could possibly include a table stating the disease, the mutation, and the symptoms for viewers after a more easy, accessible format.

Throughout the topics covered, a lot of jargon is utilized, however a full glossary has not been provided. A glossary should definitely be included for terms such as ‘gastrulation’, ‘kinases’ or ‘cardiogenesis’ in order to satisfy criteria 4. Additionally, the page does not cater for viewers interested in further reading up on the topic. To ensure criteria’s 4 and 5 are met, links to interesting facts or articles could be provided so the audience has access to more information if they would like to further their understanding. With a few improvements this Wiki page can definitely prove helpful in understanding the pathway!

===Group 2 Peer Assessment===

Your project is quite good and seems to be on the right track. All your references have been done in-text and have made it really easy to make one’s way to the research article to read more about certain points. Not only that, you have appropriately abbreviated your terms by using the full name initially, and I can see that you have a glossary section which should be beneficial in the future when more terms are added. Your history section is well presented but, it might be important to add references to the papers of the main points of discovery in your history section as to allow people to easily access and find the full article regarding the discovery.

The fact that you have added pictures is quite handy when it comes to using it as an aid to accompanying passage. With regards to the image legend, maybe add more information to it or transfer the description of the image present when clicking into the image onto the legend as to better represent what the image is about while having the passage right next to it. Furthermore, maybe it would be beneficial to add other forms of media such as videos to compliment the passages as well, and help better engage the reader in the topic.

With regards to your section on the canonical pathway, I’ve noticed that the specific genes that are targeted by Notch have been left out and I feel that it is important to mention those genes targets explicitly there as well. That being said, they are mentioned in the proceeding section so it isn’t imperative that you do this. Maybe also try seeing if there is any literature on how the NOTCH receptors come about, such as what genes transcribe it and how the protein is processed and expressed before signalling in the pathway can occur.

I think for the most part there is very little to improve with your wiki page given the quality of it albeit a few minor corrections that I have mentioned above. It is very concise and at no times do I feel that I am reading a wall of text that is disengaging. Thus I feel that as long as such quality is maintained then your wiki page will be quite good when finished.

===Group 2 – Notch Signaling Pathway ===
First impressions of Group 2’s page on the notch-signaling pathway are all positive. Subheadings are very well defined. They have chosen to include a brief yet informative introduction on the pathway, a simple table outlining the major scientific developments over the last 100 years, the molecular mechanisms of the pathway, its specific role in embryonic development (which they have further defined as cardiovascular and CNS), role in animal development, abnormalities relating to this pathway and a glossary. I think another positive aspect of this project, is that they have identified additional subheadings for which they are still to do research on; a particularly important one is current areas of research which not many groups have included. Furthermore, additional positive aspects of this project include the addition of images on the canonical notch signaling pathway and its role in cardiovascular development (which both appear also to be appropriately added to the website), which support the text nicely. It might also be useful to find a relevant video to include just to break up some of the text, and help make the page more interactive. It appears this group has widely researched their topic using both primary and review articles, which are all appropriately referenced using in-text citations. All of these aspects help to clearly convey the necessary information to the reader, and fulfill much of the required criteria of this project. In terms of their written information, Group 2 has included really detailed information on its role in embryonic cardiovascular development, as well as identifying some of the major research articles that have lead to these discoveries and a little bit about them (which then the reader if they are interested it can go read thanks to the inclusion of the in-text citations.) They do include a section of the roles of this pathway in animal development, which is really interesting and goes beyond the normal scope of this course.

Some negative aspects of the project include that, as part of the criteria being that the project has an “element of teaching at a peer level using the student's own innovative diagrams, tables or figures and/or using interesting examples or explanations” perhaps it would be useful to consider including a hand drawn image when researching the non-canonical pathway or transcriptional regulation of notch signaling, or even of some of the receptor/ligands involved in this signaling pathway. Furthermore, on a similar note it may be important to summarise the receptor subtypes involved in the different pathways, their role in embryonic development and abnormalities of the receptor subtype specifically relating to embryonic development in a table or dot point format. Additionally perhaps more information on its role in the CNS (or other systems during embryonic development) even if its not as detailed as cardiovascular, may help to inform the reader of all of its various roles.

In conclusion, it appears that this project is one of the strongest, it has very clear and informative subheadings separating well researched written material, supported by images sourced from the Internet. The main criticisms were just including your own innovative diagrams or explanations, videos to help make it more interactive and table or dot points summarizing the different receptor subtypes involved in each pathway. Following the completion of this, and the subheadings yet to be researched (and glossary) it appears that this project is going to be very successful in informing peers about the said pathway.

===Group 2 Peer Assessment===

Positive Assessment:

So far this page looks great and very organised. I am really impressed by the set out of the information and the way the headings are arranged. It made it really easy for me to navigate around for particular information and not have to look for around aimlessly when I was looking for something in particular. Furthermore I think that the actual categories/sub headings used so far are very concise and effective. For example, I appreciate the brief introduction along with an overview of the molecular mechanisms involved in notch signalling before introducing its roles in embryonic development. This way I was able to have a understanding of what is really involved before understanding how it is important in embryonic development.

The references are also very neatly and correctly done and many times when I did not fully understand a concept I clicked on the citations which took me to the relevant articles and my understanding was clarified. I also really enjoyed the commentary on the specific research papers, for example cardiomyocyte specification and differentiation where you guys actually compared information from separate studies to make the information more whole and relevant.

In the abnormalities section, I think it was really awesome you guys included so many statistics and symptoms and not just a description of the abnormality.

Critical Assessment:

Although everything looks really amazing a couple of improvements that I personally think could be made would make this page really useful to students.

The introduction, although very informative can be simplified a bit more to address criteria 4 and make it a bit easier to understand. This can be done through including an interesting or very simplified diagram to engage the student from the beginning. I would also generally include more diagrams and drawings that are personally drawn as the pictures used although effective, can be difficult to understand when you are learning for the first time. It would also be nice if more words are included in the glossary because there was a quite few words I did not know the meaning of.
Lastly I think it would be a great addition to your page to include another subheading which outlines how the abnormalities are treated as this is something that I was intrigued to discover.

Overall I think your page is going great guys keep it going !

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 07:15, 23 September 2016 (AEST) Hey no worries I hope you feel better soon! I won't be able to do as much during the midsem break which is why I've been doing lots this week. I'll have a look at that link thanks! Also I tried to make a Glossary but am having trouble with the coding side of it but I'll try fix it later to make it a proper glossary :)

 
[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 21:00, 22 September 2016 (AEST) I'm so sorry I've been terrible with putting stuff on our page!! I know I said I would get some done this week but I've been sick these past few days and still recovering so I probably won't make it to the lab tomorrow. I'm having some trouble finding an image that summarises Notch signalling that we can use (most of the good ones I've found don't allow reuse) but I'll keep looking. I'll definitely do a lot more during the midsem break. sorry again! I also found this link: http://www.omim.org/entry/190198 that looks like it has some really good references for different roles in development.

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 14:27, 16 September 2016 (AEST) looks like a great picture so I think we can leave it. I just altered the formatting of the image a little bit, but feel free to change it back if you prefer the previous placement! also I think having the copyright info on the file page is enough. thanks for doing that, I'm still hunting out pictures as well. I've been busy this week with another group project but now that it's out of the way I should be able to do a lot more here. :)

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 09:21, 16 September 2016 (AEST) I found one that didnt look too complicated and also found that it allowed unrestricted use so I've added it into the cardio section of our page (we can always delete it later if need be), not 100% about my formatting though and whether I should include the legend/copyright notice directly on our page? If you click the image though its all there :)

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 23:08, 12 September 2016 (AEST) I've been looking around and found some images, but most of the time they involve a lot more detail than we need and I don't want to overcomplicate it. will keep researching this week and see if I can find some simpler diagrams. :) The images in that book look really good but yeah I'm not sure what the copyright details for that are. if it comes to it I think if we reproduce it by hand and credit the source then it's fine!

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 13:50, 12 September 2016 (AEST) Has anyone found any good images to use for our page? I have found a couple of diagrams on cardiac development and Notch in this book chapter: http://www.sciencedirect.com.wwwproxy0.library.unsw.edu.au/science/article/pii/S0070215310920115 but am still looking to the copyright restrictions etc and thought I'd check everyone's opinion

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 09:22, 9 September 2016 (AEST) Hey everyone, I have added some of my notes to the main page, they are still a work in progress though! I'll continue to research the roles of Notch in the development of the different systems

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 10:04, 2 September 2016 (AEST) Just found this online textbook titled 'Notch Signaling : Methods and Protocols' http://www.springerprotocols.com.wwwproxy0.library.unsw.edu.au/BookToc/doi/10.1007/978-1-4939-1139-4

This one also looks like it might be helpful: http://www.sciencedirect.com.wwwproxy0.library.unsw.edu.au/science/bookseries/00702153/92

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 14:27, 26 August 2016 (AEST) Here are some reviews I have found that could be a helpful starting point:

Notch signalling at a glance: http://jcs.biologists.org/content/joces/126/10/2135.full.pdf

Signalling pathways for neural development: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4369499/pdf/WJSC-7-437.pdf

Notch's role in diabetic neuropathy https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3677813/pdf/nihms473246.pdf

Notch in cardio development and disease http://circres.ahajournals.org/content/118/1/e1.full

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 14:28, 26 August 2016 (AEST) here's the embryology site page for [[Developmental Signals - Notch|Notch signalling]]!

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 14:31, 26 August 2016 (AEST) I also found a review article for Notch signalling in the common fruit fly, which could be a good idea for a subsection: PMID 12369105 ''General outlines of the molecular genetics of the Notch signalling pathway in Drosophila melanogaster: a review''.

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 14:44, 26 August 2016 (AEST) and here's some more reviews: PMID 22397947 ''Non-canonical Notch signaling: emerging role and mechanism'' and PMID 21828089 ''Notch signaling: simplicity in design, versatility in function''.

[[User:Z5014803|Z5014803]] ([[User talk:Z5014803|talk]])
https://www.researchgate.net/publication/264164124_Introduction_to_Notch_Signaling This seems like a good link for the history and discovery of the pathway

[[User:Z5014803|Z5014803]] ([[User talk:Z5014803|talk]])
https://embryo.asu.edu/pages/notch-signaling-pathway-embryogenesis Can be used for introduction

[[User:Z3462474|Z3462474]] ([[User talk:Z3462474|talk]]) 12:41, 19 August 2016 (AEST) Signalling in neural embryonic development looks interesting! Particularly the paper about NSCs and psychiatric disorders.

[[User:Z5015014|Z5015014]] ([[User talk:Z5015014|talk]]) 21:59, 18 August 2016 (AEST): Hedgehog signalling (specifically SHH signalling) sounds really interesting!

[[User:Z5014803|Z5014803]] ([[User talk:Z5014803|talk]]) 22:39, 18 August 2016 (AEST): Bone Morphogenetic Proteins (BMP) signalling in development looks interesting

[[User:Z3491219|Z3491219]] ([[User talk:Z3491219|talk]]) I think looking at how in utero exposure to cigarette smoke affects fetal ovarian development signalling would be interesting.

Talk:2016 Group Project 3

2016-10-06T18:01:23Z

Z3417363: /* Peer Review */

{{ANAT2341Project2016discussionheader}}

==Peer Review==

===Group 3===

You guys have made a good start on your project! I particularly liked how the headings were subdivided appropriately into smaller subheadings as it effectively broke down the FGFR pathway and made the page easy to navigate. Though you have included a short and succinct introduction, I think it should address all the sections being discussed to give the reader a better overview of your project. In addition, the use of a table to explore the timeline of research of the FGF pathway was an excellent idea but I think the text above the table could be incorporated into the table itself and a more extensive timeline could be provided.

Though it was good that you provided a brief overview of the FGFR pathway, you’ve only discussed the components of the pathway rather than the pathway itself. Furthermore, when discussing signal transduction, I think you should be more specific when explaining the process, for example when you mentioned ‘which leads to changes in gene transcription through interactions with DNA’, it causes changes in transcription in which genes and through interactions with which DNA? In saying this, it was wonderful to see the inclusion of a hand-drawn diagram which represents not only your understanding of the pathway but also aids readers understanding of the FGFR pathway.

A good overview has been provided to explain the role of FGFs in embryonic development. The only suggestion I can make is to provide explanations or full names of the abbreviations to aid understanding of the concepts explored. For example, what is ETV1 and EWSR1? By explaining what these abbreviations are the reader will gain better understanding on how they function to help maintain FGF10 expression. In terms of the section on abnormalities, a succinct and coherent introduction was provided. There was a good description of the morphological changes produced by these mutations along with the cause of these abnormalities. There isn’t much I would change in this section except for maybe explaining FGFR2 mutation.

Overall, you guys have done a fantastic job! I thought the inclusion of a quiz was particularly innovative as it makes your project interactive and thus, aids the learning process. Everything was well cited and referenced and it was wonderful to see the use of an original diagram. It was also good to see all groups members contributing to the discussion page which indicates effective communication within the team.

===Group 3 Critical Assessment===
A great introduction to the topic, allowing the reader to slowly transition into the more in-depth points! I particularly like how you have broken down the different constituents of the pathway such as the receptors and protein subtypes and provided a succinct table outlining their function and clinical significance before moving onto the mechanism. Although the ‘FGF Subtype’ table has proven to be effective and helpful, the table on ‘History’ does not seem to be thorough and is very limited. Possibly extending the table by researching more developments in the field of FGF Signalling could make it appear more complete.

Effort has been made to include a hand drawn image of the signalling pathway, which serves as a great source of aid in understanding how the pathway works whilst reading the text beside it. In saying that, effort should be further made to include a complete glossary and ensure terms such as ‘receptor dimerization’ ‘morphogenesis’ are broken down for the reader in order to satisfy criteria 4. This is not only seen in the ‘Signal Transduction’ section but also throughout the other sections. As you have included a fantastic image on bone development to represent the information visually, it would also be a good idea to post up images covering the other areas of embryonic development, such as kidney and inner ear development! You could even consider including short clips explaining these processes to make the page more interactive.

It is clear a decision has been made to talk about ‘Animal Models’. As well as including text on the topic, a possible option could be including a table briefly outlining which animal model has contributed to what knowledge in relation to the pathway in order to simplify the information.

A particular highlight of the Wiki page is the use of a quiz. It is great to see viewers can test their understanding of the topic towards the end and challenge themselves! For the correct option to each question a link to a supporting article or particular section of the page can be provided so the viewer can revisit the information should they have answered the question incorrectly. Overall a great use of tables, images and interactive components!

===Group 3 Peer Assessment===

With regards to your project I have noticed there are many forms of educational tools employed or being planned other than text, which to me is a big plus with regards to your project. The usage of the table to summarises the different FGFR sub-types is really easy to read and understand, and presents the information in a better way than you could’ve with just a wall of text. Your planned multiple choice section seems like it would be a nice addition to your page where it should help solidify the knowledge of the reader, allowing to check what they know. When doing the quiz section not only would it be good if you added explanations for the correct answers, but maybe also if possible explanations of why the other answers are wrong. There seems to be no issues with your citations given that all of them are in-text and multiple. Also the link between signal transduction, embryonic development and abnormalities is quite smooth and within context of their respective preceding parts, making the page read very well.

With regards to your usage of images, it seems mostly good and compliments the passages well, but I feel that it would benefit with adding more information to the legend, possibly by moving some of the description when clicking into the image into the legend. Also since your first image contains mainly abbreviations, maybe it would be good to collate all abbreviations and add it to the glossary such that the reader can easily refer to what the abbreviations mean.

With respect to your signal transduction section, all the components of the pathway seem to have been included, but for the most part how each factor interacts with one another has been left out. Elaborating on how each factor interacts and activates one another such as how FRS2 recruits GRB2 and SHP2, and how those events actually promote activation of RAS. I feel adding this will really improve the depth of this section, and make it less about a bunch of different components and more about how the work together in the context of their individual functions. Also I feel that the history section could be expanded on, maybe to include more time points or critical areas of discovery for the FGFR pathway.

Overall I think your project is shaping up quite well, and that with the addition of the suggestions made above, would make your project quite good. Having used many images, a table, and including the quiz has really made your page quite interactive and engaging which has really benefited your page. Also your subheadings and included passages have appeared to cover most important topics within your signalling pathway.

===Group 3 Peer Assessment===
Positive aspects of the project and improvements:

The group project looks terrific at the initial glance. You can clearly see all the headings and subheadings. In particular, it is great to see a range of subheadings such as “limb bud formation”, “bone development”, “kidney development”. This shows that there was a lot of research put into this project. Also by doing so you have made it clear that your project is about the Fibroblast Growth Factor Receptor Pathway (FGFR). The page is also very easy to navigate as well which was nice to see.
It is also great to see that there is addition of tables, images, and diagrams as it kept the read a lot more interesting and captivating. This allowed you to successfully satisfy criteria 2. It is also good to see correct in text citations and references as this allowed the reader to search for additional information if interested or necessary. Although you haven’t made up any multiple choice questions it is excellent to see a MCQ section. This is a great way to test the readers’ knowledge and in turn you can reflect if you have provided accurate and sufficient information to answer these questions.

It was great to see that you added an abnormalities section and in particular different types of syndromes and disorders. This meant that you went over the minimum information required and put in extra effort to create a coherent project. This satisfied criteria 5 and thus a better project. Overall there are many positives in this report and with minor amendments such as adding information to sections such as “Apert syndrome”, “Animal models”, “Kidney development”, “external genitalia development” etc, a very articulate and well rounded project will be created.

Negative aspects of the project and improvements:

Although there are many positives in the project, there should be some amendments to the project just to ensure all bases are covered. Firstly, it would be advised to increase the amount of information to the introduction and history sections. As these sections are lacking information, the reader may not have enough information to carry on reading as their base on this topic isn’t really strong and lacks information. This can easily turn off new readers and inhibit further exploration of the topic/ project. By adding additional dates in the history section, a better overall knowledge and background of the signalling pathway can be developed which can only enhance learning.

Overall, there are not many negatives and I believe as a reader your project was a great example of progress so far and with the aforementioned minor tweaks, your group is well on their way to achieving extremely high marks.

===Group 3 Peer Assessment===

Positive Assessment

Wow this is a very professional looking page and one that I was immediately drawn to. The introduction is very clear and simple and I was able to understand the basic of FGFR straight away which made it so much easier for me to try to understand the rest of the information. I absolutely love the use of the tables to introduce the sub-types of FGFR as this is so much easier to read than blobs of information. The dot points are concise and to the point and introduce each sub-type along with its abnormality.

The signal transduction in any signalling pathway is probably the most confusing and hard to understand part. However this part of your project is my favourite and I was surprised as to how quickly I managed to understand the molecular mechanisms of FGFR. The hand drawn diagram is amazing and really clearly displays all the key elements in play for FGFR. What I really like about your page is that it is really user and student friendly. It really invites learning and encourages it. The use of a quiz is a great example of this and really does allow the student to reflect on their knowledge.

Critical Assessment:

The page is absolutely amazing but in my opinion there are a few ways that it could be made even more amazing.

Sometimes the information is a bit overwhelming, in that there is too much of it. For example in the sections Limb Bud formation and Bone development, for information that complicated it would probably be better to employ the use of dot points or tables just to make the information more digestible. Although the hand drawing of the signal induction is extremely useful, I think it could be made even better by being accompanied with some specific step by step commentary which matches with the drawing. As a student this would make learning about FGFR a lot more engaging and easier.

The section on bone development although very informative could be more relevant to embryology and lastly a section outlining the treatments available for the abnormalities would be very interesting.

Overall great work guys !

==Comments by Group 3==
[[User:Z5015686|Z5015686]] ([[User talk:Z5015686|talk]]) 12:36, 19 August 2016 (AEST) I thought looking into how prenatal cannabis exposure influences signalling during development might be interesting

[[User:Z5017002|Z5017002]] ([[User talk:Z5017002|talk]]) 12:47, 19 August 2016 (AEST) Ooh cool idea, I agree that looks really interesting, there seems to be a lot of literature about its influence on brain development

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]])z5015544[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) Thats excellent, what about the sonic hedgehog pathway?

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]])Other ideas[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) WNT Signaling Pathway is another one to look at

How can we harvest stem cells from the embyro for use in later life - z5015337

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]])z5015544[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) Ok guys I created a couple of subheadings and provided a brief history. Make sure to use primary research articles that are peer-reviewed because I just spoke to Dr Hill and noticed he stressed that a lot.

[[User:Z5015337|Z5015337]] ([[User talk:Z5015337|talk]]) I have scoured through the projects of old to get a better idea about what is expected from us for this project - [[User:Z5015337|Z5015337]] ([[User talk:Z5015337|talk]])

[[User:Z5015337|Z5015337]] ([[User talk:Z5015337|talk]])I found this giant slab of text regarding the structures of the receptors involved from a journal article and I am working through culling it down for a usable structure definition: FGF receptors and FGF signal transduction. FGFRs are modular proteins comprising 3 immunoglobulin domains (IgI, IgII and IgIII). IgI and IgII are separated by an acidic box (AD). IgII contains a heparin binding domain (HBD). The IgIII domain is followed by a unique transmembrane (TM), a juxtamembrane (JM) and a kinase domain (KD) interrupted by an interkinase domain (IKD). FGF ligands linked to heparin sulfate proteoglycan (HSPG) bind to IgII and IgIII of FGFR. This results in the dimerization and the subsequent transactivation by phosphorylation of specific tyrosine residues. The main two transduction pathways involve the phospholipase C-γ (PLCγ) and the Ras/MAP kinase. The SH2 domain of the PLCγ interacts with the phosphorylated Y766 of the activated receptor. The activated PLCγ hydrolyzes the phosphatidyl-inositol-4,5-diphosphate (PIP2) to inositol-1,4,5-triphophate (IP3) and the diacylglycerol (DAG). IP3 releases Ca2+ while DAG activates the protein kinase C-δ (PKCδ). Activated PKCδ activates Raf by phosphorylating its S338 and stimulates the downstream pathway in a Ras independent manner. The main pathway involves the interaction of the docking protein FRS2α with the amino-acid residues 407–433 (Xu et al., 1998). This protein is activated by phosphorylation on multiple tyrosine residues and subsequently interacts and activates Grb2 linked to Sos, a nucleotide exchange factor involved in the activation of Ras. Activated Ras then activates Raf which stimulates MEK which in turn phosphorylates the MAP kinase ERK. This last activated component translocates to the nucleus and phosphorylates specific transcription factors of the Ets family which in turn activate expression of specific FGF target genes. P: phosphorylation

[[User:Z5015686|Z5015686]] ([[User talk:Z5015686|talk]]) 18:31, 14 September 2016 (AEST) Hey guys I've just changed our subheadings so we can better allocate something for each of us to write on this week. More then happy to change them! Just came across these while I was researching. Did everyone maybe want to put their name next to something they are able to research or chuck in new subheadings that interest them?

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 14:31, 16 September 2016 (AEST)Hey guys, here is the link for omim. Type in the name of the gene and it will give you different articles about it: http://www.omim.org/[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 14:31, 16 September 2016 (AEST)

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 14:41, 16 September 2016 (AEST)Things to include: Flow diagram of the FGFR pathway, 3D diagram of the FGF protein (can be hand-drawn)[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 14:41, 16 September 2016 (AEST)

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 23:22, 28 September 2016 (AEST)Hey guys, hope you're all enjoying the break. Just thought I would let you know I've added a hand drawn diagram and a table too. If anyone finds more information about specific receptor functions in embryo development please add it to the table.[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 23:22, 28 September 2016 (AEST)

[[User:Z5015686|Z5015686]] ([[User talk:Z5015686|talk]]) 12:35, 29 September 2016 (AEST) '''just moved this from our main page''' Extra Resources
Useful review articles that may be worth a read through:
http://onlinelibrary.wiley.com/doi/10.1002/wdev.176/full
http://www.nature.com.wwwproxy0.library.unsw.edu.au/nrd/journal/v8/n3/pdf/nrd2792.pdf
http://www.sciencedirect.com.wwwproxy0.library.unsw.edu.au/science/article/pii/S0012160605006184
http://www.nature.com.wwwproxy0.library.unsw.edu.au/nrm/journal/v14/n3/full/nrm3528.html
http://onlinelibrary.wiley.com.wwwproxy0.library.unsw.edu.au/doi/10.1002/jcp.24649/full
http://genesdev.cshlp.org/content/29/14/1463.full (FGF signalling and skeletogenesis, specifically how mutations to the FGF signalling pathway may be responsible for skeletal diseases)

[[User:Z5015544|Z5015544]] ([[User talk:Z5015544|talk]]) 01:14, 4 October 2016 (AEDT)Looking really good guys, I think we should try and expand beyond what has been covered in the lectures. Maybe we can look at new research involving FGF. We can also look at FGF in animals and how it affects limb development. Let me know what you guys think

Talk:2016 Group Project 4

2016-10-06T17:59:42Z

Z3417363: /* Peer review */

{{ANAT2341Project2016discussionheader}}

=Peer review=
 
 
Group 4:
 
 
Positive aspects of the project and suggested improvements:
 
 

Group 4 has provided numerous headings related to the Hedgehog pathway, such as its involvement in organ development, neural development as well as its mechanism of signalling during embryonic development (criteria 1). The group has also used an image of the signalling pathway to help provide a visual description of the different components of Hedgehog signalling (criteria 2). The authors of this project have also provided in-text citations for all information utilised and have also included a list of references at the end of their page (criteria 3). It is also evident that the group has investigated the involvement of the Shh signalling pathway outside of the scope of human embryonic development by exploring its role in mice, chicks and fruit flies, which is excellent (criteria 5 and 6). The authors have also began to include new research and abnormalities related to the Shh pathway (criteria 1).
 
 

In order to further improve these positive aspects, the authors may provide a written description of the signalling pathway alongside the diagram utilised. This is because it is difficult to understand the signalling pathway just by looking at a diagram. Also, a suggestion would be to include a greater variety of diagrams and tables to support the descriptions already provided. Diagrams may relate to the animal models or the abnormalities described. A table may be utilised to summarise the history of the signalling pathway, such as different components of the pathway that were discovered and the year in which they were discovered. Additionally, whilst it appears that most of the information is correctly referenced, the authors have not correctly referenced the diagram that has been utilised to describe the signalling pathway, which is a breach of copyright laws. Therefore, a suggestion would be to ensure that all diagrams are referenced when added to the page.
 
 

Negative aspects of the project and suggested improvements: 
 
 

Whilst there were positive aspects to this project, a key negative aspect of the project is that the authors have not provided an introduction describing what the Hedgehog signalling pathway is. The introduction may include an overview of the nature and role of the hedgehog signalling pathway in embryonic development, thereby introducing headings in your page. It is also evident that the authors have not met criteria 2 completely, in that a small number of subheadings were utilised. Take for example the heading, “organogenesis”, no subheadings have been created under this heading. A suggested improvement would be to include subheadings relating to specific organs formed by the actions of the Shh pathway, accompanied by an in-depth description and diagrams. It is also evident that the authors utilise complex terminology within their description that often make it difficult to grasp certain concepts. Terms include “knockout”, “autocrine”, “appendage” and “paracrine” for example. A suggestion for improvement would be to include a table of glossary terms at the end of the page, defining these terms.
 
 
It also appears that the authors have not provided a history regarding the Hedgehog signalling pathway and its discovery. A suggestion would be to include a timeline regarding the discovery of this signalling pathway, as it provides the audience with a background of how Shh came to be known.
 
 

===Group 4===

A good start has been made to the project with the appropriate selection of headings and subheadings which provide a brief overview of what is to be discussed in terms of the Hedgehog signalling pathway. By breaking down the mechanism of the pathway, it made the foreign concept much easier to understand. In saying this, this section is quite text-heavy and may benefit with the relocation of the included diagram or even inclusion of other diagrams and flowcharts to engage readers. With the introduction of a fairly new concept, the inclusion of visual or audio stimuli and maybe even a short quiz may encourage interaction with readers.

The discussion of this pathway in mammals exposed readers to the diversity of the Hh signalling pathway but in saying this, the inclusion of a table may be useful to compare and contrast the differences between the pathways in mammals and insects. Overall, this section was well written. On the other hand, when considering the section on animal models, it provided insight into the role of Hh signalling pathway on embryological development and offered a brief introduction to the abnormalities caused by disruptions of this pathway. Once again, the inclusion of diagrams would be useful in this section to provide visual insight into the research being performed.

Though there has been significant exploration of the mechanism and animal models utilised in this pathway, more work is needed to link this pathway to embryological development and this could provide a good leeway into understanding the abnormalities associated with disruption of this pathway. This project can be significantly improved simply by focusing on making it more interactive ad engaging with the inclusion of a variety of stimuli like tables, diagrams, quizzes and even videos. In addition, all information has been well cited and referenced and there has been substantial communication between group members, allowing team members to provide feedback and suggestions thus, ultimately increasing the quality of the work produced.

===Group 4 – Hedgehog Pathway ===
Positive aspects of this project include that Group 4 appear to have well defined subheadings, which function well to help the reader navigate through the page. The information is appropriately referenced using in-text citations, appearing to be from both primary and review articles. There is a significant amount of research on the mechanisms of the pathway but less of a focus on the role of this pathway in embryonic development, which I think is really important in order to relate it back to what we are leaning in both the lectures and tutorials. I think the inclusion of current research is a very important aspect to include in this project, as it identifies the current direction in which this research is heading. This might be also interesting to link to its clinical significance and abnormalities in the signaling pathway.

However, some negative aspects of the page include the lack of an introduction as this essentially establishes your page. You need to include a brief outline of the signaling pathway, a summary of its role in development and the other aspects of it you are looking to discuss. Furthermore, the inclusion of an image outlining the signaling pathway without any information inducing or explaining it should be corrected. The project appears to be very informative but isn’t very interactive and lacks images. Perhaps sourcing images of results from some of the primary articles, which you have referenced or include videos outlining the signaling pathway, might be a useful addition. It might be a good idea to include a glossary at the bottom of the page to help readers to better understand some of these more difficult terms. Also under the subheading of history, like in some of the other projects, a table could be a useful addition, just summarizing all the scientific advances regarding this pathway since it was first discovered, this helps set up how far we have come and then may be helpful when talking about the direction in which we are heading under current research.

In conclusion, this looks like it’s on its way to being a successful project. In summary though, a greater emphasis on its role in embryonic development and conscious effort to make the page more interactive and engaging for the reader will go a long way.

===Group 4 Peer Assessment===

Positive aspects of the project and improvements:

At initial glance I can see a range of headings and subheadings which just made it easier to navigate from one aspect of the project to another. This satisfied the requirements for criteria 1 and 2. This also allowed me to recognise the main topic of the project is the Hedgehog signalling pathway. There is also an addition of an image of the pathway which was great to see as it outlines the main components of the pathway and in general educates the reader about the signalling pathway. This provided a visual stimulus/ description which in turn engaged the reader to find out more about the topic.

It was also good to see correct in text citations and a references list at the end which in turn satisfied criteria 3. To satisfy criteria 5 it was excellent to see information that was well beyond the required information. An example of this is when discussing the role of the pathway in not only humans but also in mice, chicks and fruit flies. The group also began to include new research and abnormalities related to the Shh pathway which aided in rounding off criteria 1.

In order to improve the already positives of this project it would be advised to add a description to the image just so the reader can have some sort of summary about the main points of the image/ pathway. Also, addition of diagrams or tables in some of the subheadings would be good as it will keep the reader interested and in general provide a visual aid. Also it is necessary to cite and provide a reference of the image as it breaches the copyright laws.

Negative aspects of the project and improvements:

Although there are positives to the project, there are a few negatives that can easily be fixed. It is crucial to put in an “Introduction” heading and providing relevant information. This in turn will create a coherent project as it flows from one aspect to another whilst simultaneously providing a brief overview of the Sonic Hedgehog Pathway. Although you have explored the mechanism in animal models it is imperative to link this to embryological development. Also, addition of diagrams, interactive quizzes and tables is necessary to satisfy criteria 3, since 1 image is not enough.

Adding a glossary of terms at the end of the project is needed to clarify any words or phrases that have not been previously encountered such as “organogenesis”, “paracrine”, “dephosphorylation” etc. Overall, the project is coming along nicely and with the recommended amendments, a high mark is definitely in order.

===Group 4 Peer Assessment===

Positive Assessment:

I am very impressed with the level and depth of information provided in this page so far. It is quite evident that you guys have gone to great effort and lengths to research and find relevant information regarding hedgehog signalling. The research conducted is also further solidified with the correct use of citations which link the information with their articles and allow the user to learn more if required. There is almost 34 references already provided which is a testament to the work that has been put in by the group. Well done!

I love the very detailed explanation of animal models used to investigate hedgehog signalling and there is an abundance of information provided for this where as I’ve noticed other groups tend to very lightly touch this topic.

Critical Assessment:

The page is looking very good so far but in my opinion there are a few ways in which it can be improved.

Although the information is in-depth and thorough it can be a little intense at times. I would recommend using more dot points or look into using tables to categorise information into a more user friendly structure. This can also be achieved by using more subheadings to further dissect the information and make it less imposing when reading as this content can be difficult to understand at first. I would also have a nice and clear introduction at the beginning of your page as it essential for the students entering your page to be able to familiarise themselves with Hedgehog signalling before diving into the more complicated information.

I would also make better use of the subheadings, so that they reflect more of the marking criteria in particular hedgehog signalling role in embryology. I didn’t see too much content outlining and explaining this and this is a major part of the project. It would also be a good idea to draw a picture rather than using one to explain the mechanism as simplified visual aids always help. Lastly, try including a glossary as there were many terms that I was very unfamiliar with, such as organogenesis.

[[User:Z5019880|Z5019880]] ([[User talk:Z5019880|talk]]) 14:58, 26 August 2016 (AEST) Hey guys, I have added some sub-headings for the hedgehog signalling pathway, feel free to add any headings that might be useful for the topic, or suggest a different topic.

Hey guys, I've started doing some research on the animal models for the Hedgehog signalling pathway. I'm currently finding it a little difficult understanding some of the terms when researching the experiments done on Drosophila melanogaster so I was wondering if you had any suggestions as to how much detail to include. Also I have included some links that maybe useful for those researching mechanism and history:
<ref><pubmed>17925578</pubmed></ref>
<ref><pubmed>26839340</pubmed></ref>

Also, I thought I would just put in writing here that we want everyone to have completed their parts by the end of mid semester break so that we can meet up the following week to fix any issues with formatting and work on the introduction, conclusion etc. Thanks guys!!
P.S. Did Mark mention that we shouldn't use research articles?

[[User:Z5019880|Z5019880]] ([[User talk:Z5019880|talk]]) 23:30, 12 September 2016 (AEST) Hey, what you have added is really good! To be honest I'm not completely sure about how technical we have to be, because I have also found my self that there is a lot of chemistry when it comes to the signalling pathways etc. which makes it incredibly difficult for me to understand. At this point I reckon what you have now is enough, but we can always revisit it when we have added more to the page, to keep the content at a consistent depth. Also with regards to the research articles, I'm not entirely sure what Mark said, but I'm sure it would be alright to see what is written, and click into the citations to get further information, and just cite that. Anyway I've added a small piece on the processing of the Hh protein, but am unsure if It would be necessary to go more into the chemistry behind how the auto-cleavage occurs.

So I've read what you have written and I think it sounds coherent and it's also very easy to understand so good job! I think I'll do a little more research, I think it might be a little tedious to add information of Shh knockout mice considering the experiments on the chick embryo were quite similar but I'm open to suggestions. I was also thinking it would be useful to include a link to a short youtube video of some sort that would be able to visually explain the Hh signalling pathway (something like this https://www.youtube.com/watch?v=w1xXD9kss2w but unfortunately this video has no audio but has some good visual and written cues). In regards to an image, I actually found a pretty decent image of this pathway but I'm not too sure if we can use it due to copyright. It says we can if it's not for commercial use so I think we should be ok.

Please let me know what you think of the diagram. I've just added something that looks like it could be helpful but feel free to edit/remove it if you don't think it is appropriate :)

[[User:Z5019880|Z5019880]] ([[User talk:Z5019880|talk]]) 22:28, 26 September 2016 (AEST) The diagram looks great! I reckon as long as we include all the copyright it should be fine. We can probably eventually move the image next to the mechanism of signalling section when it is finished. I have started it, and will continue finishing it tomorrow, so if there are any issues with how I'm going about it, please don't hesitate to tell me.

Good idea! I've added some information on Shh Knockout mice so any feedback on what I have written will be greatly appreciated! I'm also thinking of looking into videos that we could link as part of our assignment to make the concept easier to understand.

[[User:Z5019880|Z5019880]] ([[User talk:Z5019880|talk]]) 21:30, 28 September 2016 (AEST) Hey, I've read your section on Shh and it seems good. In terms of what you could add, do you reckon it is appropriate to talk about the advantage of using each of the models over one another, and possible problems you might encounter with each model. I get that it's probably hard to find material on that, so don't worry if that's the case. Also a video would be a great idea, although we should first check with Mark with regards to what sources would be appropriate. Anyway I have finished the first part of the mechanism part regarding the general pathway for Hh proteins that have specifically been studied in the fruit fly, and will continue onto vertebrates later. I was wondering from what you guys have studied on this pathway if I have covered most of the areas sufficiently and in a coherent matter. Any other feedback is also appreciated.

I've read what you've written and you've successfully made it really easy to understand. From the knowledge I've gained through reading articles on this pathway, I don't think you have missed anything thus far. The only improvement I could suggest is maybe referencing an image in your explanation so that readers have a visual stimulus to refer to to ease understanding of the pathway. In regards to looking at the differences between the models, I genuinely tried to find a comparison between the animal models but was met with no such information unfortunately. I'll try and have a look sometime soon. Also, I was thinking maybe we could include a quiz of some sort to make our project more interactive. We could do this by adding a quiz after each section or just one quiz at the very end. Our project is also quite text heavy so I think we should find more images and other stimuli to make it more interesting. We should create a timeline of events for the history of the pathway in the form of a table. Also, should we create a glossary?

Also, quick question, who's handling history, function and current research?

Talk:2016 Group Project 5

2016-10-06T17:58:06Z

Z3417363: /* Group 5 Peer Assessment */

{{ANAT2341Project2016discussionheader}}
=Peer Review=
 
 
Group 5: 
 
 

Positive aspects of the project and suggested improvements:
 
 
Upon reviewing this page, it is clear that group 5 has provided numerous headings and subheadings related to Tbx-genes ranging from origins of the genes, their function in embryonic development, abnormalities, history and animal models (criteria 1 and 6). In doing so, the group has also ventured to provide an in-depth explanation of each subheading. Take for example the subheading named, “limb development”, the authors have provided an in-depth description into the role of T-box transcription factors in limb development whilst utilising a diagram to reinforce this description (criteria 2). It also appears that in-text citations have been correctly used to reference the sources of data in most cases (criteria 3). The authors have utilised diagrams and a table to describe various components of the T-box gene ranging from the different types of T-box genes to its mechanisms in embryonic development (criteria 4). The extensive use of diagrams allows the audience to develop a holistic understanding of the various subheadings included, as these diagrams convey the description provided in a visual manner (criteria 5). It is also evident that the group has conducted research into animal models and evolution of the T-box gene, thus demonstrating that the group has investigated areas of research beyond formal teaching activities (criteria 5).
 
 
Improvements which may be made to this page would be to include a timeline regarding the history of the T-box family, as this will display the information in a much more organised and appealing manner. Another improvement which may be made would be to include a YouTube video to introduce the signalling process in development, such as in cardiac and limb development for example. In order to make the wikipage interactive, a further improvement which may be made would be to include a set of multiple choice questions at the end of the page which ask questions about the content covered.
 
 
Negative aspects of the project and suggested improvements:
 
 

Alongside the various positive aspects of this project, there are few negative aspects. A negative aspect identified includes the use of images from Wikipedia pages more than once. It was stated that only one Wikipedia page was allowed to be included as a source. Therefore a suggestion would be to obtain images and data from research articles rather than from Wikipedia pages, as research articles are often a more reliable source of data. It was also noticed that images were not utilised to describe different abnormalities associated with the TBX gene, hence a possible improvement would be to include images depicting such abnormalities. These images may make this section of the page more appealing and engaging to audiences.
 
 

It was also noticed that the image titled “Evolution of the T box gene family”, was incorrectly referenced. Therefore, it is suggested that the authors of the project ensure that the original author of the image are correctly referenced to ensure that copyright laws are not breached. The final negative aspect of the project was that the “Ancient origins and evolution of the T-box gene family” subheading appeared out of place in the page. Therefore a possible improvement would be to include evolution of the T-box gene under the “Origins of the T-box gene” subheading at the beginning of the page as this will create a sense of consistency in the page.
 
 

===Group 5===

First of all, well done on making significant progress on your project! You have addressed all aspects of the pathway involving T-Box genes through subdivision into various headings and subheadings. I particularly liked how there was an inclusion of the specific T-Box gene affected in each of the abnormalities in the subheading itself. The only suggestion I would make is to combine the ‘Ancient origins and evolution of the T-Box gene family’ section with the origins of the ‘T-Box genes’ section to provide a more coherent description of the history of these pathway. You could even form a table to create a timeline of events. In addition, I think it would be beneficial to include the ‘What does T-Box mean?’ as an introduction to the ‘origins of the T-box genes’ section as there is overlap between these sections.

The use of a table to describe the main T-box genes was helpful in providing a brief overview of the components of the pathway and their influence in embryological development. In addition, the link between T-Box genes and embryonic development has been explored considerably. In saying this, greater attention to detail must be paid to explaining abbreviations to aid one’s understanding of the concepts being discussed. For example, what is NKX2-5, Shh and OFT? Though you’ve explained that RA stands for retinoic acid in the ‘Organisms used in animal models for T-Box’ section, this same explanation is not provided in the ‘Limb development’ section where you have discussed that ‘RA and Shh both induced Tbx2’. These small changes will significantly improve the quality of your work.

The inclusion of abnormalities provides great insight into the role of T-Box genes in development. In saying this, though you have explored the effect of the mutation of these genes in animal models, more information is required to explain the effect of these mutations in humans and how they come about. Furthermore, under the heading of ‘Animal models’ there has been discussion mainly of the ‘brachyury gene’ which seems unrelated to animal models due to the lack of a proper introduction. I found the following section (organisms used in animal models for T-Box) to be a better introduction to the topic of animal models. In addition, there has been mention of a number of animal models ‘Drosophila, Xenopus, zebrafish, avians, and mice’ yet only marsupials and amphioxus has been discussed. This could be potentially misleading to readers.

Overall, a fantastic effort has been made. Not only have you touched upon nearly every section of the project, but have included some excellent diagrams and tables which aid understanding of this pathway. In saying this, it is noted that two Wikipedia images have been used though it has been suggested that only one of the images utilised can be from Wikipedia. All information provided was also appropriately referenced and cited. In addition, I think it would be useful to utilise the discussion page to encourage interaction between group members as it allows individuals to provide feedback and suggestions. Hope this helps!

===Group 5 Critical Assessment===
Well done on constructing a thorough Wiki page on the topic of T-box Genes and their Signalling! Viewing the page it is evident numerous headings and subheadings have been provided to accommodate for the large amount of information gathered. Starting off with the introduction I like how you have included a section on what T’-box exactly means, however the information provided in this section talks about the history significantly, hence to turn this into a positive I would suggest adding a table or timeline outlining the major events and discoveries in the past to present this information in a complete, meaningful way. This issue is also seen with the section ‘Origins of the T-box genes’ where major discoveries are highlighted and in which year they occurred. This information can also merge with the history timeline/table.

Within the ‘What does T-box mean?’ section you have also added information on which animal studies were undertaken for the discoveries. To avoid spreading of information and causing confusion for the reader, you could either construct a table to show which animal study was completed in which year, and what discovery it led to as 3 columns, or bring this information down to the section ‘Animal Models’. In saying that, you have attempted to utilize a table and the table works very well with the topic of the different T-box genes, and would prove great help for the viewer.

It is great to see T-box genes and Signalling has been explored further in the field of embryonic development. Extensive information is provided with good use of in- text citations, allowing the reader to navigate to relevant articles. The ability to navigate could be further improved by providing an accessible link to the ‘Abnormalities’ section in a case where you are directing the reader to the section for further information, instead of plain text. Beneath each section for e.g. ‘Limb Development’ Pubmed links have been provided to relevant articles, which is a fantastic idea, however the links have no indication whatsoever of what the article is about. You could add a sentence each next to the links briefly stating what the article is exploring in relation to limb development.

Overall with a few more images, possibly some interactive components such as clips, and a knowledge testing short exam or quiz this Wiki page will stand out. Remember to ensure your information flows well by placing it within appropriate sections!

===Group 5 Peer Assessment===

It is quite clear that what has been provided in your wiki page is extensive and well researched. The inclusion of tables summarizing the different T-box genes although extensive, is very concise and easy to read. I feel that this table really links all the elements of your page together, where you have included its function and related it to embryological development and abnormalities which you go on later to elaborate in other sections. I feel this really complements the introduction and gives a good feel for what’s to come in the rest of the page. The addition of what the term T-box means also is a nice touch, giving context and some history regarding the name.

Your origins section of T-box is quite well outlined, but as mentioned in your page, having a timeline with critical points of discovery with regards to the genes would probably be more beneficial as it would be a lot easier to read a see the time points as a whole. That being said, having the timeline alongside your outline would probably work well, as your outline can serve to elaborate on the timeline. With regards to your subheadings, it seems to they are quite extensive and cover practically all the key components of the T-box genes, and it is also good to see that there is a glossary subheading in place. Content wise there seems to be limited to no issues, but with regards to abbreviations, I have found that the usage hasn’t always been after the fact of providing the full name first. For example, bone morphogenic protein’s abbreviation is used consistently throughout the first part of the wiki page, but it is only described by its full name and then abbreviation later on. This is something you should check out and fix by either adding the full name the first time the abbreviation is used, or adding all these terms to the glossary.

With regards to the pictures they all seem to compliment the sections well and are quite plentiful. That being said though the picture in the “Marsupial forelimb development” does not appear to have the copyright information regarding to its usage, and referencing does not appear to be in full. This is also the same for the picture under the subheading “Organisms used in animal models for T-box”. Other than that the referencing is perfectly fine within the text.

Overall this project is really good and without any major flaws when it comes to the content. A few touch ups here and there with regards to my suggestion above, and your project should be good to go along as the quality is kept at this level.

===Group 5 – T-Box===
First impressions alone it is extremely clear that Group 5 has thoroughly researched this topic have tried hard to include many diagrams and tables to help separate their information up in order to more successfully convey the information across to the reader. Positive aspects of this project include the well-defined subheadings, making the navigation through the page very easy. The introduction is informative and introduces the following subheadings of the project well. The inclusion of what does T-Box mean is also interesting, setting you apart from the other projects. One of the best aspects of the project would have to be the summary table of the main T-box genes, which includes its main expression sites, its function and abnormalities relevant to the specific gene. You have made a note to include a timeline for the history of the T-Box gene, which I think would be successful in summarizing the scientific advances since its discovery, and also help to break up paragraphs of writing. The project appears to be referenced correctly using in-text citations, only query is whether the links to the PMID articles say in the bottom of cardiac and limb development are references or just articles in which you haven’t written on yet and will be referenced appropriately when you do later. The inclusion of a glossary is also a good idea just to help define and explain some of the more difficult terms mentioned.

As for negative aspects of the project, there wasn’t too many. Like for every project, in terms of making it more interactive it might be a good idea to include a YouTube video or animation of the signaling pathway or its role in a specific developmental process, as well as your own hand-drawn image just to fulfill the necessary criteria of this assignment. Furthermore, with some of the smaller images that don’t go the full width of the page, it might be a nice idea to align them to the right as a thumbnail next to their relevant text, so readers see them whilst reading about it. Also remember to make a reference the image you have chosen in your text to emphasise its importance to what you are actually talking about. Although the subheading “good places to look” might just be something for you guys while researching, I think that you could utilize this by including various links with more information on the relevant topics of which you have discussed. This would help to make you page more interactive as well.

This project appears to be extremely well done and is definitely one of the strongest. Most of the criticisms are regarding the formatting of the page and making it more interactive for the reader. All in all this is very well researched project!

===Group 5 Peer Assessment===

Positive aspects of the project and improvements:

Upon reviewing the page, it is evident that there has been a lot of research put in this project. Initially, there is evidence of a range of headings and subheadings which allowed the navigation from one aspect of the project to another extremely easy. This allowed me to confirm that the project is about T-box genes and their signalling. Secondly, it was excellent to see a range of images, tables and graphs as they provided visual aids to learn more about the topic and in general made it easier to accumulate information. Also it was good to see that these tables and images were correctly cited and referenced at the end which meant that there was no breach of copyright laws.

Also, throughout the project there was sufficient amount of information in each subheading which meant that the reader gained all relevant information pertaining to the section that they are reading. It was also great to see a range of abnormalities being added to the project. This meant that you have went above and beyond the scope of the assessment and researched that extra bit to provide additional information about the signalling pathway and complications arising from any mutations. This meant that you successfully satisfied criteria 5 and thus a more rounded project.

Negative aspects of the project and improvements:

This project certainly contains a range of positives but there were minimal negatives that can easily be amended in order to achieve a very high mark. I noticed that there was more than 1 image being used from Wikipedia and the criterion clearly says that a maximum of 1 was allowed. This is not a big deal but just in case there is harsh marking and penalties, it is advised to replace the additional image with another image. In addition, it would be useful to add a glossary of all the terms that one may find confusing such as “homologues”, “heterozygous”, “homology”, “notochord” etc. This in turn will provide the reader with enough information to understand the context of the project and in turn keep them engaged.

Another negative aspect of the project was that the subheading “Ancient origins and evolution of the T-box gene family” randomly appearing nearing the end of the project. This looked a bit out of place and not flowing with the rest of the passage. To correct this it would be advised to add this to the start of the page with the “Origin of the T-box genes” section just so the information clearly flows from one topic to another without creating confusion. Overall, this project is coming along quite nicely. It is evident that a lot of research has been put into constructing a coherent and succinct project but also have the visual cues to back up the main aspects. To maximise marks, it is recommended to reflect on the feedback and correct the minor mistakes.

===Group 5 Peer Assessment===

Positive feedback:

This is a very well, put together and organised page. Everything is very simple and straight-forward make it extremely student friendly and something I would definitely use to learn about T-Box genes.

The introduction along with explanation of the actual meaning of T-box is both informative and also interesting and gives students a good chance to take a break from the heavy load of information and actually indulge in some interesting facts.Following this, the table is one of the most useful things on the entire page and is extremely concise and structurally pleasing. It provides the key and relevant information and allowed me to make connections with T-box genes and their functions straight away.

There is also the use of many pictures throughout the page which definitely aids in visual learning and the more the used the better. The chronological structuring of the page is also very impressive. As I was reading the page I felt like the information that I was gathering was carrying on and helping me understand what was talked about in the next sections.

Critical Assessment:

The chronological structuring although very impressive did fall a little out of place when the sub-headings “Ancient origins and evolution of the T-box gene family” appeared at the end of the page when it seems like this is something that should be included in the start. It would be thoroughly recommended to utilised hand drawings to explain some of the concepts, especially when introducing the signalling because this would really compliment your already easy to understand introductions and really enhance learning/understanding.

Lastly I think it would be a good addition to your project to include some information on the current research that is being conducted on t-box genes and also if there are treatments for the abnormalities. This page is looking amazing so far so keep up the good work!

Some searches to get us started:
[http://www.ncbi.nlm.nih.gov/sites/gquery?term=t-box ''T-box'']
[http://www.ncbi.nlm.nih.gov/sites/gquery?term=tbx ''tbx'']

[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 14:44, 26 August 2016 (AEST)
PMID 25294936 - a relatively recent article that provides background info on the T-box gene family
PMID 16285859

[[User:Z3516832|Z3516832]] ([[User talk:Z3516832|talk]]) 14:52, 26 August 2016 (AEST)
http://www.columbia.edu/itc/hs/medical/humandev/2007/HD15/HD15.pdf

[[User:Z5020373|Z5020373]] ([[User talk:Z5020373|talk]]) 11:32, 16 September 2016 (AEST) Does anyone know how to draw up a table on the page? Thanks.

Talk:2016 Group Project 5

2016-10-06T17:57:10Z

Z3417363:

Talk:2016 Group Project 6

2016-10-06T17:54:44Z

Z3417363:

{{ANAT2341Project2016discussionheader}}
=Peer Review=
 
 
Group 6:
 
 
Positive aspects of the project and suggested improvements:
 
 
The authors of group 6 have created a variety of subheadings related to the TGF-beta signalling pathway, including the nature of the growth factor, its mechanism of action, history and emerging research (criteria 1). Authors have also provided two diagrams related to TGF-beta signalling which reinforces the description of TGF signalling provided (criteria 2). These diagrams allow for a much simpler interpretation of the signalling process described and assist in teaching at the peer level (criteria 4). It appears that the authors are beginning to conduct investigations into new research surrounding TGF-beta signalling, thus indicating that they are attempting to research beyond formal teaching activities (criteria 5).
 
 

Whilst it is excellent that multiple subheadings have been provided, a possible improvement would be to include a much larger variety of subheadings which cover the scope of TGF-beta’s role in embryonic development, abnormalities, types of TGF receptors and animal models. This may allow audiences to understand the big picture surrounding this signalling pathway which will assist in the understanding of the information already provided. Another improvement to this page would be to include more images under different subheadings. One example would be to include an image or table showing the history of discovery surrounding discovery of this signalling pathway.
 
 

Negative aspects of the project and suggested improvements:
 
 

Although there were positive aspects of this project, there were also numerous negative aspects which may be improved. One key negative feature of the page was that the authors did not discuss the role of TGF-beta signalling in the context of embryonic development, hence meaning they failed to meet criteria 6. To ensure that this criterion is met, authors may conduct research into the involvement of TGF-beta in specific processes that occur during embryonic development, perhaps organ development and growth of different primitive structures. In addition, whilst the authors have provided a history of the TGF-beta signalling pathway, the history appears to be very brief. Thus an improvement which may be implemented would be to include a more extensive background regarding the history of discovery of the pathway. It was also noticed that no tables were utilised within the page. A possible improvement would be to include a table describing different abnormalities and their causes in the context of disruption of the TGF-beta pathway. A table may also be utilised to describe different subtypes of TGF-beta receptors as well as their functions during embryonic development. Tables may be utilised as they will assist in the process of teaching at the peer level (criteria 4), particularly because they convey information in an orderly and organised manner.
 
 

The authors of this project also failed to meet criteria 3, in that only one source was referenced in the process of the signalling pathway and also no in-text citations were provided. In addition, authors failed to reference the image file named, “Process of TGF-beta signalling pathway 01”. It is vital that all sources are referenced correctly in order to ensure that the copyright laws regarding the use of information are adhered to. The final negative aspect of the project was that the page did not flow very well, in that subheadings were arranged in a disorderly fashion. An example of this is the inclusion of the subheading labelled, “history of TGF-beta signalling pathway”, towards the end of the page. Since such a subheading provides a background surrounding the pathway, a possible improvement would be to include this subheading at the beginning of the page. By ensuring the orderliness of the page, this creates a sense of coherency between subheadings, thus making the page more appealing and engaging.

===Group 6===

It was good to see some progress being made on the project with the development of some subheadings and the inclusion of an image. In saying this, a better selection of headings and sub-headings could be developed to break down the topic of TGF beta signaling pathway. I think the sub-headings provided under the general heading of ‘Introduction’ could form the main headings of this research project and they could then be further broken down into various subheadings. In addition, the subheading of TGF-beta could be eliminated and this definition could be incorporated into the glossary or general introduction of the topic instead. Furthermore, more focus is needed on the influence of this pathway on embryological development and the abnormalities caused by mutations to the pathway and its components. For example, there has been mention of the effect of TGF-beta in ‘development of the embryo and adult organism, as well as cell growth, immune function and hormone secretion’ but further discussion has not been pursued.

Though a good description of the ‘process of TGF-beta signaling pathway’ has been provided, it could be further improved by referencing the images included in this section in your text (e.g. refer to Figure 1) to aid one’s understanding of the concept being explored. In addition, a timeline of events could be provided to explore the history of this pathway and it would be appropriate to begin with the discovery of TGF-beta. To a reader, information on ‘transformed or malignant cells’ seems unrelated to the TGF-beta signaling pathway even though it may be in fact be related, due to lack of discussion of this pathway or TGF-beta in this description. In regards to the section on ‘Limitations’, what types of limitations are you trying to explore? Limitations in research? This could be better defined by appropriately allocating subheadings to each of the sections.

Though you are heading in the right direction, spending time to produce a basic layout of your project by creating appropriate headings and subheadings would be useful in breaking down the concepts needed to be explored in this pathway. This could be achieved by communicating with group members through the discussion page and providing feedback and suggestions. In addition, greater focus is required in referencing and citing your work to ensure researchers and authors are acknowledged for their work. Also, by exploring animal models of the TGF-beta pathway and the effect of this research in understanding this pathway in humans and its influence on embryological development, you could greatly increase the quality of your work. You could also try and make your project more interactive and engaging through the inclusion of tables, images and diagrams. I hope this helps! Good luck!

=== Group 6 Critical Assessment===
It is great to see an in-depth overview of the TGF beta Signalling pathway and it’s mechanism of action. The page is off to a great start and with a few key improvements it can turn into a successful one! Firstly, I like the use of images to aid the reader in understanding the content better should they be a visual learner. By reading the text on the way the pathway works, it is clear it is a complicated process hence a suggestion would be to add short clips/animations to simplify it for the viewer.

Under your introduction an attempt has been made to briefly highlight the main features of the pathway, however a negative of this section is that a lot of the content is basically listed. For example it is mentioned TGF-beta is part of a larger TGF superfamily comprising of different members such as activins and GDFs. Instead of listing, try presenting your information in a different format such as a table with s few columns stating the member, it’s function, and possibly what a mutation could lead to.

Additionally it is also stated that TGF beta has certain functions such as controlling angiogenesis however doesn’t expand on the ‘other’ functions it has. To turn this into a positive, dedicate different sections on how the pathway is involved in angiogenesis, hormone secretion, proliferation etc. If there are too many functions to fit onto the page, you could shift a few to your ‘Further Reading’ section as an option if the reader would like to explore further into the pathway’s functions, or construct a hidden table that the viewer can expand if they wish to.

In order to satisfy criteria 3, in text citations should be incorporated within text so the viewer has an option to access the article if they find the statement interesting. A reference list has been posted with a few references however ensure they are cited in the correct format. Overall a great start!

===Group 6 Peer Assessment===

In this project page a good start has been made with the inclusion of images to compliment the signalling pathway description. Appropriate abbreviations appear to be used, where the full name is used first. Also the additions of a glossary and further reading subheading is a nice touch, which should allow for better understanding of the topic should the reader want more information. In terms of the diversity of the subheadings though, it seems that a lot more could be added, such as animal models used to research the signalling pathway and also possibly abnormalities that may arise from the errors or mutations in the pathway. It is probably wise to also add a section regarding embryological development and what role TGF beta signalling pathway has in it, which should help provide context to the abnormalities section when added.

The usage of pictures is appropriate for the section it has been put in, and compliments the signal transduction pathway description well, but the picture labelled “Process of TGF-beta signalling pathway” does not appear to be referenced or have the appropriate copyright under it. There is also no legend for this picture to briefly describe it. Also for most of the page there are limited to no references, where for the signalling pathway section, it appears your groups has used websites rather than peer reviewed articles as a source. The websites are probably good starting points to get a general idea of the pathway, but it is probably better if you find peer reviewed papers to cite, which potentially the websites you have used have cited. Also when citing it is best to use in text citation such that the reader can easily see which paper you are referring to when describing certain facts.

Also in your groups signalling section, it is mentioned that SMAD when activated, recruits various transcriptional regulators that control expression of numerous genes. This is quite vague and is probably a good idea to mention some of these factors, and also what genes they regulate and the importance of such genes. Doing this should also provide your group with a good link to the embryological development section with regards to TGF-beta signalling.

It still seems that overall there is a lot of work to be done on your groups page, but a good start and effort has been made to include various images and also subheadings. I feel that if your group incorporates some of the suggested subheadings described above, and other feedback mentioned, the page should be greatly improved.

===Group 6 – TGF-beta===
You guys have made a good start to the project identifying some important subheadings introducing the TGF-beta signaling pathway, outlining its history, current research and limitations (which may be more appropriately labeled as abnormalities.) However, I do think the structure of these should be revised, what I mean by this is that you should create more levels of headings (as currently all the headings are located under the larger heading of introduction.) Furthermore, it terms of the headings, I think you need to introduce the signaling pathway, then discuss the history of its discovery, then discuss the specific mechanisms behind the pathway, its role in embryonic development (which is a very important aspect in order to relate your project back to what we are learning in the lectures and tutorials), then animal models and abnormalities. You have chosen to include some images which appear to be useful for explaining the signaling pathway, however I think it is important to refer to them in your text, as well as appropriately referencing them with the copyright from the original source (as the larger one is missing this information.)

Some negative aspects of this project are the lack of appropriate references, there are no in-text citations and the identified sources that have been used appear to be websites. Remember that most of the information, if not all should be acquired from primary research articles (supplemented with the occasional review article.) Furthermore, similar to other projects, in order to make your page more engaging you could look into including tables (say for the history or summary of receptor subtypes), more images, YouTube links or animations, or an interactive quiz.

In conclusion it seems that there is still a lot of work to be completed on this page before it is to be submitted, however you have made a successful start. The main criticisms are regarding revisiting the subheadings and including the role of embryonic development as I think this is really critical to the project, as well as adding more information to the page in general. In saying that it appears you guys are heading in the right direction!

===Group 6 Peer Assessment===

Positive aspects of the project and improvements:

Upon reviewing the page I can see a number of headings and subheadings such as the nature of the growth factor, its mechanism of action, history and emerging research. But to be critical, a range of other headings is necessary to ensure all bases are covered when researching and providing the relevant information. This will help in satisfying the requirements for criteria 1 and 2. It is good to see the addition of a diagram to your project as it is a requirement for criteria 2. This provided a visual aid that kept me interested to find out more about the topic while simultaneously making it easier to understand the theory behind the process of TGF Beta signalling pathway. It is also great to see the current research and limitations as this shows that you are going beyond the scope of the required criteria and researching ahead to provide that extra bit of information. This in turn is a great way to satisfy criteria 5.

Negative aspects of the project and improvements:

Although there are a few positives in the project, there are a number of negatives which can be improved upon to ensure a coherent project is created. Firstly, it is advised to put the heading “history of TGF- beta signalling pathway at the top” with the introduction as this is an introductory section and should be addressed initially on the page. This will allow you to create a more flowing page which also looks nice. Secondly, it would be advised to add more images and tables as it is a requirement for criteria 2. By doing this you will keep the reader engaged and wanting to find out more about the chosen topic. In saying that, you already have a couple of images but not referenced. It is imperative to correctly cite and reference these images as failure to do so will result in a breach of copyright laws.

Another critique is to add more subheadings with a range of different aspects of the signalling pathway being explored. It is recommended that you dedicate a chunk of your project to describing the pathway in detail and its role in embryonic development and abnormalities relating to mutations caused by the pathway. This will ensure you answer criteria 6 as it is a great deal of the report. It is also advised to put the history section under the introduction section as placing it in the middle of the project is a bit out of place and inhibits the flow of the information from one subheading to another. Also, the history section can be improved by adding more information as there have been more findings in this research topic since the 1970s. An addition of glossary is also needed for terms such as “peptide”, “cytokine”, “angiogenesis”, “protein kinase” etc. This will aid readers understand terms that they previously have not encountered and allow them to correctly understand the context of the information.

It has also come to my attention that there are little to no references or in text citations. By adding information without correctly giving the authors credit is a breach of copyright laws and must be done urgently. Overall, the project shows signs of progress with a number of positives. By reflecting on the negative aspects and acting upon it, it is certain that high marks are in order.

===Group 6 Peer Assessment===

Positive Feedback:

The introduction is very simple and clear making it a perfect way to familiarise with the TGF-beta signalling before diving into any more information.
The process is also described very well as it is aided with two pictures which were excellent choices. Together the information and the pictures collate to create a stable understanding of how TGF-beta signals actually communicates.

Critical Feedback:

You guys can definitely focus on explaining its role specifically in embryo development which is a key criteria for this project. This can be done in many was such as tables or pictures if there is too much information. It would also be a good idea to talk about the things that could go wrong with TGF-beta signalling pathway and the current research being done to rectify this as this is something that is very interesting and also relevant.

You guys should also start referencing early as it can become very problematic later on to keep track and doing very quick citations would go a long way later when editing the project. Lastly it would possibly help if everyone brainstormed some subheadings and categories that you further want to talk about e.g. animal models and that way you know what information you are looking for.
This page has a very strong start and if that quality is carried through to the rest of the content it will be a very successful page.

Talk:2016 Group Project 6

2016-10-06T17:54:09Z

Z3417363: /* Peer Review */

{{ANAT2341Project2016discussionheader}}
=Peer Review=
 
 
Group 6:
 
 
Positive aspects of the project and suggested improvements:
 
 
The authors of group 6 have created a variety of subheadings related to the TGF-beta signalling pathway, including the nature of the growth factor, its mechanism of action, history and emerging research (criteria 1). Authors have also provided two diagrams related to TGF-beta signalling which reinforces the description of TGF signalling provided (criteria 2). These diagrams allow for a much simpler interpretation of the signalling process described and assist in teaching at the peer level (criteria 4). It appears that the authors are beginning to conduct investigations into new research surrounding TGF-beta signalling, thus indicating that they are attempting to research beyond formal teaching activities (criteria 5).
 
 

Whilst it is excellent that multiple subheadings have been provided, a possible improvement would be to include a much larger variety of subheadings which cover the scope of TGF-beta’s role in embryonic development, abnormalities, types of TGF receptors and animal models. This may allow audiences to understand the big picture surrounding this signalling pathway which will assist in the understanding of the information already provided. Another improvement to this page would be to include more images under different subheadings. One example would be to include an image or table showing the history of discovery surrounding discovery of this signalling pathway.
 
 

Negative aspects of the project and suggested improvements:
 
 

Although there were positive aspects of this project, there were also numerous negative aspects which may be improved. One key negative feature of the page was that the authors did not discuss the role of TGF-beta signalling in the context of embryonic development, hence meaning they failed to meet criteria 6. To ensure that this criterion is met, authors may conduct research into the involvement of TGF-beta in specific processes that occur during embryonic development, perhaps organ development and growth of different primitive structures. In addition, whilst the authors have provided a history of the TGF-beta signalling pathway, the history appears to be very brief. Thus an improvement which may be implemented would be to include a more extensive background regarding the history of discovery of the pathway. It was also noticed that no tables were utilised within the page. A possible improvement would be to include a table describing different abnormalities and their causes in the context of disruption of the TGF-beta pathway. A table may also be utilised to describe different subtypes of TGF-beta receptors as well as their functions during embryonic development. Tables may be utilised as they will assist in the process of teaching at the peer level (criteria 4), particularly because they convey information in an orderly and organised manner.
 
 

The authors of this project also failed to meet criteria 3, in that only one source was referenced in the process of the signalling pathway and also no in-text citations were provided. In addition, authors failed to reference the image file named, “Process of TGF-beta signalling pathway 01”. It is vital that all sources are referenced correctly in order to ensure that the copyright laws regarding the use of information are adhered to. The final negative aspect of the project was that the page did not flow very well, in that subheadings were arranged in a disorderly fashion. An example of this is the inclusion of the subheading labelled, “history of TGF-beta signalling pathway”, towards the end of the page. Since such a subheading provides a background surrounding the pathway, a possible improvement would be to include this subheading at the beginning of the page. By ensuring the orderliness of the page, this creates a sense of coherency between subheadings, thus making the page more appealing and engaging.

===Group 6===

It was good to see some progress being made on the project with the development of some subheadings and the inclusion of an image. In saying this, a better selection of headings and sub-headings could be developed to break down the topic of TGF beta signaling pathway. I think the sub-headings provided under the general heading of ‘Introduction’ could form the main headings of this research project and they could then be further broken down into various subheadings. In addition, the subheading of TGF-beta could be eliminated and this definition could be incorporated into the glossary or general introduction of the topic instead. Furthermore, more focus is needed on the influence of this pathway on embryological development and the abnormalities caused by mutations to the pathway and its components. For example, there has been mention of the effect of TGF-beta in ‘development of the embryo and adult organism, as well as cell growth, immune function and hormone secretion’ but further discussion has not been pursued.

Though a good description of the ‘process of TGF-beta signaling pathway’ has been provided, it could be further improved by referencing the images included in this section in your text (e.g. refer to Figure 1) to aid one’s understanding of the concept being explored. In addition, a timeline of events could be provided to explore the history of this pathway and it would be appropriate to begin with the discovery of TGF-beta. To a reader, information on ‘transformed or malignant cells’ seems unrelated to the TGF-beta signaling pathway even though it may be in fact be related, due to lack of discussion of this pathway or TGF-beta in this description. In regards to the section on ‘Limitations’, what types of limitations are you trying to explore? Limitations in research? This could be better defined by appropriately allocating subheadings to each of the sections.

Though you are heading in the right direction, spending time to produce a basic layout of your project by creating appropriate headings and subheadings would be useful in breaking down the concepts needed to be explored in this pathway. This could be achieved by communicating with group members through the discussion page and providing feedback and suggestions. In addition, greater focus is required in referencing and citing your work to ensure researchers and authors are acknowledged for their work. Also, by exploring animal models of the TGF-beta pathway and the effect of this research in understanding this pathway in humans and its influence on embryological development, you could greatly increase the quality of your work. You could also try and make your project more interactive and engaging through the inclusion of tables, images and diagrams. I hope this helps! Good luck!

=== Group 6 Critical Assessment===
It is great to see an in-depth overview of the TGF beta Signalling pathway and it’s mechanism of action. The page is off to a great start and with a few key improvements it can turn into a successful one! Firstly, I like the use of images to aid the reader in understanding the content better should they be a visual learner. By reading the text on the way the pathway works, it is clear it is a complicated process hence a suggestion would be to add short clips/animations to simplify it for the viewer.

Under your introduction an attempt has been made to briefly highlight the main features of the pathway, however a negative of this section is that a lot of the content is basically listed. For example it is mentioned TGF-beta is part of a larger TGF superfamily comprising of different members such as activins and GDFs. Instead of listing, try presenting your information in a different format such as a table with s few columns stating the member, it’s function, and possibly what a mutation could lead to.

Additionally it is also stated that TGF beta has certain functions such as controlling angiogenesis however doesn’t expand on the ‘other’ functions it has. To turn this into a positive, dedicate different sections on how the pathway is involved in angiogenesis, hormone secretion, proliferation etc. If there are too many functions to fit onto the page, you could shift a few to your ‘Further Reading’ section as an option if the reader would like to explore further into the pathway’s functions, or construct a hidden table that the viewer can expand if they wish to.

In order to satisfy criteria 3, in text citations should be incorporated within text so the viewer has an option to access the article if they find the statement interesting. A reference list has been posted with a few references however ensure they are cited in the correct format. Overall a great start!

===Group 6 Peer Assessment===

In this project page a good start has been made with the inclusion of images to compliment the signalling pathway description. Appropriate abbreviations appear to be used, where the full name is used first. Also the additions of a glossary and further reading subheading is a nice touch, which should allow for better understanding of the topic should the reader want more information. In terms of the diversity of the subheadings though, it seems that a lot more could be added, such as animal models used to research the signalling pathway and also possibly abnormalities that may arise from the errors or mutations in the pathway. It is probably wise to also add a section regarding embryological development and what role TGF beta signalling pathway has in it, which should help provide context to the abnormalities section when added.

The usage of pictures is appropriate for the section it has been put in, and compliments the signal transduction pathway description well, but the picture labelled “Process of TGF-beta signalling pathway” does not appear to be referenced or have the appropriate copyright under it. There is also no legend for this picture to briefly describe it. Also for most of the page there are limited to no references, where for the signalling pathway section, it appears your groups has used websites rather than peer reviewed articles as a source. The websites are probably good starting points to get a general idea of the pathway, but it is probably better if you find peer reviewed papers to cite, which potentially the websites you have used have cited. Also when citing it is best to use in text citation such that the reader can easily see which paper you are referring to when describing certain facts.

Also in your groups signalling section, it is mentioned that SMAD when activated, recruits various transcriptional regulators that control expression of numerous genes. This is quite vague and is probably a good idea to mention some of these factors, and also what genes they regulate and the importance of such genes. Doing this should also provide your group with a good link to the embryological development section with regards to TGF-beta signalling.

It still seems that overall there is a lot of work to be done on your groups page, but a good start and effort has been made to include various images and also subheadings. I feel that if your group incorporates some of the suggested subheadings described above, and other feedback mentioned, the page should be greatly improved.

===Group 6 – TGF-beta===
You guys have made a good start to the project identifying some important subheadings introducing the TGF-beta signaling pathway, outlining its history, current research and limitations (which may be more appropriately labeled as abnormalities.) However, I do think the structure of these should be revised, what I mean by this is that you should create more levels of headings (as currently all the headings are located under the larger heading of introduction.) Furthermore, it terms of the headings, I think you need to introduce the signaling pathway, then discuss the history of its discovery, then discuss the specific mechanisms behind the pathway, its role in embryonic development (which is a very important aspect in order to relate your project back to what we are learning in the lectures and tutorials), then animal models and abnormalities. You have chosen to include some images which appear to be useful for explaining the signaling pathway, however I think it is important to refer to them in your text, as well as appropriately referencing them with the copyright from the original source (as the larger one is missing this information.)

Some negative aspects of this project are the lack of appropriate references, there are no in-text citations and the identified sources that have been used appear to be websites. Remember that most of the information, if not all should be acquired from primary research articles (supplemented with the occasional review article.) Furthermore, similar to other projects, in order to make your page more engaging you could look into including tables (say for the history or summary of receptor subtypes), more images, YouTube links or animations, or an interactive quiz.

In conclusion it seems that there is still a lot of work to be completed on this page before it is to be submitted, however you have made a successful start. The main criticisms are regarding revisiting the subheadings and including the role of embryonic development as I think this is really critical to the project, as well as adding more information to the page in general. In saying that it appears you guys are heading in the right direction!

===Group 6 Peer Assessment===

Positive aspects of the project and improvements:

Upon reviewing the page I can see a number of headings and subheadings such as the nature of the growth factor, its mechanism of action, history and emerging research. But to be critical, a range of other headings is necessary to ensure all bases are covered when researching and providing the relevant information. This will help in satisfying the requirements for criteria 1 and 2. It is good to see the addition of a diagram to your project as it is a requirement for criteria 2. This provided a visual aid that kept me interested to find out more about the topic while simultaneously making it easier to understand the theory behind the process of TGF Beta signalling pathway. It is also great to see the current research and limitations as this shows that you are going beyond the scope of the required criteria and researching ahead to provide that extra bit of information. This in turn is a great way to satisfy criteria 5.

Negative aspects of the project and improvements:

Although there are a few positives in the project, there are a number of negatives which can be improved upon to ensure a coherent project is created. Firstly, it is advised to put the heading “history of TGF- beta signalling pathway at the top” with the introduction as this is an introductory section and should be addressed initially on the page. This will allow you to create a more flowing page which also looks nice. Secondly, it would be advised to add more images and tables as it is a requirement for criteria 2. By doing this you will keep the reader engaged and wanting to find out more about the chosen topic. In saying that, you already have a couple of images but not referenced. It is imperative to correctly cite and reference these images as failure to do so will result in a breach of copyright laws.

Another critique is to add more subheadings with a range of different aspects of the signalling pathway being explored. It is recommended that you dedicate a chunk of your project to describing the pathway in detail and its role in embryonic development and abnormalities relating to mutations caused by the pathway. This will ensure you answer criteria 6 as it is a great deal of the report. It is also advised to put the history section under the introduction section as placing it in the middle of the project is a bit out of place and inhibits the flow of the information from one subheading to another. Also, the history section can be improved by adding more information as there have been more findings in this research topic since the 1970s. An addition of glossary is also needed for terms such as “peptide”, “cytokine”, “angiogenesis”, “protein kinase” etc. This will aid readers understand terms that they previously have not encountered and allow them to correctly understand the context of the information.

It has also come to my attention that there are little to no references or in text citations. By adding information without correctly giving the authors credit is a breach of copyright laws and must be done urgently. Overall, the project shows signs of progress with a number of positives. By reflecting on the negative aspects and acting upon it, it is certain that high marks are in order.

===Group 6 Peer Assessment=== z3417363

Positive Feedback:

The introduction is very simple and clear making it a perfect way to familiarise with the TGF-beta signalling before diving into any more information.
The process is also described very well as it is aided with two pictures which were excellent choices. Together the information and the pictures collate to create a stable understanding of how TGF-beta signals actually communicates.

Critical Feedback:

You guys can definitely focus on explaining its role specifically in embryo development which is a key criteria for this project. This can be done in many was such as tables or pictures if there is too much information. It would also be a good idea to talk about the things that could go wrong with TGF-beta signalling pathway and the current research being done to rectify this as this is something that is very interesting and also relevant.

You guys should also start referencing early as it can become very problematic later on to keep track and doing very quick citations would go a long way later when editing the project. Lastly it would possibly help if everyone brainstormed some subheadings and categories that you further want to talk about e.g. animal models and that way you know what information you are looking for.
This page has a very strong start and if that quality is carried through to the rest of the content it will be a very successful page.

User:Z3417363

2016-10-06T15:39:57Z

Z3417363:

User:Z3417363

2016-10-06T15:09:55Z

Z3417363:

User talk:Z3417363

2016-09-23T04:53:27Z

Z3417363:

this is a draft page people cant see this

peer assessment, what is missing what should be in it?

User:Z3417363

2016-09-23T03:27:39Z

Z3417363:

User:Z3417363

2016-09-23T03:12:26Z

Z3417363: /* Lab 7 Duchenne muscular dystrophy */

Talk:2016 Group Project 1

2016-09-16T04:53:31Z

Z3417363: /* Notes */

{{ANAT2341Project2016discussionheader}}

To all group members:
*More info on pathway focusing on fetus development, and which pathway it is majorly part of - focus research on those body parts
*Make your section presentable
*At least one picture per section

==Notes==
Just trying to simplify and understand the process and these are some of my notes !(z3417363)

The inactive Wnt Pathway In a normal cell:

In most normal cells the Wnt pathway is inactive. In the cytosol , the destruction complex is formed from the proteins beta catenin, GSK3 beta, Axin,APC, Ck1-alpha. The ubiquitin ligase beta TRCP is able to bind to beta catenin and transfer short ubiquitin peptides to beta-catenin. In other words the beta-catenin is phosphorolated and this beta catenin can then bound and be by a complex of protease (proteasome) . Thus a low level of cellular beta catenin is achieved.
Therefore no beta catenin reaches the nucleus and the transcription factor of the TCF LEF family along with other proteins (groucho) binds to DNA and inhibits gene expression.
So essentially when WnT is inactive, beta canenin is destroyed and does not reach nucleus and transcription is inhibited.

The Active Wnt Pathway in a normal cell.

Extracellular(outside cell) Wnt binds with the membrane receptor frizzled (FZD). The wnt pathway is activated and activates the cytosolic protein "dishevelled"(DSH) which induces dissociation of the protein destruction complex. Because the protein complex is destroyed beta- catenin is no longer modified by unbiquitin peptides/phosporolated and is not destroyed. Since the supply of beta catenin continues the level of beta catenin rises, first in the cytosol and later in the nucleus. Once the beta catenin reaches the nuclue it binds to the TCF LEF transcription factor which changes them from a transcriptional repressor into an activator. TCF itself activates an RNA polymerase which induces gene transcription.
So essentially WnT starts gene transcription by allowing beta catenin to reach the nucleus.

This is actually very similar to a tumour cell where the mutation of the protein complex also inhibits the destruction of beta catenin and allows it to grow in quantity and reach the nucleus and start gene expression. However this is not uncontrolled and can be compared to a car travelling with no brakes. Ultimately this abnormal proliferation leads to malignant adenocarcinoma (cancer).

Use pubmed, biomedcentral journals==you can find it on pubmed just plug in the title and you will get the pubmed number>BMC developmental biology journal, journal of cell biology(cant use last 6months of research), proceeding national academy of science(can only use after 6months), public library of science omim
include research labs, animations
use the help tab
where the terminlogy came from
this is a student drawn image , based upon and give the reference

User:Z3417363

2016-09-16T04:23:11Z

Z3417363:

User:Z3417363

2016-09-16T04:03:12Z

Z3417363:

User:Z3417363

2016-09-16T03:38:13Z

Z3417363: /* lab attendance */

2016 Group Project 1

2016-09-15T02:40:14Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

2016 Group Project 1

2016-09-15T02:39:47Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?===z3417363

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

2016 Group Project 1

2016-09-15T02:39:15Z

Z3417363: /* How does it work in a foetus (skin formation) - Arsalan */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?====z3417363

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

2016 Group Project 1

2016-09-15T02:38:57Z

Z3417363: /* How does it work in a foetus (skin formation) - Arsalan */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?=====z3417363

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

2016 Group Project 1

2016-09-15T02:38:27Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?=== z3417363

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

Talk:2016 Group Project 1

2016-09-12T10:44:28Z

Z3417363:

Talk:2016 Group Project 1

2016-09-12T10:43:45Z

Z3417363:

{{ANAT2341Project2016discussionheader}}

To all group members:
*More info on pathway focusing on fetus development, and which pathway it is majorly part of - focus research on those body parts
*Make your section presentable
*At least one picture per section

Just trying to simplify and understand the process some of my notes !(z3417363)

The inactive Wnt Pathway In a normal cell:

In most normal cells the Wnt pathway is inactive. In the cytosol , the destruction complex is formed from the proteins beta catenin, GSK3 beta, Axin,APC, Ck1-alpha. The ubiquitin ligase beta TRCP is able to bind to beta catenin and transfer short ubiquitin peptides to beta-catenin. In other words the beta-catenin is phosphorolated and this beta catenin can then bound and be by a complex of protease (proteasome) . Thus a low level of cellular beta catenin is achieved.
Therefore no beta catenin reaches the nucleus and the transcription factor of the TCF LEF family along with other proteins (groucho) binds to DNA and inhibits gene expression.
So essentially when WnT is inactive, beta canenin is destroyed and does not reach nucleus and transcription is inhibited.

The Active Wnt Pathway in a normal cell.

Extracellular(outside cell) Wnt binds with the membrane receptor frizzled (FZD). The wnt pathway is activated and activates the cytosolic protein "dishevelled"(DSH) which induces dissociation of the protein destruction complex. Because the protein complex is destroyed beta- catenin is no longer modified by unbiquitin peptides/phosporolated and is not destroyed. Since the supply of beta catenin continues the level of beta catenin rises, first in the cytosol and later in the nucleus. Once the beta catenin reaches the nuclue it binds to the TCF LEF transcription factor which changes them from a transcriptional repressor into an activator. TCF itself activates an RNA polymerase which induces gene transcription.
So essentially WnT starts gene transcription by allowing beta catenin to reach the nucleus.

This is actually very similar to a tumour cell where the mutation of the protein complex also inhibits the destruction of beta catenin and allows it to grow in quantity and reach the nucleus and start gene expression. However this is not uncontrolled and can be compared to a car travelling with no brakes. Ultimately this abnormal proliferation leads to malignant adenocarcinoma (cancer).

2016 Group Project 1

2016-09-12T10:25:50Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?== z3417363

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

2016 Group Project 1

2016-09-12T10:25:11Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===Things to do===
'''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''

===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>

====Non-Canonical Pathway - Caroline====
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

====WnT-Calcium Ion Pathway - Tony====

*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?

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

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

User:Z3417363

2016-09-02T03:14:25Z

Z3417363: /* lab attendance */

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)

[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13, 2 September 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

User:Z3417363

2016-09-02T03:13:57Z

Z3417363: /* lab attendance */

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)
[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]]) 1:13. 2 September 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

User:Z3417363

2016-09-02T03:13:08Z

Z3417363: /* lab attendance */

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)
[[User:Z3417363|Z3417363]] ([[User talk: Z3417363|talk]])

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

2016 Group Project 1

2016-09-02T03:06:07Z

Z3417363: /* Topic: WnT Signaling Pathway in skin of fetus */

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



==Topic: WnT Signaling Pathway in skin of fetus==

===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>

*Non-Canonical Pathway - Caroline
**The non-canonical pathway can also be referred to as the beta-catenin independent pathway. It can be divided into two pathways known as the Planar Call Polarity pathway or the 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 non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.

<pubmed>19279717</pubmed>

*WnT-Calcium Ion Pathway - Tony

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?

==Wnt-Calcium Ion Pathway==

===References===
PMID 21903638

User:Z3417363

2016-09-02T02:09:04Z

Z3417363:

{{ANAT2341Student2016}}
==lab attendance==

[[User:Z3417363|Z3417363]] ([[User talk:Z3417363|talk]]) 14:34, 5 August 2016 (AEST)

==New sub heading==
===external link===
https://my.unsw.edu.au/

===internal link===

https://embryology.med.unsw.edu.au/embryology/index.php/ANAT2341_Lab_1

[[ ANAT2341_Lab_1 |anat2341 lab 1]]

===referencing===

[http://www.ncbi.nlm.nih.gov/sites/entrez?db=pubmed&cmd=search&term=fertilization fertilization]

PMID 27486480

==Lab 1 Assessment==

<pubmed>24194470</pubmed>

Summary the cell biology of fertilisation:

Fertilisation is a complex yet essential part of life and the continuation of it. There are significant amounts of research into the components of fertilisation as we try to understand its mechanisms and establish ways of creating successful fertilization. With the introduction of gene manipulation and in vitro technology there has been a great increase in the opportunity to learn more about the molecular mechanisms that regulate the fertilization; an area that is still relatively obscure. This article explores the mechanisms of fertilisation that have been seen to be an essential part of the process, while also shedding lights on events that are not as essential.

Factors regulating fertilization: During this study, gene manipulation and fertilisation of mice was observed and many factors thought to be essential in the classical models seemed not to be as significant whereas some factors revealed themselves to be essential. For example spermatozoa from some of the mice lacked ADAM3 and this suggested that this plays a key role in the fertilisation process of the mice.
Factors regulating sperm migration: Calmegin is one of the essential genes required for spermatozoa to migrate into the oviduct. The study conducted an experiment where both wild type and calmegin disrupted spermatozoa was used observe migration habits. It was observed that only the wild sperm migrated to the oviduct while the calmegin disrupted spermatozoa (which are equally motile) remained in the uterus. This result shows the essential mechanism of calmegin in relation to successful fertilisation.
Factors regulating the zona-binding ability of spermatozoa: Many studies have postulated that carbohydrates play an imperative role in sperm-zona binding. However through this study many residues which did not contain key carbohydrates still resulted in successful fertilisation indicating that it may not play an important role as hypothesised.
This research article aims to present factors that lead to “essential” fertility. It aims to dispel misplaced enthusiasm for research into components of fertilisation that are not as important and may lead to misleading results. It is therefore important to focus gene manipulation and in vitro studies on valid candidate molecules to better understand this complex process of fertilisation

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 18 August 2016 - You have added the citation correctly and written an extensive summary of the article. Unfortunately I have had to take marks off the final assessment as you have not followed the assessment criteria "It must be a research article not a Review.", this is a review paper. '''Reviews''' are not '''research articles''', though good in providing a timely summary of a topic{s) they do not contain new research data. Please read the assessment criteria carefully.

The cell biology of mammalian fertilisation. Okabe M. Development. 2013 Nov;140(22):4471-9. doi: 10.1242/dev.090613. '''Review''' PMID 24194470
| width=100px| Assessment 3/5
|}

==Lab 2==

==Lab assessment 2==

[[File:Sperm binding test using unfertilised and fertilised human oocytes.jpg|Sperm binding test using unfertilised and fertilised human oocytes]]

Sperm binding test using unfertilised and fertilised human oocytes<ref name=PMID17147816><pubmed>17147816</pubmed></ref>

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 29 August 2016 - All information Reference, Copyright and Student Image template correctly included with the file and referenced on your page here.
| Assessment 5/5
|}

==Lab attendance 3==

{| width=95%
|-bgcolor="FAF5FF"
| [mailto:m.hill@unsw.edu.au Mark Hill] 31 August 2016 - Lab 3 Assessment Quiz - [[Lecture_-_Mesoderm_Development|Mesoderm]] and [[Lecture_-_Ectoderm_Development|Ectoderm]] development.

[[Lecture_-_Ectoderm_Development#Primary_Vesicles|Question 3 - brain vesicles]]

[[Lecture_-_Ectoderm_Development#Brain_Flexures|Question 4 - brain flexures]]

[[Lecture_-_Ectoderm_Development#Maternal_Diet|Question 5 - maternal diet]]
| Assessment 2/5
|}

==Assessment 4==

==Quiz==

<quiz display=simple>

{Which of the following cavities is formed from the coelomic cavity
|type="()"}
- pericardial
- pleural
- peritoneal
+all of the above
|| Option four is correct as all 3 major cavities are formed from this cavity.

{What can be best described as the process of recanalization.
|type="()"}
-the lengthening of the GIT tube
-the shortening of the GIT tube
-the GIT tube becoming solid then hollow and then solid again
+the GIT tube becoming hollow then solid and then hollow again
|| option four is correct, around week 6 of development the tube will go from hollow to solid(occluding) and then hollow once more as the tissue degenerates by week 8.

{What is the major artery that supplies the midgut.
|type="()"}
-Celiac artery
+superior mesenteric artery
-inferior mesenteric artery
-All of the above
|| option 2 is correct. Celiac artery supplies foregut and inferior artery supplies hindgut.

{Intestinal Malrotation is caused by which of the following
|type="()"}
-improper closure and absorption of the vitelline duct
-Incomplete recanalization resulting in parallel lumens
+ a series of bands crossing the duodenum which cause duodenal obstruction
- the lack of enteric nervous system (neural ganglia) in the intestinal tract responsible for gastric motility
|| option 3 is correct. these are fibrous bands(of Ladd) which course over the vertical portion of the duodenum causing intestinal obstruction.

</quiz>

[[Student Page]]

User:Z3417363

2016-09-02T01:46:59Z

Z3417363:

User:Z3417363

2016-09-02T01:18:35Z

Z3417363: