The Role of Wnt Signalling in Neural Crest Development.

The neural folds come together along the midline of the neural plate and fuse. The movement of the neural folds is generated by changes in the shape of the cells within the folds and the neural plate.

The fusion of the neural folds "pinches off" the neural tube as a hollow cylinder of ectoderm, lying beneath the remaining surface ectoderm. The region of the embryo that lies between the newly formed neural tube and the surface ectoderm is composed of the neural crest cells. 

Prior to neurulation, gastrulation creates the three germ layers and organises them within the embryo. Following gastrulation the surface of the embryo consists of ectoderm. Beneath the ectoderm lies the mesodermal layer and still further inside the embryo the endoderm is found.

The neural crest is found only in vertebrate embryos and is often cited as a defining characteristic of vertebrate organisms. The neural crest contains a migratory population of cells that give rise to most of the peripheral nervous system, facial skeleton and numerous other derivatives throughout the embryo.

The tissues and organs of developing embryos are ordered by cell-to-cell communication. These interactions are mediated by a moderately small number of signalling molecules. These signals are continually used at different stages of development and in different tissues of the embryo.

Experiments have shown evidence that induction of the neural crest can be initiated by interactions between the ectoderm and neural plate. The established view is that a signal originates from the ectoderm and is received in the neuroepithelium.

The addition of growth factors to naïve neural plate tissue has shown possible molecules with the capability to initiate neural crest formation. Such molecules include, transforming growth factors, (TGF-β), bone morphogenetic proteins; BMP-4 and BMP-7, and activin. These molecules are thought to be the ectodermal-inducing signal. (Liem et al. 1993)

At the border region between the ectoderm and neural plate, induction is continuous and BMP’s are expressed ephemerally in the caudal-most ectoderm. Nonetheless, most ubiquitous BMP expression at this point is in the neural folds and dorsal neural tube, with faint expression in the ectoderm. Lower expression of BMP in inducing tissue than in the responding tissue shrouds the analysis of its function. As a result, the fundamental nature of the “ectodermal inducer” is ambiguous. (Basch et al. 2000)

The Wnt family of genes encode secreted glycoproteins that function as signals in cell-to-cell communication during animal embryonic development. The segment polarity gene wingless in Drosophila and the proto-oncogene int-1 (Wingless + iNT-1 = WNT) are the best-known members of this class, but Wnt genes have also been found in animals ranging from the nematode worm C.elegans to vertebrates such as the amphibian and humans. Abnormally expressed Wnt's are suspected to be involved in certain types of tumours such as breast and colon cancer. 

Much of what is known about the functional role of Wnt signalling in early vertebrate development and neural crest induction comes from experiments with Xenopus laevis. Maternally encoded components of the canonical Wnt signalling pathway function to establish the endogenous dorsal axis. Wnt signalling in most tissues is thought to be mediated by the canonical Wnt signal transduction pathway. The extracellular Wnt ligands bind to the transmembrane receptor Frizzled (Fz) which are secreted glycoproteins that play a role in both embryonic development and tumorigenesis.  This binding activates the cytoplasmic phosphoprotein Dishevelled (dsh). Activated Dishevelled (dsh) inhibits GSK3β-mediated degradation of β-catenin. β-catenin protein therefore accumulates and in association with transcription factors (Tcf-3) regulates gene transcription in the cell nucleus. 

The Wnt signalling system is one of only a limited number of signalling systems used during embryonic development to pattern the body plan. Wnt signalling is therefore used several times during development, both in different tissues and at different stages.  

In the development of the nervous system, Wnt1 and Wnt3a are expressed on the dorsal neural tube shortly after its closure. If these genes are mutated, some neural crest derivatives are diminished, but neural crest induction is not affected. Amphibian embryos require a combination of Wnt’s, fibroblast growth factors or the inhibition of BMP’s to induce neural crest formation. Until recently, relatively little information had been found on the specific role of Wnt signalling pathways in the formation of the neural crest. (Ikeya et al. 1997)

Recent research by the Californian Institute of Technology examined the role of Wnt molecules on induction of chick neural crest cells both in vivo and in vitro. Initially, various Wnt family members were examined using in situ hybridisation, to determine candidates for neural crest induction. It was found that Wnt5a, Wnt5b and Wnt8c were not expressed in the ectoderm but showed evidence in the caudal-most region of the neural plate. Wnt6 was expressed in the ectoderm nearby to neural folds but was elusive in the neural folds and plate. This discovery is coherent with a potential ectoderm neural inducer. BMP-4 however showed some disparity, showing considerable expression in the dorsal neural tube and neural folds but weakly in ectoderm tissue directly adjacent to the neural plate. (Garcia-Castro et al. 2002)

Inhibitory vertebrate Wnt ligands can be designed based approximately on the homologuous sequence in other Wnt molecules produces a secreted Wnt protein with inhibitory activity. These molecules inhibit the signalling activity of endogenous Wnt ligands in a cell non-automnomous way and can therefore be used to study the requirement for endogenous Wnt function in different processes in development.

The researchers extended their investigation by examining the effect of Wnt inhibitors on Wnt signalling pathways in vivo. The dominant negative Wnt1 construct, DnWnt1 is known to have a 71-amino acid carboxy-terminal, which is known to block Wnt signalling by possible binding to Wnt receptors. The expression Slug is a known assay to indicate the formation of the neural crest. When the inhibitor was injected adjacent to the neural plate, a significant decrease in slug expression was observed. Interestingly, when the inhibitor was mixed with Wnt1 expressing cells and injected in the same regions, there was partial expression of Slug. (Hoppler et al. 1996)

The next step in the experiment was to monitor the effect of Wnt inhibitory on the migration of neural crest cell 36 hours after the initial injection by using an antibody HNK-1. It was shown that DnWnt-1 distorted neural crest migration compared to control cells. (Garcia-Castro et al. 2002)

The Wnt canonical pathway, upon activation, causes β-catenin to enter the nucleus and, in conjunction with T cell factors (TCF’s), it acts a transcription factor. The presence of β-catenin was found in the nuclei of neural folds of the open neural plate which conforms to the role of Wnt signalling in neural crest induction. Earlier studies in Xenopus show that β-catenin copies the effects of Wnt over-expression by instigating surplus neural crest formation. (Chang et al. 1998)

As the study progressed the researchers used naïve neuroepithelium to test the ability of Wnt signalling in neural crest induction. Drosophila S2 cells infected with a Wnt-1 homolog, a known trigger of Wnt signals, were used in a conditioned medium. Results gave evidence that the infected medium gave rise to robust neural crest formation compared to control plates. (Phanot et al. 1998)

BMP-4 has been shown itself to induce neural crest formation in similar assays, but in the absence of certain additives the induction is stopped. In contrast, it has been shown that Wnt signalling can trigger neural crest formation even in the absence of these same additives. (Shultheiss et al. 1997)

Wnt6 is expressed in the ectoderm close to the region where the induction of the neural plate takes place. It would be plausible to say that this Wnt signal, in this specific region, is a ectodermal neural crest inducer. These results support the notion that Wnt signalling could be a widespread mechanism in neural crest induction. (Garcia-Castro et al. 2002)   

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