Neural crest
The neural crest is a ridge-like structure that is formed transiently between the epidermal ectoderm and neural plate during vertebrate development. Neural crest cells originate from this structure through the epithelial-mesenchymal transition, and in turn give rise to a diverse cell lineage—including melanocytes, craniofacial cartilage and bone, smooth muscle, dentin, peripheral and enteric neurons, adrenal medulla and glia.
After gastrulation, the neural crest is specified at the border of the neural plate and the non-neural ectoderm. During neurulation, the borders of the neural plate, also known as the neural folds, converge at the dorsal midline to form the neural tube. Subsequently, neural crest cells from the roof plate of the neural tube undergo an epithelial to mesenchymal transition, delaminating from the neuroepithelium and migrating through the periphery, where they differentiate into varied cell types. The emergence of the neural crest was important in vertebrate evolution because many of its structural derivatives are defining features of the vertebrate clade.
Underlying the development of the neural crest is a gene regulatory network, described as a set of interacting signals, transcription factors, and downstream effector genes, that confer cell characteristics such as multipotency and migratory capabilities. Understanding the molecular mechanisms of neural crest formation is important for our knowledge of human disease because of its contributions to multiple cell lineages. Abnormalities in neural crest development cause neurocristopathies, which include conditions such as frontonasal dysplasia, Waardenburg–Shah syndrome, and DiGeorge syndrome.
Defining the mechanisms of neural crest development may reveal key insights into vertebrate evolution and neurocristopathies.
History
The neural crest was first described in the chick embryo by Wilhelm His Sr. in 1868 as "the cord in between" because of its origin between the neural plate and non-neural ectoderm. He named the tissue "ganglionic crest," since its final destination was each lateral side of the neural tube, where it differentiated into spinal ganglia. During the first half of the 20th century, the majority of research on the neural crest was done using amphibian embryos which was reviewed by Hörstadius in a well known monograph.Cell labeling techniques advanced research into the neural crest because they allowed researchers to visualize the migration of the tissue throughout the developing embryos. In the 1960s, Weston and Chibon utilized radioisotopic labeling of the nucleus with tritiated thymidine in chick and amphibian embryo respectively. However, this method suffers from drawbacks of stability, since every time the labeled cell divides the signal is diluted. Modern cell labeling techniques such as rhodamine-lysinated dextran and the vital dye diI have also been developed to transiently mark neural crest lineages.
The quail-chick marking system, devised by Nicole Le Douarin in 1969, was another instrumental technique used to track neural crest cells. Chimeras, generated through transplantation, enabled researchers to distinguish neural crest cells of one species from the surrounding tissue of another species. With this technique, generations of scientists were able to reliably mark and study the ontogeny of neural crest cells.
Induction
A molecular cascade of events is involved in establishing the migratory and multipotent characteristics of neural crest cells. This gene regulatory network can be subdivided into the following four sub-networks described below.Inductive signals
First, extracellular signaling molecules, secreted from the adjacent epidermis and underlying mesoderm such as Wnts, BMPs and Fgfs separate the non-neural ectoderm from the neural plate during neural induction.Wnt signaling has been demonstrated in neural crest induction in several species through gain-of-function and loss-of-function experiments. In coherence with this observation, the promoter region of slug contains a binding site for transcription factors involved in the activation of Wnt-dependent target genes, suggestive of a direct role of Wnt signaling in neural crest specification.
The current role of BMP in neural crest formation is associated with the induction of the neural plate. BMP antagonists diffusing from the ectoderm generates a gradient of BMP activity. In this manner, the neural crest lineage forms from intermediate levels of BMP signaling required for the development of the neural plate and epidermis.
Fgf from the paraxial mesoderm has been suggested as a source of neural crest inductive signal. Researchers have demonstrated that the expression of dominate-negative Fgf receptor in ectoderm explants blocks neural crest induction when recombined with paraxial mesoderm. The understanding of the role of BMP, Wnt, and Fgf pathways on neural crest specifier expression remains incomplete.
Neural plate border specifiers
Signaling events that establish the neural plate border lead to the expression of a set of transcription factors delineated here as neural plate border specifiers. These molecules include Zic factors, Pax3/7, Dlx5, Msx1/2 which may mediate the influence of Wnts, BMPs, and Fgfs. These genes are expressed broadly at the neural plate border region and precede the expression of bona fide neural crest markers.Experimental evidence places these transcription factors upstream of neural crest specifiers. For example, in Xenopus Msx1 is necessary and sufficient for the expression of Slug, Snail, and FoxD3. Furthermore, Pax3 is essential for FoxD3 expression in mouse embryos.
Neural crest specifiers
Following the expression of neural plate border specifiers is a collection of genes including Slug/Snail, FoxD3, Sox10, Sox9, AP-2 and c-Myc. This suite of genes, designated here as neural crest specifiers, are activated in emergent neural crest cells. At least in Xenopus, every neural crest specifier is necessary and/or sufficient for the expression of all other specifiers, demonstrating the existence of extensive cross-regulation. Moreover, this model organism was instrumental in the elucidation of the role of the Hedgehog signaling pathway in the specification of the neural crest, with the transcription factor Gli2 playing a key role.Outside of the tightly regulated network of neural crest specifiers are two other transcription factors Twist and Id. Twist, a bHLH transcription factor, is required for mesenchyme differentiation of the pharyngeal arch structures. Id is a direct target of c-Myc and is known to be important for the maintenance of neural crest stem cells.
Neural crest effector genes
Finally, neural crest specifiers turn on the expression of effector genes, which confer certain properties such as migration and multipotency. Two neural crest effectors, Rho GTPases and cadherins, function in delamination by regulating cell morphology and adhesive properties. Sox9 and Sox10 regulate neural crest differentiation by activating many cell-type-specific effectors including Mitf, P0, Cx32, Trp and cKit.Image:GRN Crest.png|thumb|center|700px|alt=Neural Crest.|Putative neural crest gene-regulatory network functioning at the neural plate border in vertebrates. Red arrows represent proven direct regulatory interactions. Black arrows show genetic interactions based on loss-of-function and gain-of-functions studies. Gray lines denote repression. Adapted from Bronner-Fraser 2004
Migration
The migration of neural crest cells involves a highly coordinated cascade of events that begins with closure of the dorsal neural tube.Delamination
After fusion of the neural folds to create the neural tube, cells originally located in the neural plate border become neural crest cells. For migration to begin, neural crest cells must undergo a process called delamination that involves a full or partial epithelial–mesenchymal transition. Delamination is defined as the separation of tissue into different populations, in this case neural crest cells separating from the surrounding tissue. Conversely, EMT is a series of events coordinating a change from an epithelial to mesenchymal phenotype. For example, delamination in chick embryos is triggered by a BMP/Wnt cascade that induces the expression of EMT promoting transcription factors such as SNAI2 and FOXD3. Although all neural crest cells undergo EMT, the timing of delamination occurs at different stages in different organisms: in Xenopus laevis embryos there is a massive delamination that occurs when the neural plate is not entirely fused, whereas delamination in the chick embryo occurs during fusion of the neural fold.Prior to delamination, presumptive neural crest cells are initially anchored to neighboring cells by tight junction proteins such as occludin and cell adhesion molecules such as NCAM and N-Cadherin. Dorsally expressed BMPs initiate delamination by inducing the expression of the zinc finger protein transcription factors snail, slug, and twist. These factors play a direct role in inducing the epithelial-mesenchymal transition by reducing expression of occludin and N-Cadherin in addition to promoting modification of NCAMs with polysialic acid residues to decrease adhesiveness. Neural crest cells also begin expressing proteases capable of degrading cadherins such as ADAM10 and secreting matrix metalloproteinases that degrade the overlying basal lamina of the neural tube to allow neural crest cells to escape. Additionally, neural crest cells begin expressing integrins that associate with extracellular matrix proteins, including collagen, fibronectin, and laminin, during migration. Once the basal lamina becomes permeable, neural crest cells can begin migrating throughout the embryo.
Migration
Neural crest cell migration occurs in a rostral to caudal direction without the need of a neuronal scaffold such as along a radial glial cell. For this reason the crest cell migration process is termed "free migration". Instead of scaffolding on progenitor cells, neural crest migration is the result of repulsive guidance via EphB/EphrinB and semaphorin/neuropilin signaling, interactions with the extracellular matrix, and contact inhibition with one another. While Ephrin and Eph proteins have the capacity to undergo bi-directional signaling, neural crest cell repulsion employs predominantly forward signaling to initiate a response within the receptor bearing neural crest cell. Burgeoning neural crest cells express EphB, a receptor tyrosine kinase, which binds the EphrinB transmembrane ligand expressed in the caudal half of each somite. When these two domains interact it causes receptor tyrosine phosphorylation, activation of rhoGTPases, and eventual cytoskeletal rearrangements within the crest cells inducing them to repel. This phenomenon allows neural crest cells to funnel through the rostral portion of each somite.Semaphorin-neuropilin repulsive signaling works synergistically with EphB signaling to guide neural crest cells down the rostral half of somites in mice. In chick embryos, semaphorin acts in the cephalic region to guide neural crest cells through the pharyngeal arches. On top of repulsive repulsive signaling, neural crest cells express β1and α4 integrins which allows for binding and guided interaction with collagen, laminin, and fibronectin of the extracellular matrix as they travel. Additionally, crest cells have intrinsic contact inhibition with one another while freely invading tissues of different origin such as mesoderm. Neural crest cells that migrate through the rostral half of somites differentiate into sensory and sympathetic neurons of the peripheral nervous system. The other main route neural crest cells take is dorsolaterally between the epidermis and the dermamyotome. Cells migrating through this path differentiate into pigment cells of the dermis. Further neural crest cell differentiation and specification into their final cell type is biased by their spatiotemporal subjection to morphogenic cues such as BMP, Wnt, FGF, Hox, and Notch.