Specification of neural crest cell formation and migration in mouse embryos

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1 Seminars in Cell & Developmental Biology 16 (2005) Review Specification of neural crest cell formation and migration in mouse embryos Paul A. Trainor Stowers Institute for Medical Research, 1000 E. 50th Street, Kansas City, MO 64110, USA Available online 25 July 2005 Abstract Of all the model organisms used to study human development, rodents such as mice most accurately reflect human craniofacial development. Collective advances in mouse embryology and mouse genetics continue to shape our understanding of neural crest cell development and by extrapolation the etiology of human congenital head and facial birth defects. The aim of this review is to highlight the considerable progress being made in our understanding of cranial neural crest cell patterning in mouse embryos Elsevier Ltd. All rights reserved. Keywords: Mouse; Neural crest; Craniofacial; Induction; Migration Contents 1. Introduction Mouse neural crest cells: formation, migration, differentiation Specification of mouse neural crest cell formation Specification of mouse neural crest cell migration Specification of multipotency versus restricted potency of mouse neural crest cells Conclusions Acknowledgements References Introduction The craniofacial complex is anatomically the most sophisticated part of the body and to function properly it requires the orchestrated integration of the viscerocranium and neurocranium, the central and peripheral nervous systems, facial muscles, connective tissue, vasculature and dermis. Not surprisingly this integration often goes awry such that craniofacial abnormalities are the most common congenital malformation constituting at least a third of all birth defects. Neural crest cells, are a migratory stem cell population that Tel.: ; fax: address: pat@stowers-institute.org. form very early during craniofacial development and they generate the majority of the cartilage, bone, connective and peripheral nerve tissue in the head (Fig. 1). Not only are neural crest cells crucial to head development but they are also synonymous with vertebrate craniofacial evolution. Craniofacial abnormalities are largely attributed to defects in the formation, migration and differentiation of neural crest cells and the origins of particular congenital syndromes can be traced back specifically to problems in one or more of these phases of neural crest cell development. For example, First Arch syndrome broadly describes craniofacial abnormalities characterized by malformation of the eyes, ears, lower jaw and palate. Treacher Collins syndrome and Pierre Robin syndrome are two of the more extreme examples falling into this /$ see front matter 2005 Elsevier Ltd. All rights reserved. doi: /j.semcdb

2 684 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) Fig. 1. Migration and differentiation of neural crest cells. Sox10 in situ hybridization (purple stain) labels the distinct segregated streams of neural crest cells as they leave the neural tube of mouse embryos at 8.5 dpc (A) and as they condense to contribute to the cranial ganglia at 9.5 dpc (B). Neurofilament immunohistochemistry highlights the differentiation of neural crest cells in the cranial ganglia into sensory neurons that project axons into the branchial arches at 10.5 dpc (C). Neural crest cells also give rise to the majority of the cartilage (blue) and bone (red) that constitute the viscerocranium and neurocranium (D, sagittal view; E, superior view). category. The clinical abnormalities associated with Treacher Collins and Pierre Robin syndromes are thought to arise due to defects in the migration of neural crest cells. In contrast, craniosynostoses, which are characterized by the premature fusion of the bony plates in the skull are related to problems with neural crest cell differentiation [1 3]. Consequently it is important to understand the distinct mechanisms, which regulate the formation, migration and differentiation of neural crest cells. 2. Mouse neural crest cells: formation, migration, differentiation Murine neural crest cells are generated transiently along almost the entire vertebrate axis at the interface between the surface ectoderm and the neural plate of the embryo, in a region that is referred to as the neural plate border. During this induction process, neuroepithelial cells undergo an epithelial to mesenchymal transformation at which point they delaminate and begin to emigrate from the neural tube, a process that requires significant cytoarchitectural and cell adhesive changes. The induction of the neural crest cells is typically assayed by the expression of members of the Snail (Snail and Slug) zinc-finger transcription factors gene family [4,5], which play key roles in the epithelial to mesenchymal transformation process by repressing the cell adhesion molecule E-cadherin [6]. Snail/Slug transcription factors are among the earliest known markers of neural crest cell formation and the onset of their expression has been used to study the spatial and temporal competence of the neural plate to initiate neural crest induction in response to different signals. Murine neural crest cell formation and migration commences at approximately the 4 5 somite stage in the region of the caudal midbrain and rostral hindbrain [7] and proceeds simultaneously as a wave rostrally towards the forebrain and caudally towards the tail (Fig. 1). In avian embryos neural crest cell migration commences after neural tube closure however this is not the case in mammalian embryos such as mice where neural crest cell formation and migration commences well before fusion of the bilateral halves of the neural plate. Typically there is a narrow temporal window during which neural crest cells are induced to delaminate and emigrate from the dorsal neural tube and although this period varies between species, in mice it typically lasts mice 7 9 h at each axial level [8]. The neural crest can be subdivided rostrocaudally into at least four distinct major axial populations; cranial, cardiac, vagal and trunk, each of which migrates along unique pathways, contributing to specific cell and tissue types that are characteristic of their axial level of origin. The cranial neural crest, which is the focus of this review can be divided into forebrain, midbrain and hindbrain domains of migrating neural crest cells. Cranial neural crest cells do not appear to migrate randomly, rather they follow precise, species and region specific pathways moving subectodermally over the surface of the cranial mesoderm [8 10].

3 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) Cranial neural crest cells typically migrate in discrete segregated streams, the pattern of which is highly conserved in vertebrate species as disparate as amphibians, teleosts, avians, marsupials and mammals (reviewed in [11]). Briefly, forebrain and rostral midbrain neural crest cells colonise the frontonasal and periocular regions. Caudal midbrain derived neural crest cells populate the maxillary component of the first branchial arch [9,10]. The hindbrain is divided into seven distinct segments known as rhombomeres [12] and neural crest cells emigrate from each of the rhombomeres, but predominantly from rhombomeres 2, 4 and 6 in discrete segregated streams that populate the first, second and third branchial arches respectively [9,10]. Exquisite fate mapping analyses particularly in avians using the quail-chick chimera system, have revealed that neural crest cells derived from each axial region of the cranial neural plate and in particular from each individual rhombomere generate specific and unique components of the craniofacial complex including the viscero- and neurocraniums as well as the peripheral nervous system [13 16]. Despite the generation of lineage fate maps in mice [9,10], the limitations of mouse embryo culture prevented a long-term differentiation analysis of the extensive fates of murine neural crest cells although obvious parallels were drawn with avians. Recently however, genetic recombination experiments finally permitted the long-term tracing of murine neural crest cells. Initially P0-cre mice were used in combination with chicken -actin conditional reporters, which confirmed much of what was expected in terms of the fates of mammalian neural crest cells [17]. However, P0-cre mediated recombination only occurred in subsets of neural crest cells and exhibited some ectopic expression leaving many questions in mammalian neural crest differentiation unanswered. Shortly thereafter, a more specific neural crest cell promoter from the Wnt1 gene was used in combination with ROSA26-lacZ conditional reporter mice to indelibly mark the progeny of the cranial and cardiac neural crest cells during embryonic, fetal and post-natal development [18 20]. Murine cranial neural crest cells contribute to the formation of the condensed dental mesenchyme, dental papilla, odontoblasts, dentine matrix, pulp, cementum, periodontal ligaments, chondrocytes in Meckel s cartilage, mandible, the articulating disk of the temporomandibular joint and the branchial arch nerve ganglia [20]. In the neurocranium, the meninges and frontal bones are derived from neural crest cells as is the suture mesenchyme, which separates the bony plates [18]. In the cardiac region, neural crest cells contribute to the aorticopulmonary septum, conotruncal cushions, and adult derivatives of the third, fourth and sixth pharyngeal arch arteries [19]. Not surprisingly, the overall patterns of migration and long-term differentiation are highly conserved between mammalian and avian embryos. Further refinements to lineage and fate determination in mammalian embryos in terms of using axial level specific promoters such as those that delineate a single rhombomere subpopulation of neural crest cells are no doubt in progress. These results are eagerly anticipated as they will substantially aid comparative analysis of neural crest cell patterning in diverse organisms and further our understanding of neural crest cell and craniofacial evolution. Collectively these analyses performed in mice together with those from avians, fish and frogs demonstrate that cardiac, vagal and trunk neural crest cells produce neurons, glial cells, secretory cells and pigment cells contributing to the peripheral nervous system, enteric nervous system, endocrine system and skin. Cranial neural crest cells however exhibit an even more surprising diversity of derivatives, giving rise to pigment cells, nerve ganglia, smooth muscle, and connective tissue, as well as most of the bone and cartilage of the head (Fig. 1). 3. Specification of mouse neural crest cell formation Neural crest cell formation requires contact mediated signals between the neural plate and paraxial tissues such as the surface ectoderm and/or the mesoderm [21]. Three key signaling pathways (BMP, FGF, Wnt) intersect at the neural plate border, each of which is critical for neural crest cell induction. Previously, BMP4/7 signaling in the ectoderm and neuroepithelium in the form of a morphogen gradient was considered to be the key factor in inducing neural crest cell formation [5,21 23]. However, more recently, the emphasis has shifted in favour of Wnt signaling from the surface and in chick embryos Wnt6 is key, since it is widely expressed in domains of epithelial to mesenchymal transitions during embryogenesis [24]. In contrast in zebrafish, Wnt8 signaling from the surface ectoderm is the main protagonist of neural crest cell induction [25]. In Xenopus, although Wnt signaling is also critical for neural crest cell induction, the situation is more complex since FGF signaling from the underlying mesoderm also independently induces neural crest cell formation [26 28]. The specifics of neural crest cell induction in these species are discussed in detail elsewhere in this volume, however, the contact mediated induction is considered to be a conserved feature of vertebrate neural crest cell formation as it has been rigorously tested in chick and frog embryos due to their amenability to tissue manipulation, recombination and explant culture. In the case of mammalian embryos, there is currently a paucity of data linking these different signaling pathways in neural crest cell induction mechanisms. Part of the problem is that many of the Wnt, Bmp and Fgf genes described as playing roles in neural crest cell induction in non-mammalian embryos also play key roles during gastrulation and/or neural plate induction. Therefore, many knockout models of these genes exhibit embryonic lethality prior to the onset of neural crest cell formation or no phenotype at this specific stage due to functional redundancy. For example, genetic ablation of Wnt1 or Wnt3a in mouse embryos failed to demonstrate a conserved role for Wnt signaling in murine neural crest cell induction. Wnt1 was shown to be important for mid-

4 686 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) brain patterning while Wnt3a was required for formation of the paraxial mesoderm [29,30]. However, in the absence of both Wnt1 and Wnt3a there is a marked deficiency in neural crest derivatives. Double mutant mice exhibit skeletal defects and a significant reduction in cranial and spinal sensory neurons as well as melanocytes. [31]. Further evidence of a role for Wnt signaling in neural crest cell determination came from targeted inactivation of downstream components of the Wnt signaling pathway. Inactivation of -catenin, in the dorsal neural tube of mouse embryos [32,33] or null mutations in APC [34] result in severe defects in cranial neural crest derivatives including the cranial and dorsal root ganglia and the craniofacial skeletal elements. To date there is no literature available describing a null mutation in Wnt6. This implies that Wnt signaling in mouse embryos appears to be more important for the lineage specification of neural crest cell differentiation rather than neural crest cell induction. In support of this idea, Wnt/ -catenin signal activation in emigrating neural crest stem cells (NCSCs), regulates cell fate decisions by promoting the formation of sensory neural cells in vivo at the expense of other neural crest derivatives [35]. Bmp4 and Bmp7 have been implicated in neural crest cell induction in avians [22,36], however mutations in Bmp4 or Bmp7 do not produce obvious deficiencies in murine cranial neural crest development [37 39]. Functional redundancy may explain the absence of an effect, however even mouse embryos mutant for both Bmp7 and Bmp5 also exhibit normal patterns of neural crest cell formation and migration [40]. Furthermore, Bmpr1a (Alk3) and Bmpr1b (Alk2), which are expressed in the neural tube of mouse embryos at the time of neural crest cell induction, do not affect the formation of neural crest cells when conditionally inactivated [41,42]. They do however affect the later differentiation of neural crest cells. In contrast to chick embryos, Bmp4 expression is not detected in the dorsal neural tube or surface ectoderm of mouse embryos during the formation of neural crest cells [43]. Rather Bmp2 is expressed in a small region of the surface ectoderm that abuts the neural plate suggesting that it may play a role in murine neural crest cell patterning [43,44]. In order to disrupt BMP signaling in premigratory and migratory neural crest cells, without the problems of functional redundancy and early embryonic lethality potentially, Xenopus noggin was expressed under the control of the Hoxa2 promoter in the hindbrain of transgenic mouse embryos [44]. Hoxa2 is expressed in the neural tube and in the second and third branchial arch mesenchyme. Consequently, noggin overexpression in the Hoxa2 domain led to a spatially specific depletion of second and third branchial arch neural crest derivatives which included components of the cranial ganglia, the stapes and styloid process, greater horn and body of the hyoid bone, thyroid and cricoid cartilages [44]. Underlying the malformation or absence of these skeletal elements was the observation that the migration of cranial neural crest cells was nearly completely abolished in the caudal branchial arch region. A role for BMP signaling and BMP2 in particular in neural crest cell formation in mice is further supported by analyses of Bmp2 null mutant mice which can survive to the somite stage [45]. These embryos exhibit no evidence of migrating neural crest cells as assayed by the expression of the neural crest cell marker Crabp1 [44]. We cannot conclusively rule out that neural crest cells formed but failed to migrate because markers of neural crest cell formation such as Snail were not used in this study. However, an absence of neural crest cell formation seems to be the most likely scenario, which suggests that roles for the surface ectoderm and also BMP signaling in neural crest cell formation are conserved even in mouse embryos. Interestingly, recent work in avians has reported an integration between BMP and Wnt signaling with the cell cycle during avian trunk neural crest cell induction [46]. The overexpression of noggin in the neural tube inhibits G1/S transition, which in turn abrogates BMP-induced neural crest cell delamination. Similarly, interfering with -catenin and LEF/TCF also inhibits G1/S transition and neural crest cell formation. Exogenous BMP can stimulate Wnt1 transcription, which implies that BMP signaling regulates G1/S transition and consequently, neural crest cell formation through the canonical Wnt signaling pathway [46]. It remains to be seen however if this mechanism also holds true in the mouse during neural crest cell induction. Apart from the Bmp2 mutants, currently there is at least one other clear example of murine neural crest cell induction failure and that occurs in a restricted domain in the Hoxa1/b1 double null mutant mice [47]. Hoxa1/b1 double mutants lack all second branchial arch skeletal elements and exhibit striking external and middle ear malformations. Lineage tracing in cultured double mutant embryos together with analyses of neural crest cell markers such as Sox10 revealed a complete absence of second branchial arch neural crest cells. What is significant about this particular study was the demonstration that wild type cells transplanted into the double mutant embryos could migrate into the second branchial arch. This suggested that there was no inherent defect in the endogenous migration pathway but rather that the neural crest cells at this axial level failed to form [47]. It is unlikely that Hox genes play a universal role in neural crest cell induction because they are not expressed throughout the anterior regions of the head where neural crest cells clearly form. Hox genes are clearly well recognized for their roles in anterior posterior patterning, however new roles are slowly being uncovered for these genes in dorso-ventral patterning as well [48].In the Hoxa1/b1 double mutants it therefore seems likely that the absence of neural crest cell formation was the secondary consequence of dorso-ventral patterning transformation defects which respecifies the dorsal neural plate border territory to a non-neural crest identity. Overall, there are surprisingly and disappointingly few mouse mutants that exhibit a clear absence of migrating neural crest cells. Snail/Slug are widely used as indicators of neural crest cell formation since they mark the epithelial to

5 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) mesenchymal transition that neural crest cells undergo during their formation and emigration from the neural tube. By directly repressing E-cadherin, Snail/Slug promotes the formation of neural crest cells and the onset of their migration from the neural tube [6]. Unfortunately, null mutations in Slug demonstrate that Slug is not required for neural crest cell generation [49]. This may possibly be explained by functional redundancy with Snail, however to date there is no literature describing a null allele of Snail or a phenotype with a defect in neural crest cell formation or migration. Although it is widely believed that the same contact mediated inductive mechanisms underlie murine neural crest cell formation, interactions between the neural ectoderm and surface ectoderm or the neural ectoderm and paraxial mesoderm have yet to be definitively tested because of the technical difficulty of manipulating mouse embryos due to their small size and complex in vitro culture requirements. 4. Specification of mouse neural crest cell migration One of the key steps in neural crest cell development is their migration from the neural tube through the extracellular matrix to their final destinations throughout the body [50]. The formation of neural crest cells is concomitant with the epithelial to mesenchymal transition of neuroepithelial cells and their emigration from the neural tube. Therefore, it is extremely difficult to distinguish between a failure in neural crest cell induction versus a complete failure of neural crest cell migration largely because the latter depends on the former. Currently, there does not appear to be a situation where neural crest cells are induced to form but fail to migrate and consequently most of our analyses of the specification of neural crest cell migration in mice relate to aberrant streams of neural crest cells in vivo and in vitro cell culture assays. As described above Snail/Slug are key regulators of both neural crest cell induction and migration. The inhibition of Slug blocks neural crest migration in Xenopus [51,52]. Similarly, antisense oligonucleotides directed against Slug in avian embryos also blocks the ability of neuroepithelial cells to emigrate from the neural tube [4]. Conversely, over-expression of Slug in the chick neural tube leads to an increased production of migrating neural crest cells [53]. The caveat of these analyses is that Slug/Snail are expressed in neural crest cells during their formation and also during their migration. What is still critically missing from analyses of neural crest cell determination in mammalian embryos is an assessment of the function of Slug/Snail during the migration of neural crest cells completely independently of their formation. To achieve this will require conditional or inducible inactivation of Slug/Snail in migrating neural crest cells in a strict spatiotemporally controlled manner. Neural crest cell delamination and migration from the neural tube is aided by the down-regulation of cell adhesion molecules such as NCAM, N-cadherin, and cadherin-6b and the up-regulation of cadherin-7 and cadherin-11 [6,54 57]. A breakdown of the cytoskeletal components in the basement membrane surrounding the neural tube where the crest cells emigrate is also required [58]. Hindbrain derived cranial neural crest cells migrate in distinct segregated streams adjacent to the even numbered rhombomeres and into the branchial arches in mouse embryos [10]. Consequently, neural crest free zones exist adjacent to rhombomeres 3 and 5 (Fig. 2). In contrast to the even numbered rhombomeres, we know from lineage tracing in mouse embryos that generally fewer neural crest cells emigrate from the odd numbered rhombomeres and also that rather than migrating laterally these neural crest cells migrate anteriorly or posteriorly to join the adjacent even rhombomere neural crest streams [43]. The segregation of these neural crest cell populations is critical to prevent fusions of the cranial ganglia and skeletal elements and also to prevent mixing of neural crest cells with different genetic constitutions, which as described below are critical for axial neural crest and craniofacial patterning. The presence of neural crest free zones and the segregation of neural crest cells into discrete streams is not an intrinsic property of the vertebrate hindbrain or the neural crest cells. In ErbB4 null mutant mice neural crest cells from rhombomere 4 acquire the ability to migrate through the dorsal mesenchyme adjacent to rhombomere 3, which is normally free of neural crest cells to join the first branchial arch stream of neural crest cells (Fig. 2) [59]. Consequently, this leads to fusions of the trigeminal and facio-acoustic ganglia. This aberrant rostral migration is not autonomous to the neural crest cells, since wild type neural crest cells transplanted into Erbb4 mutants also exhibit this aberrant migration. Conversely, when ErbB4 mutant neural crest cells are homotopically transplanted into wild type embryos they emulate the normal endogenous neural crest migratory pattern from rhombomere 4 into the second branchial arch [60]. The aberrant migration of neural crest cells in ErbB4 mutants is due to as yet unidentified changes in the paraxial mesenchyme environment. Since ErbB4 is normally expressed in rhombomeres 3 and 5 this phenotype reflects defects in signalling between the hindbrain and the adjacent environment branchial arches in mutant embryos. Fusions of the cranial ganglia have also been described in the neuregulin and Krox20 null mutant mice [61 63], however, currently no neural crest cell lineage tracing or gene expression data is available to confirm the suspicion that neural crest cells also migrate aberrantly in these mice. Interestingly, neuregulin is a ligand for ErbB4, so it is not surprising that it too may also play a role in regulating the segregated pathways of cranial neural crest cell migration. Coincidentally, Krox20 is expressed in rhombomeres 3 and 5, which overlaps with ErbB4. To date no direct link between Krox20 and ErbB4 has been established, but given that Krox20 is one of the earliest determinants of rhombomere 3 identity, it seems plausible (although not yet proven) that ErbB4 expression is disrupted in the Krox20 mutants which

6 688 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) Fig. 2. Normal and abnormal patterns of neural crest cell migration. Lineage tracing of neural crest cells in mouse embryos with DiI (fluorescent red dots) demonstrates that rhombomere 2 derived neural crest cells contribute to the first branchial arch (A and B) whereas rhombomere four derived neural crest cells contribute to the second branchial arch (C and D). Normally cranial neural crest cells migrate as distinct segregated streams into the adjacent branchial arches (E). Mixing between first and second branchial arch streams is observed dorsally adjacent to the neural tube in ErbB4 mutants (F). Extensive mixing of neural crest streams is observed in Twist mutants (G). Mixing is also observed in Tbx1 mutants but only ventrally in the vicinity of the first pharyngeal pouch (H). would account for the same ganglia fusions as observed in ErbB4 null mutant mice. Twist mutant embryos also exhibit aberrant neural crest cell migration such that second branchial arch neural crest cells stray from their subectodermal route towards the second branchial arch and invade the normally neural crest free mesenchyme adjacent to rhombomere 3 (Fig. 2) [64,65]. Consequently, Twist mutant mice also display fusions of the trigeminal and facio-acoustic ganglia, similar to the ErbB4 mutants [65]. Interestingly, the expression pattern of ErbB4 is normal in the Twist mutant hindbrain, which implies that Twist could act downstream of ErbB4 or be part of an additional important pathway that regulates the segregated migration pathways of cranial neural crest cells. It remains to be determined whether the mesodermal or neural expression domain of Twist regulates the migration patterns of cranial neural crest cells, but conditional Twist mutants will eventually answer this question. Previously it has been proposed that the cranial mesoderm influences the migration pathways of cranial neural crest cells in mouse embryos [43] and consequently this implies that not all craniofacial malformations arise due to defects intrinsic to the neural crest. Rather, primary defects in the cranial mesoderm or other tissues that the neural crest cells contact could lead to secondary abnormalities in both the migration and patterning of cranial neural crest cells [43,66]. This idea appears to be supported by neural crest cell migration anomalies in Tbx1 mutant mice (Fig. 2). In Tbx1 mutant embryos, patterning of the second and more caudal branchial arches are disrupted resulting in poor colonisation by neural crest cells. Consequently, a stream of Hoxa2 expressing neural crest cells originating from rhombomere four migrate aberrantly into the first branchial arch, which results in inner ear abnormalities [67]. Tbx1 is not expressed in neural crest cells but rather in the paraxial mesoderm and core regions of the branchial arches. This suggests that Tbx1 is acting noncell autonomously in the branchial region during craniofacial morphogenesis. This data also provocatively suggests that craniofacial malformations such as those seen in DiGeorge syndrome in which Tbx1 mutations are implicated may occur as a secondary consequence of defects in neural crest cell migration. The primary defect possibly lies in the mesoderm or endoderm, which are the main regions of Tbx1 expression. These results demonstrate that ErbB4, Twist and Tbx1 may all be acting non-cell autonomously in directing the migration pathways of cranial neural crest cells and to date surprisingly few molecules that intrinsically influence the path finding of murine cranial neural crest cells have been identified. Evidence obtained primarily from analyses in frog embryos suggests that bi-directional Eph/ephrin cell signalling plays an important intrinsic role in keeping the neural crest cell streams segregated [68]. This is also supported by a recent study in mouse embryos, where it was recently shown that ephrinb1 acts cell autonomously in neural crest cells to regulate craniofacial development [69]. EphrinB1 deficient mice exhibit high degree of cleft palate and tympanic ring defects and the neural crest cells from the post-otic region of the hindbrain display aberrant migration by invading territories

7 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) normally devoid of neural crest cells which ultimately led to nerve fasciculation and branching defects. Each of the studies detailed above describe subpopulations of neural crest cells migrating aberrantly but they do not reveal the underlying mechanism that controls neural crest cell migration along there normal pathways. This instead appears to be a morphogen regulated event. BMPs are not only critical for neural crest induction, but they are also involved in neural crest migration. In mouse embryos in which Alk2 (Bmpr1b) is conditionally deleted from neural crest cells, the initial formation and migration of neural crest cells remains unaffected. However, the later migration of mutant neural crest cells to the outflow tract of the heart is dramatically impaired [41]. The effect of BMP signaling on changes in cell adhesion and the cytoskeletal components within the neural tube remains to be clearly established, however one target of BMP signaling is the GTPbinding protein, rhob [70]. Ectopic expression of noggin in the avian neural tube, leads to a reduction in the expression of rhob as well as cadherin-6b which prevents neural crest emigration from the dorsal neuroepithelium [71]. Collectively, these studies support a role for BMP in neural crest migration from the neural tube through the regulation of rhob and cadherins. Although a role for FGF signaling in murine neural crest cell formation has not been determined, in vitro migration assays have revealed that exogenous FGF2 (basic FGF) and FGF8 exhibit a chemo-attractive activity that influences the migration of mesencephalic mouse neural crest cells [72]. Anti-FGF2 and anti-fgf8 neutralising antibodies inhibit the chemotaxic response. In vivo, FGF2 activity is detected predominantly in target regions such as the mandibular mesenchyme, which is colonised by mesencephalic neural crest cells. This characteristic distribution supports the notion that FGF2 acts as a chemo-attractant in the mouse embryo that directs mesencephalic neural crest cell migration. The domains of FGF8 activity are largely restricted to the branchial arch epithelium suggesting it may not be involved in guiding neural crest cell migration, however FGF2 activity can be promoted by FGF8. This has led to the hypothesis that FGF8 activity in the mandibular arch epithelium is a prerequisite for the differential localisation of FGF2 and that in turn, the distribution of FGF2 is essential for chemotaxis of mesencephalic neural crest cell migration [72]. In vivo this is not the case since the conditional inactivation of Fgf8 in the branchial arch epithelium does not disrupt the migration of cranial neural crest cells into the branchial arches. Instead FGF8 activity in the branchial arch surface ectoderm appears to play an important functional role in neural crest cell survival [73]. Further evidence for a role of FGF signaling in neural crest cell migration has come from analyses of the Fgfr1 null mutant mice. Fgfr1 is expressed in multiple cellular components of the branchial arches [74] and recent studies have shown that perturbation of Fgfr1 function during branchial arch development results in failure of the neural crest to enter the second arch [75]. The hindbrain derived neural crest cells migrate toward the second branchial arch, but failing to enter, they instead accumulate in a region proximal to the second branchial arch. While this defect in neural crest migration does not reflect a neural crest cell specific function of Fgfr1, the second branchial arch exhibited by these mutants appear to result from abnormal patterning during early development of the branchial arch region. The initial generation of neural crest and the segmentation and patterning of the hindbrain appear normal in these mutants. Taken together, this suggests Fgfr1 patterns the branchial arch region creating permissive environments that enable neural crest cell migration. Cranial neural crest cells are also susceptible to retinoic acid-mediated teratogenesis [76] and in vitro and in vivo studies have demonstrated that retinoic acid interferes with neural crest migration [77 81]. Targeted inactivation of the mouse retinaldehyde dehydrogenase 2 (Raldh2), the enzyme responsible for early embryonic retinoic acid synthesis, leads to prenatal death from a lack of aorticopulmonary septation [82]. Raldh2 null embryos exhibit impaired development of their posterior (third to sixth) branchial arches. Post-otic neural crest cells fail to establish segmental migratory pathways and are misrouted caudally. The disruption of cardiac neural crest cell migration leads to the absence of outflow tract septation. Similar defects in vagal neural crest leads to agenesis of the enteric ganglia, a condition reminiscent of Hirschprung s disease in humans. Raldh2 expression is restricted to the posterior most pharyngeal mesoderm, highlighting the potential importance of the mesoderm during the migration phase of neural crest cell development. In support of these genetic studies, treating headfold-stage mouse embryos with a pan- RAR antagonist in vitro and in vivo results in a complete absence of the third and fourth branchial arches [83]. Consequently, neural crest cells normally destined for the third and fourth arches migrate ectopically. Further roles for retinoic acid in the migration of neural crest cells have been observed in a stage-dependent manner in rat embryos [80]. Early-stage retinoic acid treatment induced an ectopic caudal migration of the anterior hindbrain (rhombomeres (r) 1 and 2) crest cells into the second branchial arch and acousticofacial ganglion. In contrast, late-stage treatment did not disturb the segmental migration pattern of hindbrainderived crest cells, although it did induce branchial arch fusions. Anterior hindbrain-derived neural crest cells populated the anterior half of the fused arch while neural crest cells derived from the pre-otic hindbrain (r3 and r4) occupied its posterior half [80]. Similar treatments of mouse embryos with retinoic acid in culture result in the ectopic migration of second arch neural crest cells into the first arch [84], which interestingly mimics the Tbx1 mutant neural crest cell migration phenotype. One of the potential targets of retinoic acid is the c-jun N-terminal kinase (JNK) pathway [85]. Cardiac neural crest migration is inhibited when retinoic acid blocks JNK phosphorylation, which is critical for neural crest outgrowth. Fur-

8 690 P.A. Trainor / Seminars in Cell & Developmental Biology 16 (2005) thermore, the use of dominant-negative constructs to perturb upstream and downstream components of the JNK pathway also reduce cardiac neural crest outgrowth, implying that JNK is not only a target for the action of retinoic acid, but is also critical for the migration of cardiac neural crest cells in vitro. Neural crest cells express retinoic acid receptors (RARs) and cellular retinoic acid binding proteins (CRABPs) however the primary source of retinoic acid comes from the paraxial mesoderm. Collectively, these studies demonstrate that appropriate migration may not be an intrinsic property of neural crest cells but rather is directed non-cell autonomously in an extremely intricate fashion that requires complex interactions between signals from the neuroepithelium, together with the paraxial mesoderm, ectoderm and endoderm tissues [43,59]. 5. Specification of multipotency versus restricted potency of mouse neural crest cells One of the many intriguing features of neural crest cell is their pluripotency. A single neural crest cell will differentiate into any of several distinct cell types depending on its location within the embryo. However, it is still not certain whether most individual neural crest cells that leave the neural tube are pluripotent or whether the majority of the population is already restricted to certain fates. Some of the strongest evidence for pluripotency comes from single neuroepithelial cell labeling in chick embryos, which revealed that progeny of a single neural crest cell could become sensory neurons, melanocytes, adrenomedullary cells and also glia [86,87]. Similar results were obtained from in vitro clonal assays in mice [88], where it was demonstrated that these cells have the capacity to self renew and form neurons, glia and smooth muscle [89]. Consequently they have been termed neural crest stem cells [90]. In contrast analyses in zebrafish provide equally strong evidence for restricted lineage determination since similar labeling experiments revealed that most neural crest cells differentiate into only one cell type [91,92]. Evidence in mouse embryos suggests that some populations of neural crest cells are committed at the time of emigration from the neural tube or very soon after since they express transcription factors that constrain the cell types they can produce [93,94]. Using the Cre recombinase system to permanently mark subsets of neural crest cells that transiently expressed the transcription factor Ngn2 or Wnt1 revealed that Ngn2 positive progenitors were four-fold more likely than Wnt1 positive cells to contribute to sensory and sympathetic ganglia [95]. Within the dorsal route ganglia however, both Ngn2 and Wnt1 positive populations were equally likely to generate neurons or glia. Hence this suggests that very early in neural crest migration, Ngn2 may mark a sub-population that is already fate biased. Furthermore, this data also implies that in the trunk neural crest cells become restricted to sensory or autonomic lineages before committing to either neuronal or glial fates. This issue of multipotency versus lineage restriction has also been addressed with respect to melanocytes. A subpopulation of cells in the dorsomedial domain of the neural tube, which expresses the receptor tyrosine kinase, Kit, migrates exclusively into the developing dermis [96]. Consequently, these cells activate definitive melanocyte lineage markers indicating they are melanocyte progenitor cells. This particular sub-population is generated predominantly at the midbrain hindbrain junction and at the cervical and lower trunk levels. Other cells within the dorsal neural tube that are Kit negative, express p75 and migrate ventrally giving rise to neurons and glia. Interestingly, the p75 positive cells are located ventrolateral to the Kit positive cells, suggesting that there is in vivo neural crest cell lineage segregation within the mouse neural tube [96]. This poses an important question that currently remains unresolved and that is what are the signaling pathways that establish this lineage segregation during the period of neural crest cell formation? Is the interface between FGF, WNT and BMP signaling at the neural plate border only stimulating the induction of neural crest cells or do each of these signaling pathways have the capacity to specify sublineages within the neural crest. 6. Conclusions Mammalian neural crest cells are a mulitpotent migratory stem cell population that generates an impressively broad array of cell and tissue types during craniofacial development. Currently, BMP2 signaling specifically from the surface ectoderm immediately adjacent to the neural plate appears to be the key player in murine neural crest cell induction. This is somewhat different to chick, frog and zebrafish embryos where the emphasis lies with Wnt signaling. Collectively, BMP, Wnt and FGF signaling all play key roles in regulating either early or late steps in neural crest cell migration. Surprisingly, these signaling pathways also mediate major aspects of neural crest cell differentiation. Hence, repeated use of the same signaling pathways elicits completely different effects on neural crest cell formation, migration and differentiation. The studies described in this review highlight the complexity in regulating neural crest cell patterning and the balance between signals acquired in the neuroepithelium during formation versus signals received from the extrinsic tissue environments during migration. This intricate regulation affects not only the migratory properties of neural crest cells but also dramatically influences their differentiation and has been crucial to vertebrate neural crest and craniofacial evolution. Acknowledgements P.A.T. is supported by research funds from the Stowers Institute, a Basil O Connor Research Scholar Award (#5- FY03-16) from the March of Dimes and grant RO1 DE from the National Institute of Dental and Craniofacial Research.

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