2016 Group Project 1

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2016 Group Project Topic - Signaling in Development

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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.[1]

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

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.[2] The Fz receptors are a family of seven-transmembrane spanning proteins and are coupled with G-proteins at their intracellular domain.[3] 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).[2]

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. [4]
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. [5]

In the absence of Wnt
Alt
Diagram of Wnt/β-catenin signalling pathway
  1. 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.[6]

In the presence of Wnt

  1. 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 [7]. Therefore, the kinase complex used to negatively regulate β-catenin destruction also seem to surprisingly have a contradictory role of positively regulating β-catenin
  2. 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:
    1. Wnt-induced Axin dissociation
      Dephosphorylation of PP1 on Axin causes Axin-GSK3 dissociation
    2. 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
    3. Axin degradation:
      Overexpression of activated Wnt receptor may cause Axin degradation
    4. 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).
  3. 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.[7] [8]


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. [9]

Alt
Diagram of PCP pathway leading to gastrulation in embryonic development

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. [4]


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. [10][11]

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.[12]

Alt
Wnt calcium ion signalling pathway and its main components.

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). [13]
  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.[14]
  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. [15]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.[16] CaMKII may also activate other transcription factors such as CREB, ATF-1 and ELK-1 leading to other activities in the cell.[12]
    • CaCN
      CaCN is a protein regulated by calcium/calmodulin, Fe3+ and Zn2+ ions together[17]. 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).[18] NF-AT may further activates AP1 or GATA transcription factors.[19] NF-AT itself may be exported out of the nuclear by glycogen synthase kinase 3beta (GSK3beta), jun N-terminal kinase (JNK) or p38.[20] 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.[21] As a result, all classical PKCs and PKC delta, which belongs to the novel PKCs, can be activated by wnt/calcium ion signalling pathway.[22] 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.[13]


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.

  1. 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.[7]
  2. 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 [23]
  3. 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.
  4. 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. [24]
  5. 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. [25]

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. [9][10]

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. [26] [27] [28]

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?

  1. 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.[29]
    • 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.[13]
  2. 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. [30]
  3. 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.[31]
  4. 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. [32]

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. [33] 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. [34] 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.[35] Therefore Wnt signalling is permanently active and b-catenin violently accumulates, allowing over and unnecessary transcription, proliferation and differentiation leading to formation of tumours.[36] 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.[37]

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. [38] 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. [39] These observations were both observed in the studies taken with mice cells [40] and also human cells [41]. 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. [42]

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.[43]. 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 [44] . 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. [45]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.[46]

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. [47] 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.[48]

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.[49]

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[50]. 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

1. Please pick the correct statement about the function of wnt calcium ion signalling pathway from the following choices.

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.

2. 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.

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.

3. What is the role of WnT11 in the PCP pathway.

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

4. Which of the following statement(s) are true?

  1. Statement 1: Glycogen and CSK3 can only down regulating β-catenin in cellular cytoplasm.
  2. 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.
  3. Statement 3: β-catenin enhances the function of DNA-bound transcription factor family known as 'TCF/LEF' that would normally transcript DNA genes.
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

5. Which of the following cancers is most linked to the mutation of the APC complex?

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

6. Which of the following models has the most similarity to the human Wnt pathway system?

Xenopus
Zebrafish
Mice
This is correct, both humans and mice have 19 Wnt glands and 10 frizzled(Fz) receptors.
Drosophila

Your score is 0 / 0


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