INDUCTION OF THE NEURAL CREST: A MULTIGENE PROCESS

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1 INDUCTION OF THE NEURAL CREST: A MULTIGENE PROCESS Anne K. Knecht and Marianne Bronner-Fraser In the embryo, the neural crest is an important population of cells that gives rise to diverse derivatives, including the peripheral nervous system and the craniofacial skeleton. Evolutionarily, the neural crest is of interest as an important innovation in vertebrates. Experimentally, it represents an excellent system for studying fundamental developmental processes, such as tissue induction. Classical embryologists have identified interactions between tissues that lead to neural crest formation. More recently, geneticists and molecular biologists have identified the genes that are involved in these interactions; this recent work has revealed that induction of the neural crest is a complex multistep process that involves many genes. NEURAL TUBE A cylindrical structure that runs through the midline of the embryo; it expands in the head to form the brain and in the trunk to form the spinal cord. NEURAL FOLDS Tips of invaginating ectoderm that will close to form the dorsal portion of the neural tube. Division of Biology , California Institute of Technology, Pasadena, California 91125, USA. Correspondence to M.B.-F. mbronner@gg.caltech.edu doi: /nrg819 A central question in developmental biology is how a single cell the egg can give rise to many tissues. A principal mechanism for generating such complexity is induction, whereby one set of cells influences another, causing those cells to adopt a different fate. Although induction is usually represented by a single arrow in diagrams, recent advances in the molecular genetics of development have shown that induction involves several signals. These include inducers, which activate a new genetic programme that leads to conversion to a different cell type; competence factors, which control the time at which cells can respond to these inducers; maintenance factors, which maintain the induced developmental programme, perhaps through many intermediate stages; and cellsurvival or proliferation signals, which regulate growth. Likewise, recent research has identified several genes that are involved in the induction of the neural crest, including, in particular, secreted growth factors of the bone morphogenetic protein (BMP), fibroblast growth factor (FGF) and Wingless/INT-related (WNT) families. However, the precise functions of these genes remain unclear. This review describes recent findings about the molecular basis of neural crest induction, with the goal of elucidating the steps and signals that are involved. Early development of neural crest The neural crest is a transient, migratory population of cells found in all vertebrate embryos, including the ancestral vertebrates, such as lampreys (BOX 1). The cells that form neural crest are induced at the border between the neural plate, which forms the central nervous system, and the non-neural ectoderm, which forms the epidermis (FIG. 1). As the neural plate folds over itself to form the NEURAL TUBE, border regions (NEURAL FOLDS) from opposite sides of the ectoderm come together and later fuse. In this way, neural crest progenitors come to lie in, and/or immediately adjacent to, the dorsal neural tube 1. During or after neural tube closure (depending on the species), neural crest cells leave the neural tube and migrate throughout the body, where they differentiate into neurons, cartilage, melanocytes and many other types of cell (BOX 2). This review focuses only on the early formation of neural crest progenitors; neural crest migration and differentiation are discussed elsewhere 1. Although neural crest progenitors lie in the neural plate border, this region does not give rise exclusively to neural crest. If a single cell in the neural folds is labelled before neural tube closure, then the labelled derivatives can later be found in the neural crest, neural tube and epidermis 2. Even after neural tube closure, cells in the dorsal neural tube in the chick 2 and in the frog 3 can generate both neural tube cells and neural crest cells. After emigration from the neural tube, neural crest cells do not normally contribute to the neural tube, but if these cells are injected into the ventral neural tube, they can adopt the fates of their NATURE REVIEWS GENETICS VOLUME 3 JUNE

2 Box 1 Evolution of the neural crest Vertebrate evolution has been intimately linked to the evolution of two embryonic cell populations the neural crest and the cranial ectodermal PLACODES. These cell populations together give rise to many of the defining characteristics of vertebrates, including a well-defined head with teeth and paired sensory organs. Both neural crest and placodes are migratory populations that form at the border between neural plate and epidermis. They generate some of the same cell types, such as sensory neurons. Although they share many characteristics, there are also some differences between them; for example, placodes are confined to the head, whereas neural crest cells arise from most of the anteroposterior axis. Unlike placodes, neural crest cells form melanocytes and AUTONOMIC NEURONS, and produce mineralized matrices like bone. These differences might reflect differences in their evolutionary origin 74. Neural crest evolved soon after the split of cephalochordates (amphioxus) and vertebrates. Amphioxus, the closest living invertebrate relative of the vertebrates, shares some characteristics with vertebrates, such as segmented muscles, but lacks definitive neural crest 75,76. By contrast, structures that are derived from the neural crest, such as PHARYNGEAL DENTICLES, are present in the earliest vertebrate fossils, and the most basal extant vertebrates, hagfish and lampreys, have well-developed structures that are derived from the neural crest. Lampreys are jawless fish that represent the most primitive extant vertebrates for which it is feasible to obtain embryos, and these clearly have neural crest cells 77. Because of the genome-wide duplications associated with vertebrate evolution, one possible way to explain the evolution of neural crest is that, in the vertebrate lineage, new genes were formed by duplication and this facilitated diversification of gene function, which led to the origin of a new cell type. However, many homologues of vertebrate neural crest markers have been cloned in both amphioxus and lampreys, which indicates that the same complement of genes exists in both species, even though the former lacks a definitive neural crest Although the functions of these genes have not been studied sufficiently for us to be certain that they act the same way in different species, in general, it seems more likely that the evolution of the neural crest was accompanied by the use of old genes in new ways rather than by the invention of new genes for a new cell population. Further molecular characterization and embryological analysis are necessary to gain a better understanding of the evolutionary origin of neural crest cells and placode cells. PLACODES Thickenings in the vertebrate cranial ectoderm that invaginate and form parts of cranial sensory ganglia and paired sensory organs. AUTONOMIC NEURONS Nerve cells of the peripheral nervous system that innervate the viscera, smooth muscles and exocrine glands. PHARYNGEAL DENTICLES Dense structures on the surface of pharyngeal arches of early vertebrates that are thought to be pressure sensitive. GASTRULATION Morphogenetic movement that transforms a single-layered embryo into an embryo with three germ layers. HAMBURGER HAMILTON STAGES Stages that describe the age of chick embryos; stage 2 refers to the time before gastrulation. neighbours and form floor plate and motor neurons 4. Such results show that, although induction at the neural plate border leads to the formation of neural crest cells, there are many steps in between, in which cells show remarkable flexibility in their cell-fate determination. Because of this, it is difficult to apply traditional developmental terms, such as commitment or specification, to neural crest. A tissue is operationally defined as specified for a certain fate if it continues to adopt that fate when explanted away from other external signals. By this definition, the neural folds are specified to form neural crest, as explants from this region produce neural crest derivatives 2 ; however, as described above, not all cells in this region form neural crest, as the neural, epidermal and neural crest lineages are not yet segregated. Furthermore, if early neural folds represent a heterogeneous population of cells, including both neural and epidermal cells, signalling might occur between cell types in explants, and therefore the prospective neural crest is not truly isolated. Given these issues, it is difficult to say when neural crest induction really occurs. In recent experiments, researchers have focused on expression of the transcription factor Slug as one of the earliest indicators of neural crest induction, owing to its expression in regions of the neural folds that have the potential to form neural crest 5,6. For simplicity, this review refers to the induction of Slug as neural crest induction, but with the understanding that this might actually represent an early step in an ongoing process. Models of neural crest induction Neural induction. An obvious first step in the formation of the neural-plate border is the formation of the neural plate. During GASTRULATION, ectoderm is induced to form neural tissue by signals from a specialized region of mesoderm known as the organizer. A model of the molecular basis of neural induction, which is referred to as the neural default model (reviewed in REF. 7), has been developed during the past decade on the basis of experiments carried out in the frog Xenopus laevis. According to this model, the default fate of ectoderm is to form neural tissue; however, before neural induction, all of the ectoderm produces the growth factor BMP4, which suppresses the formation of neural tissue and promotes the formation of epidermis. To overcome this suppression and, therefore, to generate the neural plate, the organizer secretes BMP antagonists, such as noggin, chordin and follistatin, which bind to BMPs and prevent signalling through their receptors 7. Although this model is well supported by experiments in Xenopus, it remains unclear how well it applies to other vertebrates, and whether other factors might be involved. Mouse knockouts of BMPs and BMP antagonists were largely uninformative 8, probably owing to functional redundancy between genes. In the chick, Streit et al. 9 questioned whether BMP antagonism is necessary for neural induction for the following reasons. First, they showed that BMP4 and BMP7 expression in the chick disappears from the ectoderm before gastrulation (HAMBURGER HAMILTON STAGE 2) 9.However,Faure et al. 10 found that BMP signalling, which is manifested by activation of the downstream signalling molecule SMAD1, is reactivated throughout the ectoderm at stage 3 of chick development and is subsequently downregulated in the prospective neural plate at stage 4. So, as in Xenopus, reduction of BMP signalling correlates with neural induction. Second, Streit et al. also found that the addition of BMPs to prospective neural plate in stage-3 chick embryos does not block neural induction, and application of chordin to ectoderm cannot induce neural tissue. However, these experiments might have been carried out too late in development, after the initial neural specification. Although neural tissue is not specified in Xenopus before gastrulation, Wilson et al. 11 showed that explants that are cultured from chick embryos before gastrulation are already specified to express neural markers, and Streit et al. 12 found the expression of a neural marker before gastrulation. At these early stages, the entire EPIBLAST shows BMP signalling 10, which supports the idea that BMP inhibition is not necessary for initial neural specification. In culture, however, prospective neural explants downregulate BMP4 expression 11, as in Xenopus. 454 JUNE 2002 VOLUME 3

3 EPIBLAST A term for the embryonic layer in chicks, mice and humans from which the embryo proper arises during gastrulation. a b c d nc np nf np nt nt Figure 1 Regions that form neural crest during neurulation in a hypothetical vertebrate embryo. a Open neural plate (np) stage, after gastrulation. b Closing neural folds (nf). c Closed neural tube (nt). d Migrating neural crest (nc). Non-neural epidermis (epi) is shown in grey; np and nt in orange; and np border, nf and migrating nc are shown in yellow. Interestingly, this downregulation occurs in the absence of detectable BMP antagonists; instead, these explants express FGF3, and FGF signalling is required for BMP downregulation 11. Several studies in the chick have shown that FGF can act as a neural inducer 12,13 and that FGF signalling is required for neural induction 12. Furthermore, members of the WNT family have also been shown to have a role in avian neural induction. WNT3A and WNT8C are expressed in prospective epidermis, and WNT3A can block neural induction in explants by blocking their ability to downregulate BMPs in response to FGFs 14. In summary, although neural induction is likely to be more complex than the neural default model involving other signals, such as FGFs and WNTs the absence of BMP activity in prospective neural tissue remains a key feature of the developing neural plate that is conserved in both the frog and the chick. A variation on the neural default model is that neural induction divides the ectoderm into not two but three fates: epidermis, neural plate and neural plate border (reviewed in REF. 15). If diffusion of BMP antagonists through the ectoderm creates a gradient of BMP activity, in which high activity specifies epidermis and low activity specifies neural plate, then intermediate levels of BMP activity could specify the neural plate border. Because the role of BMPs in neural induction remains controversial in the chick and in the mouse, it is unclear how this model relates to these vertebrates, especially given that BMP4 is most highly expressed in early neurula chick embryos at the neural plate border, epi epi epi epi which it seems to maintain 16. This model is, however, supported by BMP-pathway mutants in zebrafish (swirl/bmp2b, snailhouse and somitabun) that have either reduced or expanded domains of neural crest progenitors, depending on the precise alteration of the BMP-signalling levels 17. Also, injection of BMP4 RNA into Xenopus embryos leads to a reduction in neural crest 18, whereas injection of BMP antagonists causes neural crest expansion 6,18. Importantly, the expanded neural crest domain remains contiguous with its normal domain and cannot be induced throughout the ectoderm, which indicates that other signals limit the territory that forms neural crest. Although these in vivo results indicate an important role for BMP signalling in neural crest induction, the mesoderm is also affected in these mutants and, for this reason, in vitro experiments are necessary to determine whether BMP alone is sufficient and whether its action is direct. In Xenopus ectodermal cells in vitro, intermediate BMP levels can induce some genes that are normally expressed in the anterior regions of neural plate border (XAG1 (REF. 19) and cpl1 (REF. 20)). Furthermore, in vivo, some genes that mark the neural plate border in Xenopus, such as Snail 21 and Pax3 (REF. 22), are expressed at the developing border as early as mid-gastrula stages, which indicates that this border forms during neural induction. However, in Xenopus, Slug is not expressed until later 6,23 and, in explants of Xenopus ectoderm, intermediate levels of BMP signalling alone are insufficient to induce robust expression of Slug 18. Marchant et al. 24 observed the induction of Slug in explants by intermediate BMP signalling, but Villanueva et al. 25 suggested that this result was due to overly large explants, which probably incorporated extra signals. In conclusion, although some aspects of the neural plate border might be specified during neural induction, and intermediate BMP signalling is probably an important first step towards forming the neural crest, the induction of cells with the potential to form neural crest apparently requires additional signals. Two-signal model of neural crest induction. Although intermediate levels of BMP signalling alone cannot induce neural crest in Xenopus ectodermal explants, several groups have found that the addition of a second signal in the same assay could induce Slug. First, bfgf (FGF2) 6, and later efgf 18, were shown to induce Slug in combination with noggin or chordin. The following members of the Wnt family were also found to induce Slug expression when combined with a BMP antagonist: Wnt1 and Wnt3a 26, Wnt7b 27 and Wnt8 (REF. 18).Many of these genes are endogenously expressed at the appropriate stages in candidate tissues that induce neural crest (see the next section for further discussion): efgf 28 and Wnt8 (REF. 29) are expressed in paraxial mesoderm, and Wnt7b is present throughout the ectoderm 27. FGFs might act indirectly through Wnts; efgf can induce Wnt8 expression and its ability to induce Slug in Xenopus ectodermal explants can be strongly inhibited by an inhibitor of Wnt signalling 18. Overexpression of NATURE REVIEWS GENETICS VOLUME 3 JUNE

4 Box 2 Derivatives of neural-crest cells Neural crest cells migrate throughout the body and differentiate into many different cell types (see REF. 1 for more details). Although neural crest cells are pluripotent, differences exist between cells that are generated from different anteroposterior levels: neural crest cells in the trunk form melanocytes and several neuron and glia cell types, whereas neural crest cells in the cranial (the embryonic head region) also have the potential to form mesenchymal derivatives, such as cartilage, bone and connective tissue. Cranial Trunk Neurons and glia of cranial ganglia Dorsal Pigment cells Neural tube Cartilage and bone Sensory neurons and glia Ventral Connective tissue Sympatho-adrenal cells MORPHOLINO ANTISENSE OLIGONUCLEOTIDES Modified antisense oligonucleotides that are designed to block translation by pairing with the translation start site in the 5 untranslated region. the Wnt-signalling components frizzled 3 (REF. 30) and β-catenin 18 can also induce Slug expression in this assay, and the effects of β-catenin are cell autonomous, which indicates that Wnt signalling might be a direct inducer. Recently, retinoic acid was also shown to induce Slug expression in combination with a BMP inhibitor in Xenopus ectodermal explants 25. Retinoic acid 31, FGF2 (REF. 32) and Wnt3a 33 have all been shown to function as posteriorizing signals in the anteroposterior patterning of the neural tube. Because of this, it has been proposed that these signals function in explant assays by posteriorizing the tissue that is induced by intermediate BMP signalling and that resembles anterior neural plate border 15,25. This idea is supported by evidence that the endogenous anterior neural plate border, which normally does not express Slug or form neural crest, can be induced to express Slug by FGFs or retinoic acid 25. However, this model cannot account for the ability of Wnt signalling to induce neural crest in ectodermal explants that overexpress Slug, as Slug does not induce anterior neural border 18. Moreover, it is not clear that the source of the signal that induces neural crest in vivo lies in the posterior. The epidermis is also a source of signals that induce neural crest (see the next section for further discussion), and the activities of epidermal signals might be separable from posteriorizing signals. Epidermis can induce explants of the anterior neural plate to express Slug, but cannot induce Pax3, another neural crest marker that is regulated by posteriorizing signals 22. Regardless of whether the second signal (Wnt, FGF and/or retinoic acid) posteriorizes anterior neural plate border or directly induces neural crest in cooperation with intermediate BMP signalling, it is clear that these two signals are sufficient to mediate neural crest induction. However, are these candidates necessary for neural crest induction in vivo? For some factors, this has been tested genetically. Mouse embryos that lack both Wnt1 and Wnt3a had a reduced amount of neural crest, which indicates that these genes are not required for neural crest induction but might be important for the proliferation of neural crest precursors (consistent with their relatively late expression in the dorsal neural tube) 34.For other genes, loss-of-function experiments have been carried out in non-genetic organisms (that is, in Xenopus and in the chick) by overexpressing antagonists or dominant-negative constructs to block endogenous signalling. For all three candidate second signalling pathways, such experiments have resulted in the inhibition of Slug expression, which indicates that FGF, Wnt and retinoic acid signalling are all required to some extent for neural crest formation 18,25,26,30,35. A caveat of these experiments is that they generally inhibit signalling by several members of the targeted gene family. A more-specific approach is to use MORPHOLINO ANTISENSE OLIGONUCLEOTIDES to block translation of a particular gene. When frizzled 3 (REF. 30) and its proposed adaptor protein Kermit 36 were depleted in this way in Xenopus embryos, Slug expression was reduced, which verifies a crucial role for Wnt signalling in neural crest induction. 456 JUNE 2002 VOLUME 3

5 PARAXIAL MESODERM Mesoderm that is adjacent to the neural tube and that is destined to form somites. SOMITES Mesodermal balls of cells adjacent to the neural tube that will differentiate into the muscle, vertebrae and dermis. Sources of inducing signals There is a continuing debate about which tissue in the embryo provides the signals that induce the neural crest. After gastrulation, the region that forms the neural crest is in contact with three tissues: the neural plate, the epidermis and the underlying mesoderm (PARAXIAL MESODERM, which forms the SOMITES); all three tissues have been proposed to participate in neural crest induction. Paraxial mesoderm. One of the earliest neural crest experiments in amphibians showed that paraxial mesoderm can induce ectoderm to form neural crest 37.More recently, this experiment has been verified in vitro; explants of Xenopus prospective paraxial mesoderm and ectoderm were combined and later found to express Slug 18,24,38. In the chick, paraxial mesoderm can also induce explants of neural plate to form melanocytes, a neural crest derivative 2. It is not surprising that paraxial mesoderm has such activity, as this tissue expresses candidate inducing signals, such as efgf, WNT8 and BMP4. The crucial question is whether paraxial mesoderm is necessary for neural crest induction. Both Bonstein et al. 38 and Marchant et al. 24 tested this by excising the prospective paraxial mesoderm from Xenopus embryos at the onset of gastrulation; both found that expression of Slug was greatly reduced, which indicates a possible requirement for paraxial mesoderm in vivo. However, with such a large-scale, early dissection, it remains possible that neural crest reduction is due to indirect effects of the treatment, such as changes in the lateral ectoderm that this tissue would normally underlie. Also, the requirement for paraxial mesoderm remains to be shown in other vertebrates, in which such dissections are nearly impossible, except perhaps through genetic manipulations. Epithelium neural plate interactions. Another source of inducing signals is the interaction between neural plate and epithelium. This possibility was first indicated by experiments in which explants of epidermis or neural plate were grafted ectopically in amphibian embryos; neural crest cells were observed wherever new boundaries were created between neural and non-neural tissues 39,40. This result was also shown recently in similar grafting experiments in zebrafish embryos 41. Moury and Jacobson 42 further showed in amphibians that the induced neural crest was formed from both neural and epidermal tissues, which indicates the possibility of reciprocal signalling from both tissues. In these experiments, paraxial mesoderm remained a possible source of inducing signals; however, more recent in vitro studies have verified that Slug expression can be induced by combining only neural plate and epidermis, in the chick 2 and in Xenopus 43. It is important to note that the zebrafish experiments are done at later developmental stages than the Xenopus experiments described above, which makes it difficult to interpret these results in terms of the Xenopus twosignal model. Although Xenopus experiments have generally used relatively naive ectoderm that was explanted before neural induction, the experiments described in this section involved epidermis and neural plate that was explanted after neural induction was complete. Therefore, if intermediate BMP signalling in the twosignal model is interpreted as an extension of neural induction, then that signalling will be complete before these later experiments have begun. It might be that the neuralized ectoderm in chick neural plate explants acts like Xenopus explants that have been neuralized by BMP antagonism, so that this first signal is bypassed in the chick and induction requires only the second signal. Alternatively, it could be that neural crest induction in the chick is fundamentally different, or that the later experiments in the chick exploit a later phase in neural crest induction (FIG. 2). Recently, researchers have sought to identify the epidermal inducer. Liem et al. 44 found that both BMP4 and BMP7, which are expressed in the epidermis before neural tube closure, can mimic this epidermal signal, inducing chick ventral neural plate explants to express Slug and produce migratory neural crest cells. Furthermore, the inducing ability of epidermis in these cultures could largely be blocked by adding the BMP antagonists noggin and follistatin, which indicates that BMPs might be necessary components of the epidermal signal 45. However, the timing of this BMP requirement might be later than expected for the epidermal signal. Selleck et al. 46 showed that noggin cannot block the formation of neural crest during the open neural plate phase in chick embryos, when BMPs are expressed weakly in the epidermis and the epithelial signal has been shown to act 2. Instead, noggin was found to block neural crest induction at a later stage, in the closing neural tube, when the BMPs are expressed in the dorsal neural folds. So, Selleck et al. proposed that neural crest induction involves at least two phases: an early phase, in which neural crest is induced by an epidermal signal but is insensitive to BMP signalling; and a later phase, in which neural crest progenitors are sensitive to BMP4, which is by then present in the dorsal neural folds. According to this model, the role of BMP4 is to maintain neural crest progenitors that have been induced by an earlier signal. Why, then, were BMPs able to mimic the earlier signal in explant experiments? Isolated explants might be sufficiently labile to allow the later BMP signal to bypass the early BMP-independent phase or, alternatively, the culture medium that was used might have contained other factors. Liem et al. 44 cultured their explants in a medium that contained additives (N3 supplement that contains insulin and other hormones). When BMP4 protein was used in a medium without such factors, García-Castro et al. 47 found that this signal was unable to induce neural crest. By contrast, medium that had been conditioned with Wingless protein, a Drosophila homologue of Wnt that is active in vertebrates, can induce neural crest in the absence of additives to the medium. Moreover, injection of broad-spectrum Wnt inhibitors was shown to inhibit neural crest formation in vivo, and nuclear localization of β-catenin, which is an NATURE REVIEWS GENETICS VOLUME 3 JUNE

6 Xenopus Chick Open neural plate ect np ect ect np ect Closing neural folds Closed neural tube BMP Wnt1/3a/7b BMP Wnt1/3a Wnt BMP Figure 2 Dynamic expression patterns of Wnts and BMPs in the ectoderm of frog and chick. In the frog, bone morphogenetic protein (BMP) is expressed strongly in the ectoderm (ect) at all stages of neurulation. Wnt7b (wingless-related 7b) also is expressed in the ectoderm. After neural tube closure, BMP and other Wnts (for example, Wnt1 and Wnt3a) are expressed in the dorsal neural tube. In the chick, Wnt6 is expressed in the ectoderm at all stages during neurulation. By contrast, BMP is expressed in the ectoderm only at low levels (light green) and transiently when the neural plate (np) is open. As the neural folds elevate, BMP is downregulated in the ectoderm but upregulated in the neural folds. After neural tube closure, BMP is expressed in the dorsal neural tube, as are Wnt1 and Wnt3a. ROHON BEARD SENSORY NEURONS Early-differentiating neurons in the dorsal neural tube of fish and amphibians. indicator of active Wnt signalling, was observed in neural folds at the open neural-plate stage 47.A member of the Wnt family therefore represents a strong candidate for the early epidermal signal. Wnt6, in particular, is expressed in the epidermis at the appropriate stages 47. However, because Wnts are also expressed in prospective paraxial mesoderm, it remains possible that this mesoderm is the usual source of Wnts and that epidermis merely mimics this activity. Future experiments that involve conditional inactivation of genes in specific tissues will be necessary to resolve this issue, although it is also possible that signalling from many tissues is required. Other signals in neural crest induction Research in many systems has focused on the role of BMPs and Wnts in neural crest induction. These signals probably act at many different times in neural crest development. For example, Wnt1 and Wnt3a are candidates to regulate proliferation of neural crest progenitors 30, and BMP4 has been shown to regulate neural crest emigration from the neural tube 48. However, other signals, including Notch/Delta, Noelin and possibly Narrowminded, are also likely to be involved in neural crest formation. Notch and Delta. Signalling by the cell-surface protein Delta, and its receptor Notch, has been implicated in neural crest development 49. Notch signalling has been studied mainly as a mechanism that allows cells with equal developmental potential to adopt two different fates (reviewed in REF. 50). Such a process occurs in the dorsal neural tube, where cells can form either migra- tory neural crest or neural tube neurons. In zebrafish embryos, pre-migratory neural crest and ROHON BEARD SENSORY NEURONS are thought to form from a common progenitor pool. Zebrafish Delta mutants (deltaa missense mutation; dia d 2 ) have increased numbers of Rohon Beard cells and decreased neural crest cells, which indicates that Delta is necessary to promote the decision to form neural crest 51. Interestingly, this requirement is restricted to neural crest in the trunk, with cranial regions being unaffected. Experiments by Endo et al. 52 in the chick have indicated an earlier role for Notch signalling in neural crest induction in the cranium, as perturbations of Notch signalling in early embryos strongly reduce Slug expression. The authors propose that this effect is mediated indirectly by BMP4, which they found is genetically downstream of Delta. Both overactivation and inhibition of Notch signalling reduce BMP4 expression in the epidermis, indicating that intermediate levels of Notch signalling might be required to maintain epidermal BMP4, which might be necessary to induce neural crest. However, in these experiments, BMP4 is also downregulated in the dorsal neural tube, so a loss of Slug expression might reflect the later impairment of neural crest maintenance rather than an early effect on neural crest induction. Noelin. The neural tube is competent to produce neural crest for a limited period of time. Factors that control this competence are largely unknown, with the possible exception of the large secreted factor Noelin, which can prolong the period of neural crest production 53. However, this molecule is largely uncharacterized and the mechanism of its action is as yet unknown. 458 JUNE 2002 VOLUME 3

7 Narrowminded. A recent zebrafish screen identified the mutant narrowminded, which has reduced numbers of neural crest cells at early stages of development 54. Rohon Beard cells are also absent, which further supports the contention that these cells share a common progenitor with neural crest cells. The narrowminded mutation has been shown to function cell autonomously, which indicates that it might have a role in receiving or interpreting the signals that induce neural crest. Molecular characterization of the mutation will provide further clues to its actual function. Downstream transcription factors Inductive signals bring about the expression of transcription factors that mediate further neural crest development. Overexpression or inhibition experiments have indicated roles in neural crest formation for the following classes of transcription factors: Slug 5,18,55,Pax 56,57,Fox 58 60,Zic 61 65,Sox 66,67 and Meis 68. However, for most of these, the precise function remains unclear. Although some genes are likely to be direct downstream targets of neural crest induction, others are thought to be involved in later aspects of neural crest development. For example, Pax3 might maintain the neural crest progenitors in an undifferentiated state in the dorsal neural tube 69, and Foxd3 might cause these uncommitted progenitors to become neural crest instead of interneurons 58. The functions of Slug are more thoroughly understood. Because the promoter of the Xenopus Slug gene contains a functional binding site for Lef and β-catenin, which are downstream effectors of Wnt signalling, Slug might, therefore, be a direct target of Wnt induction 70. Accordingly, early perturbation of Slug expression in Xenopus affects the early specification of neural crest; neural crest is expanded by overexpression of Slug 18 and blocked by injection of dominant-negative Slug constructs 55.However, these studies also showed that Slug overexpression, like intermediate BMP signalling, cannot generate neural crest unless it is combined with FGFs or Wnts. So, although Slug can bypass the requirement for intermediate BMP signalling, neural crest formation requires other genes that are induced by FGFs and/ or Wnts. Although early inhibition of Slug blocks neural crest formation, its later inhibition blocks neural crest migration 55. This result agrees with those of experiments in the chick, in which application of antisense oligonucleotides against Slug allows formation of neural crest precursors but blocks their emigration from the neural tube 5. In these experiments, antisense oligonucleotides were unable to block neural crest formation. However, in recent experiments, Slug overexpression in the chick neural tube caused increased production of neural crest 71, as seen in Xenopus. Interestingly, this effect was restricted to the cranial region. Chick Slug might have more restricted activities because of the later initiation of Slug expression in closing neural folds and not at the borders of the open neural plate, as in Xenopus. Furthermore, in the mouse, Slug is expressed in migratory neural crest but not in pre-migratory cells 72,73 ; instead, the related gene Snail is expressed in the mouse in a pattern that is similar to that of Slug expression in the chick. This reversal and redundancy might explain the absence of a neural crest phenotype in mice that lack Slug 72.In summary, Slug, and probably other factors as well, might have different roles in neural crest development in different species. Table 1 Steps in neural-crest induction Stage Process Genes/proteins Induction of neural plate and neural-plate border BMPs and BMP antagonists (Solid line indicates the strong inhibition of BMPs by their antagonists, which come from the organizer region and which induce the neural plate. Dotted line represents a weaker antagonism that induces the neural plate border.) Induction of neural crest potential as assayed by Slug expression Maintenance, proliferation and non-differentiation of neural crest precursors Wnts, FGFs and retinoic acid from epidermis or lateral mesoderm BMPs, Wnt1, Wnt3a and Pax3 in the dorsal neural tube Subdivision of neural crest/neural tube lineage Notch/Delta, Foxd3 Emigration of neural crest BMPs, Slug The proposed stage at which each process functions is indicated by the diagram in the left column, whereas genes/proteins that are implicated in each process are listed in the right column. Although processes are separated for clarity, they might actually occur simultaneously in the embryo. BMP, bone morphogenetic protein; FGF, fibroblast growth factor. NATURE REVIEWS GENETICS VOLUME 3 JUNE

8 Multistep model of neural crest induction Research in the past decade has made great strides towards understanding the molecular basis of neural crest induction. Although much remains unclear, a picture is nevertheless emerging of a continuing process that involves several signals that act at different stages. A model that lists these proposed stages and signals is described in TABLE 1. Briefly, the process begins with induction of the neural plate and its border, mediated, at least in part, by inhibition of BMP signalling. This border region acquires the potential to form neural crest as a result of additional signalling (by Wnts, FGFs or retinoic acid) from the epidermis, paraxial mesoderm or both. Neural crest potential must then be maintained, perhaps through the activity of BMPs in the dorsal neural tube. Multipotent precursors proliferate in response to Wnt1 and Wnt3a, but are prevented from differentiating prematurely by Pax3. Eventually, these precursors adopt the fate of either neural crest or dorsal neurons in a process that involves Notch signalling and Foxd3. Finally, BMPs and Slug are again important as neural crest cells emigrate from the neural tube. Although this model is useful for thinking about the steps that are involved in neural crest induction, it is not intended to be complete or inarguably accurate. Too much remains to be studied and too many controversies remain unresolved. One gaping hole is the absence of solid data about when each step of the process occurs (many might occur simultaneously) and when each gene acts. Another problem is how to reconcile conflicting data from different species; although species differences do exist, it is likely that much of the process is conserved, but it is difficult to compare results when experiments use different tissues at different stages. 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Anat. 199, (2001). 79. Neidert, A. H., Panopoulou, G. & Langeland, J. A. Amphioxus goosecoid and the evolution of the head organizer and prechordal plate. Evol. Dev. 2, (2000). Acknowledgements The authors thank T. Moreno for help with the figures, and M. García-Castro and M. Albrecht for critical reading of the manuscript. Online links DATABASES The following terms in this article are linked online to: LocusLink: BMP4 BMP7 Foxd3 Pax3 Snail snailhouse somitabun swirl/bmp2b Wingless Wnt1 Wnt3a WNT3A FURTHER INFORMATION Encyclopedia of Life Sciences: Neural crest: origin, migration and differentiation Vertebrate embryo: patterning the neural crest lineage Access to this interactive links box is free online. NATURE REVIEWS GENETICS VOLUME 3 JUNE