2016 Group Project 1

2016 Student Projects
Signalling: 1 Wnt | 2 Notch | 3 FGF Receptor | 4 Hedgehog | 5 T-box | 6 TGF-Beta
2016 Group Project Topic - Signaling in Development OK you are now in a group, add a topic with your student signature to the group page.
This page is an undergraduate science embryology student project and may contain inaccuracies in either descriptions or acknowledgements.

Group Assessment Criteria
Science Student Projects The key points relating to the topic that your group allocated are clearly described. The choice of content, headings and sub-headings, diagrams, tables, graphs show a good understanding of the topic area. Content is correctly cited and referenced. The wiki 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. Evidence of significant research relating to basic and applied sciences that goes beyond the formal teaching activities. Relates the topic and content of the Wiki entry to learning aims of embryology. Clearly reflects on editing/feedback from group peers and articulates how the Wiki could be improved (or not) based on peer comments/feedback. Demonstrates an ability to review own work when criticised in an open edited wiki format. Reflects on what was learned from the process of editing a peer's wiki. Evaluates own performance and that of group peers to give a rounded summary of this wiki process in terms of group effort and achievement. The content of the wiki should demonstrate to the reader that your group has researched adequately on this topic and covered the key areas necessary to inform your peers in their learning. Develops and edits the wiki entries in accordance with the above guidelines.
More Information on Assessment Criteria | Science Student Projects

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.

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.

What is wnt ligands what is Fz receptor what is G-protein

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. The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain. 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).

Canonical Pathway

• The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
• They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
• The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
• Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
• This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

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.

The general mechanism of wnt/calcium ion signalling is described below:

1. 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).
2. 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.
3. 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. 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. CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.
   • CaCN
     
   • PKC
     

PMID 24489854 beta-Arrastin 2 (Arrb2) binds with Dishevelled (Dvl) and the β and γ subunits of trimeric G-proteins, respectively Gβ and Gγ (Gβγ) to form a functional complex to conduct signal downwards from wnt fizzeld interaction. Arrb2 can also activate and induce translocation of PKC alpha to cell membrane. This activity is down stream of Fz7 activation.

Wnt signalling in Embryonic Development

What does canonical pathway do?

• Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
• Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
• In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
• Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
• At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
• However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

What does Calcium ion pathway do?

1. Xenopus gastrulation
   • Promoting involution during 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.
   • 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.
2. Mouse Neural Crest formation and embryonic stem cell fate decision
   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.

PMCID: PMC2634250

Experimental Models

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

Diseases related with wnt dysfunction

1. 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.

Topic: WnT Signaling Pathway in skin of fetus

Things to do/ Reference

Friday 9/9/16: 1. Identify all the components of each pathway. (Can possibly look into the similarities vs differences/ interactions between the pathways. 2. Research into how each pathway contributes to fetal devleopment in different body. (Can look into which fetal part we can focus on e.g. skin) 3. Look for picture/diagrams/graphs etc.

Let's get some more information up by next Thursday, 15/9/16

Friday 16/9/16: Good resource: Omim.org (Contains alot of information about different genes and every signalling pathways. How to reference?)

The signalling pathways:

Canonical Pathway - Gloria

• • The Wnt family of secreted glycolipoproteins play an important role in the role of embryonic development and adult homeostasis.
  • They do so via the transcription of β-catenin, which accumulates in the cytoplasm and eventually gets translocated into the nucleus to act as a transcriptional coactivator of transcription factors that belong to the TCF/LEF family.
  • The canonical Wnt pathway of (Wnt/ β-catenin pathway) is the Wnt pathway that causes β-catenin to stay in the cytoplasm rather than be degraded through ubiquitination which is induced by destruction complexes. The destruction complexes which sends β-catenin to proteasome for digestion are proteins such as: Axin, adenomatosis, polyposiscoli (APC), protein phosphatase 2A (PP2A), glycogen synthase kinase 3 (GSK3) and casein kinase 1α (CK1α).
  • Once the stabilised β-catenin enters the cell nucleus it acts as a transcriptional coactivator for transcription of Wnt-target genes. The primary family of transcription factor which β-catenin associates with is the TCF/LEF family. Activation through β-catenin is mediated with compounds such as histone acetyl transferase CBP, the chromatin-remodeling SWI/SNF complex and Bcl9 bound to pygopus (Pyg).
  • This signalling pathway is crucial for deciding the fate of cells during early embryogenesis.

<pubmed>19279717 </pubmed>

Canonical Pathway: How it works - Gloria

• Wnt family of signalling proteins plays various roles in fetus development of embryogenesis.
• Wnt signals are pleiotropic (when a gene has effects on two or more seemingly unrelated phenotypic traits). The signals have effects on mitogenic stimulation, cell fate specification and differentiation.
• In the canonical pathway, Wnt ligands bind to frizzle receptors that are at the cell surface.
• Due to this activation of Wnt signalling, B-catenin, which is normally degraded within the cell, starts to accumulate in the cell and ultimately the nucleus.
• At normal levels of B-catenin, the protein binds at the intracellular side of the membrane with cadherins to promote cell adhesion and also controls cell shape through actin microfilament cytoskeletal network.
• However at elevated level of this protein, activation of transcription occurs alongside co-transcription factors such as action of Lefs/Tcfs.

<pubmed>15473860 </pubmed>

Non-Canonical Pathway - Caroline

Pathways

• The Wnt signal pathway consists of proteins that assist cell communication by passing the signal to the cell surface receptors.
• The general process of the pathway involves binding the protein to a Frizzled family receptor, this then passes the signal to the disheveled protein located on the interior of the cell
• The non-canonical pathway can also be referred to as the beta-catenin independent pathway, due to the absence of β-Catenin.
• The non-canonical pathway can be divided into two pathways, one of them is known as the Planar Cell Polarity pathway or the PCP pathway, and the other is known as the Wnt Calcium pathway

Role

• The non-canonical Wnt pathway regulates cell polarity and movements of dorsal mesodermal cells during convergent extension and later during neural tube closure.
• Studies suggest that the non-canonical pathway has an impact on the expression of early cardiac genes. The non-canonical pathway affects the histone deacetylase (HDAC) activity, which in turn is affected by CaMKII, which is necessary for the expression of the cardiac genes. Thus, any discrepancy in the non-canonical pathway would result in a discrepancy in the normal cardiac development

PCP Pathway

• The PCP pathway was discovered through genetic studies in Drosophila. It was found that mutations in Wnt signaling resulted in a randomised orientation of epithelial structures.
• During vertebrate gastrulation, the mesodermal and ectodermal cells undergo convergent extension. Polarized cells will thus intercalate along the mediolateral axis, resulting in mediolateral narrowing (convergent) and anteroposterior elongation (extension).
• The PCP signaling pathway has another role involving the cell-contact mediated neural crest cell guidance
• PTK7 is a protein coding gene and has been linked to the non-canonical Wnt PCP pathway, this allows and encourages movement of the cells and tissues

Studies

• A study shows the importance of the non-canonical pathway in the migration of the epithelial cells throughout the embryo to allow further embryonic development
• Another study has been performed on the embryos of Xenopus laevis (the clawed frog), to determine what the non-canonical Wnt signaling pathway does. Due to the size of the embryo, it could be easily manipulated to provide the answers needed.

PMID 20576942 <pubmed>19279717</pubmed> <pubmed>27101101</pubmed> <pubmed>26680417</pubmed> <pubmed>26555387 </pubmed> <pubmed> 26499793</pubmed> <pubmed>17045694</pubmed>

<pubmed> 26035863</pubmed>

<pubmed> 25732825</pubmed>

<pubmed> 25428587</pubmed>

<pubmed> 25448697 </pubmed>

<pubmed> 25540130</pubmed>

<pubmed> 25410658</pubmed>

<pubmed> 25266145</pubmed>

WnT-Calcium Ion Pathway - Tony

• PMID 21903638
  • WNT5 ligands were specifically studied since calcium ions are secondary signaling molecules of wnt5. During gastrulation, wnt/beta-catenin pathway is switched on to promote migration of cells, while wnt/calcium pathway is later turned on to stop its effect. Wnt signalling pathway is especially important in guiding the movement of embryonic cells, and wnt/calcium pathway is especially active during gastrulation process.
  • Drosophila and zebrafish models are frequently used to study wnt singalling pathway, due to the fact that wnt signaling is quite conserved across species.
  • Wnt/calcium pathway has also been know to be able to alter the signalling transduction of wnt ligand through other receptors.

• PMID 17881570
  • It is suggested that wnt3a is identified as a wnt canonical pathway ligand in hematopoietic stem cells. However, wnt5a functions in the same cell line mainly as a wnt canonical pathway antagonist since the intracellular catenin level was significantly decreased. Wnt 5a is able to increase the transcription of BCL-2 in the HSC cells and therefore, increase their life span. It seems that in HSCs, wnt5a can also induce the transcription of hes-1 gene which then activates Notch signalling pathway. Notch signalling pathway has been reported to be able to increase the re-productivity of the HSCs. Therefore, it can be seen that wnt5a also possesses a positive effect on HSCs reproduction. wnt5a induces the self-renewal of HSCs, while wnt3a induction increases the amount of c-myc whcih inhibits this action.

How does it work in a foetus (skin formation) - Arsalan

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

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

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

<pubmed>PMC3552514</pubmed>

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

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

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

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

References

PMID 21903638