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Magazine R381

Primer specification of the neural crest. between the neural crest cells and More specifically, this inductive their neighbours within the dorsal event is mediated by members of neural tube. More specifically, it the Bmp family of signalling has been found that the The neural crest molecules. Both Bmp-4 and Bmp- delamination of neural crest cells 7 are expressed by the epidermal requires a switch in the repertoire Anthony Graham ectoderm, and the factors they of cell adhesion molecules encode will elicit the production of expressed by these cells. Thus, The neural crest is a transient and neural crest cells from the neural the crest cells down-regulate N- discrete embryonic cell population plate tissue. Furthermore, it has CAM and N-cadherin, which are of profound importance to the been recently suggested that generally expressed by cells of the development and the evolution of another signalling molecule, Wnt- neural tube. They also lose the the vertebrates. This cell type 6, which is also made in the expression of cadherin-6b, which emerges from the dorsal neural epidermal ectoderm, may also be is expressed more specifically tube during early development. involved in neural crest induction. within the neural tube by the cells Subsequently, the cells migrate It should also be noted, of the dorsalmost region. The into the periphery along however, that the interaction crest cells themselves then stereotypical paths to a number of between the neural plate and the upregulate cadherin-7. sites at which these cells stop and epidermal ectoderm does not just Interestingly, this delamination differentiate into a wide variety of induce the formation of the crest process is also controlled by derivatives. These include sensory but that it also induces other Bmps, most prominently Bmp-4, neurons, autonomic neurons, glia, dorsal neural tube cell types. This which comes to be expressed at melanocytes, cells of the adrenal is in keeping with the fact that the the dorsal midline of the neural medulla and, additionally in the neural folds do not exclusively tube. If Bmp-4 function is head, bone, cartilage, connective give rise to neural crest cells but perturbed, the neural crest cells tissue and smooth muscle cells. also to the other most dorsal cell cannot escape the neural tube and, Importantly, the neural crest is a types of the neural tube. Thus, in instead, accumulate within it. At unique feature of vertebrate essence, the interaction between least in part, Bmp-4 drives embryos, and the adult structures the neural plate and the epidermal delamination through up-regulation generated from the neural crest ectoderm does not directly induce of rhoB, which encodes a small are, fundamentally, those that the formation of the neural crest, GTP-binding protein and is distinguish the vertebrates from but rather results in the induction expressed specifically in the neural their nearest relatives. of the neural folds, which in turn crest cells prior to, and just after, Consequently, it has been give rise to neural crest, as well as delamination. If RhoB function is proposed that the evolution of the other very dorsal cell types. inhibited, the ability of the neural neural crest was key to the A consequence of this inductive crest to escape from the neural evolution of the vertebrates interaction is to stimulate the primordium is severely curtailed. themselves. expression of a number of transcription factors in the neural The migration of the neural crest The formation of the neural crest folds, which help mediate the In the trunk Induction generation of the neural crest. Once the neural crest cells escape In the embryo, the neural crest These have been found to include from the neural tube, they migrate cells first become manifest as they Pax gene products, members of along stereotypical pathways detach and leave the dorsal neural the Zic family, Slug and Foxd3. towards their sites of tube. However, the event that However, of these only Slug, a differentiation, and it would seem triggers the formation of the zinc finger transcription factor, that the pathway choices are neural crest occurs earlier in and FoxD3, a winged-helix dictated to the crest cells by development, during the process transcription factor, are environmental cues. The first of neurulation (Figure 1). A exclusively made in the neural structures encountered by crest consequence of this process, crest; the others are also made in cells as they leave the neural tube which involves the folding of the other cells within the dorsal neural are the somites, and, interestingly, flat neural plate into the neural tube. It is therefore likely that it is the migratory crest cells display a tube, is that the dorsal midline of Slug and FoxD3 which help define precise relationship with the the neural tube forms from the particular cells within the dorsal compartments of the somites. The edges of the neural plate that abut neural tube as being crest cells. early, ventrally migrating neural the epidermal ectoderm — the crest cells move through the neural folds. Thus, the neural crest Delamination sclerotome, the region of the is lineally derived from these Once the neural crest cells have somite that forms the axial structures. That the neural folds been induced, they have to leave skeleton, but, importantly, only form between the neural plate and the neural tube, and to do this through the anterior half the epidermal ectoderm betrays they must undergo a transition sclerotome of each somite (Figure the fact that it is an inductive from epithelial to mesenchymal 2). Neural crest cells do not enter interaction between these two type. Clearly this entails radical the posterior sclerotome of the tissues which underlies the alterations in the relationship somites. Of the ventrally migrating Current Biology Vol 13 No 10 R382

somites. Here, the early migratory ABC cells move ventrolaterally and populate the pharyngeal arches and facial prominences, Neural generating ectomesenchymal crest Neural Neural derivatives: bone, cartilage and folds plate connective tissue. The late migrating neural crest cells stay Notochord closer to the neural tube and Current Biology generate neurons and glia of the cranial ganglia. These crest cells, Figure 1. The induction of the neural crest occurs during the process of neurulation. however, do not migrate as a During neurulation, the neural plate (light blue) progressively folds to form the neural single mass, but are organised tube (A through to C). The ventral midline of the neural plate attaches to the notochord into three streams: trigeminal, (red). The neural folds (yellow) form at the interface between the neural plate and the epidermal ectoderm (dark blue), and the interaction between these two tissues is hyoid and post-otic. The required for the induction of the neural crest. The neural folds progressively come trigeminal neural crest arises from together to form the dorsal aspect of the neural tube and the neural crest (yellow). the midbrain and anterior hindbrain and forms neurons of cells, those that move furthest Ephrin-B1 and Ephrin-B2, and the trigeminal ganglion and the stop in the vicinity of the dorsal Sema-3A, while the crest cells components of the orofacial aorta and generate the neurons make the corresponding receptors, prominences and mandibular and glia of the sympathetic Eph-B2 and Eph-B3, and arch. The second stream, the ganglia. The remaining ventrally Neuropilin-1. Furthermore, in vitro hyoid, arises primarily from the migrating cells arrest more studies demonstrated that when central region of the hindbrain and dorsally within the anterior migrating neural crest cells are forms neurons of the proximal sclerotome, close to the neural confronted with a choice between facial ganglion as well as the tube and generate the sensory a permissive substrate, such as constituents of the second neurons and glia of the dorsal root fibronectin or Ephrin-B1, they pharyngeal arch. Finally, the post- ganglia (Figure 2). In contrast, the invariably avoid the ephrin domain otic crest is generated by the late migrating neural crest cells do and migrate along the fibronectin posterior hindbrain and forms the not follow this path, but migrate area, and, similarly, they will avoid neurons of the proximal and dorsolaterally between the a Sema-3A substrate. jugular ganglia, and the dermamyotomal region of the The somites are also involved in components of the posterior somite, which gives rise to muscle controlling the emigration of pharyngeal arches. and dermis, and the overlying neural crest cells along the This segregation of the neural ectoderm. These cells give rise to dorsolateral path. During the early crest into streams is important, as melanocytes (Figure 2). phases of crest migration, the it is required for normal A consequence of the migration dermamyotome and the ectoderm oropharyngeal development and of the early neural crest cells are in close contact, and the can have drastic consequences if it through only the anterior half dermamyotome actively inhibits is experimentally altered. The sclerotome of each somite is the migration. However, as the segregation of the head neural segmental organisation of their dermamyotome matures, it moves crest is evident as soon as the neuronal derivatives, the away from the somites and crest cells are born, and it is events sympathetic and the dorsal root cells do now enter the within the developing brain that are ganglia. In fact, the motor neurons dorsolateral path. The early responsible for establishing this that exit the spinal cord also only inhibitory role of the segregation. Studies in chick have enter the anterior sclerotome of dermamyotome is again mediated shown that one process that helps each somite and, thus, are also through production of Ephrin-B to enforce the segregation of the segmentally organised. This is molecules. But the neural crest neural crest is the focal depletion important as it fixes the position cells that leave the neural tube at of crest cells via apoptosis from of the ganglia and motor nerves late stages are not inhibited by the two territories between the and thus generates a the Ephrin-B ligands, even though streams. Contrastingly, recent correspondence between them they also express the Eph-B studies in mice have suggested and the muscle and skeleton of receptors. Instead, these late born that here, while neural crest the backbone. crest cells migrate along the segregation is controlled by events At the molecular level, it can be Ephrin-B-rich dorsolateral within the hindbrain, the seen that this segmental migration pathway using this molecule as a mechanism is somewhat different, is largely due to ligand receptor migratory substrate. although the extent of this is as yet interactions between the cells of unclear. Over and above these the posterior sclerotome and the In the head considerations, there are also crest cells. More specifically, it has In the head of the embryo, the events occurring in the periphery been shown that the posterior neural crest cells pour out from which act to direct and focus the sclerotome produces a number of the developing brain into a crest streams, again through the inhibitory molecules, including periphery that is devoid of use of inhibitory cues. Magazine R383

AB controlled through environmental factors during normal development. Indeed, the key 3 issues in understanding the differentiation of the neural crest 2 are what causes these migratory multipotent cells to arrest at particular sites in the embryo, and which factors in this environment cause the cells to follow their 1 particular path of differentiation. We have practically no idea what causes neural crest to stop migrating at particular sites, but we have some insight into the environmental factors that act at

Current Biology these locations to induce particular crest derivatives. Figure 2. The migration of the trunk neural crest. (A) A schematic of a longitudinal section through the trunk. The neural tube is coloured Cues for neural crest blue, the somites orange, the notochord red and the migratory crest cells yellow. As is differentiation shown, in the trunk the neural crest cells only migrate through the anterior half sclero- With regard to neuronal tome of each somite. (B) Schematic of a transverse section through the trunk. The arrows highlight the paths taken by the crest cells. The earliest migrating crest cells differentiation, we know least follow path 1 and give rise to the cells of the sympathetic ganglion. The cells that follow about the factors that promote the path 2 enter the anterior sclerotome and generate the dorsal root ganglion. The late differentiation of sensory neurons, migrating crest cells follow path 3 and form melanocytes. and this probably reflects the absence of any good general Differentiation of the neural crest Although these studies markers for sensory neurons. It is Fate versus potential suggesting the plasticity of the apparent, however, that the The broad range of neural crest neural crest have been conducted sensory neurons generated by the derivatives poses the problem of at the level of crest populations, neural crest differentiate into post- how individual fates become they also find support at the mitotic neurons in close proximity specified during development. single cell level. Clonal analysis of to the neural tube, and it has been Importantly, it has long been single chick cranial neural crest shown that a factor(s) emanating noted that neural crest cells from cells has shown the presence of from the neural tube is required for different axial levels have different multipotent progenitors, capable the neural crest cells to fates: sympathetic neurons are of generating neurons, schwann differentiate into sensory neurons. formed by trunk neural crest, cells, melanocytes and cartilage; The precise nature of this factor(s) enteric neurons by vagal and pluripotent progenitors, which remains unclear, however. sacral neural crest, and cartilage produce more restricted clones; In contrast, we have a fairly and bone by cranial neural crest. and monopotent cells, generating good idea of the factors that This could mean that neural crest only neurons, or only glia, or only trigger the differentiation of neural cells from particular axial levels cartilage, and so on. Studies in crest cells into sympathetic and are able to generate only a limited mammals have also identified enteric neurons. The neural crest repertoire of crest derivatives. neural crest stem cells, which are cells that form the sympathetic However, experimental studies capable of self-renewal and singly ganglia settle in the vicinity of the have shown that this is not the can generate neurons, glia and dorsal aorta, and their case, and neural crest cells from smooth muscle. Also, cell lineage differentiation is triggered by this all axial levels have similar analysis in vivo has shown that blood vessel, through secretion of potentials. For example, if vagal is individual neural crest cells give Bmp-4 and Bmp-7. The neural replaced by thoracic neural crest, rise to multiple progenitors. The crest cells generating the enteric then the transplanted cells fate of individual cells can be nervous system, however, migrate populate the gut and differentiate followed in the embryo by a to the gut wall and then move into enteric neurons, whereas variety of labelling techniques, through the mesodermal their normal fate is to form and this has shown that, for mesenchyme of the gut sensory and sympathetic neurons. example, a migrating neural crest populating its entire length. This Correspondingly, and perhaps cell will give rise to both sensory migration is followed by a wave of more dramatically, trunk neural and sympathetic neurons, as well differentiation, giving rise to both crest cells will form bone and as to Schwann cells. neurons and glia, which is cartilage in vitro when grown in Taken together, these studies triggered by factors within the gut the appropriate media and demonstrate that neural crest wall mesenchyme, including contribute to cranial skeletal cells are a multipotent progenitor GDNF and endothelin-3. GDNF is elements when grafted into the population, so that the fates of made during the period of neural developing head. specific crest populations must be crest migration through this Current Biology Vol 13 No 10 R384 tissue; the neural crest cells melanogenic neural crest cells similar to that of vertebrates. It themselves express the receptors and it has been noted that any has a dorsal nerve cord, a for this factor, c-Ret and GFRα-1, neurogenic neural crest cells notochord, segmented muscle and this ligand–receptor found on this path die by blocks and pharyngeal gill slits, interaction is required for the apoptosis. Other work, however, but it lacks some key generation of the enteric neurons. suggests that the assignment of characteristics, which have their Similarly, the endothelin-3 melanogenic fates, like that of embryonic origin in the neural receptor, Endothelin receptor-B, neuronal fates, is directed by crest. These include the presence is also made by the neural crest environmental cues, the difference of sensory, sympathetic and cells, and again this being that these cues exert their parasympathetic peripheral ligand–receptor interaction is effects prior to emigration. Thus, it ganglia, schwann cells, required for enteric neuronal has shown that the specification melanocytes, teeth and the differentiation. of melanogenic fate is directed by skeletal elements of the head. The neural crest forms three members of the Wnt family of Indeed, it has been suggested distinct types of peripheral glia: the signalling molecules, which are that the evolution of the neural Schwann cells of the peripheral produced in the dorsal neural crest was pivotal to the evolution nerves; the satellite cells of the tube. Interestingly, however, the of the vertebrates. sensory and sympathetic ganglia; early-born neurogenic neural crest Interestingly, recent studies and the glia of the enteric nervous cells make a Wnt antagonist, have suggested that the genetic system. The neural crest cells that cfrzb-1, while the late-born neural framework used for the generation will give rise to Schwann cells crest cells do not, suggesting that of the neural crest in vertebrates associate with axonal projections, only these late born cells can was already in place early in and their differentiation is cued by perceive the Wnt signal. chordate evolution. It has been factors derived from the axons, Relatively little is known about found that in Amphioxus there is most notably Neuregulin-1, which the nature of the factors, tissues the Bmp2/4 gene, which is will act upon the receptor ErbB3 and molecules that direct the expressed in the non-neural made by the neural crest cells. The differentiation of the cranial neural ectoderm. Furthermore, emergence of the satellite cells of crest into cartilage, bone or orthologues of the msx and the dorsal root and sympathetic odontoblasts. But it is clear that slug/snail genes have been ganglia is seemingly more these fates are also the identified in amphioxus and found problematic, as both these and the consequence of environmental to be expressed in the neural neurons of the ganglia arise from cues acting on plastic neural crest plate abutting the non-neural neural crest cells in the same cells. More specifically, studies in ectoderm. However, there is no environment. It has been shown, zebrafish have shown that the direct evidence of neural crest however, that the choice between differentiation of neural crest into cells in Amphioxus. Rather, it is these two cell types is achieved cartilage in the pharyngeal arches likely that these genes are, as they through Notch-mediated lateral is induced by the adjacent are in vertebrates, involved more inhibition. Delta-1, the Notch pharyngeal endoderm, mediated generally in the development of ligand, is made by early neural- by members of the FGF family of the dorsal nerve cord. These crest-derived neuronal cells, while signalling molecules. Along similar results would, however, be undifferentiated neural crest cells lines, it has been shown that the consistent with the neural crest make Notch-1. If Notch signalling differentiation of the crest into evolving as part of an elaboration is activated, then that crest cell is odontoblasts is also under the of the genetic programme, which inhibited from becoming neuronal control of peripheral tissues, patterns the dorsal neural tube. and instead undergoes although in this case the However, it is still as yet unclear gliogenesis. This mechanism ectoderm is the inducing tissue. how these dorsal neural tube cells ensures a match in cell number Finally, although it has not been first began to delaminate and to between the neurons and satellite definitively demonstrated, it is migrate into the periphery and cells of the ganglia. likely that the differentiation of thus to be neural crest cells. A number of studies on the crest cells into bone is also melanogenic lineage have controlled by environmental Further reading suggested that, in contrast to the factors, with, for example, Knecht, A.K. and Bronner-Fraser, M. (2002). Induction of the neural neurogenic neural crest, these formation of the membrane bones crest: a multigene process. Nat. cells are assigned their fate prior of the skull being induced by Rev. Genet. 3, 453–461. to their emergence from the signals emanating from the brain. Le Douarin, N.M. and Kalcheim, C. neural tube. When early migrating (1999). The Neural Crest, 2nd edn. (Cambridge University Press, New crest cells are grafted into a host The evolution of the neural crest York). embryo, they only migrate It is generally accepted that the Gans, C. and Northcutt, R.G. (1983). ventrally. Contrastingly, when vertebrates evolved from Neural crest and the origin of melanoblasts are grafted into a protochordates ancestors, vertebrates: a new head. Science young host, they precociously probably from an animal akin to 220, 268–274. enter the dorsolateral pathway. modern day Amphioxus, a sessile MRC Centre for Developmental The dorsolateral path also seems filter feeder whose anatomical Neurobiology, Kings College London, unable to support non- organisation is in many ways very London SE1 1UL, UK.