Vital dye analysis of cranial neural crest cell migration in the mouse embryo.

The spatial and temporal aspects of cranial neural crest cell migration in the mouse are poorly understood because of technical limitations. No reliable cell markers are available and vital staining of embryos in culture has had limited success because they develop normally for only 24 hours. Here, we circumvent these problems by combining vital dye labelling with exo utero embryological techniques. To define better the nature of cranial neural crest cell migration in the mouse embryo, premigratory cranial neural crest cells were labelled by injecting DiI into the amniotic cavity on embryonic day 8. Embryos, allowed to develop an additional 1 to 5 days exo utero in the mother before analysis, showed distinct and characteristic patterns of cranial neural crest cell migration at the different axial levels. Neural crest cells arising at the level of the forebrain migrated ventrally in a contiguous stream through the mesenchyme between the eye and the diencephalon. In the region of the midbrain, the cells migrated ventrolaterally as dispersed cells through the mesenchyme bordered by the lateral surface of the mesencephalon and the ectoderm. At the level of the hindbrain, neural crest cells migrated ventrolaterally in three subectodermal streams that were segmentally distributed. Each stream extended from the dorsal portion of the neural tube into the distal portion of the adjacent branchial arch. The order in which cranial neural crest cells populate their derivatives was determined by labelling embryos at different stages of development. Cranial neural crest cells populated their derivatives in a ventral-to-dorsal order, similar to the pattern observed at trunk levels. In order to confirm and extend the findings obtained with exo utero embryos, DiI (1,1-dioctadecyl-3,3,3',3'-tetramethylindo-carbocyanine perchlorate) was applied focally to the neural folds of embryos, which were then cultured for 24 hours. Because the culture technique permitted increased control of the timing and location of the DiI injection, it was possible to determine the duration of cranial neural crest cell emigration from the neural tube. Cranial neural crest cell emigration from the neural folds was completed by the 11-somite stage in the region of the rostral hindbrain, the 14-somite stage in the regions of the midbrain and caudal hindbrain and not until the 16-somite stage in the region of the forebrain. At each level, the time between the earliest and latest neural crest cells to emigrate from the neural tube appeared to be 9 hours.(ABSTRACT TRUNCATED AT 400 WORDS)     

Effects of antibodies against N-cadherin and N-CAM on the cranial neural crest and neural tube.

We have examined the distribution and function of the defined cell adhesion molecules, N-cadherin and N-CAM, in the emigration of cranial neural crest cells from the neural tube in vivo. By immunocytochemical analysis, both N-cadherin and N-CAM were detected on the cranial neural folds prior to neural tube closure. After closure of the neural tube, presumptive cranial neural crest cells within the dorsal aspect of the neural tube had bright N-CAM and weak N-cadherin immunoreactivity. By the 10- to 11-somite stage, N-cadherin was prominent on all neural tube cells with the exception of the dorsal-most cells, which had little or no detectable immunoreactivity. N-CAM, but not N-cadherin, was observed on some migrating neural crest cells after their departure from the cranial neural tube. To examine the functional significance of these molecules, perturbation experiments were performed by injecting antibodies against N-CAM or N-cadherin into the cranial mesenchyme adjacent to the midbrain. Fab' fragments or whole IgGs of monoclonal and polyclonal antibodies against N-CAM caused abnormalities in the cranial neural tube and neural crest. Predominantly observed defects included neural crest cells in ectopic locations, both within and external to the neural tube, and mildly deformed neural tubes containing some dissociating cells. A monoclonal antibody against N-cadherin also disrupted cranial development, with the major defect being grossly distorted neural tubes and some ectopic neural crest cells outside of the neural tube. In contrast, nonblocking N-CAM antibodies and control IgGs had few effects. Embryos appeared to be sensitive to the N-CAM and N-cadherin antibodies for a limited developmental period from the neural fold to the 9-somite stage, with older embryos no longer displaying defects after antibody injection. These results suggest that the cell adhesion molecules N-CAM and N-cadherin are important for the normal integrity of the cranial neural tube and for the emigration of neural crest cells. Because cell-matrix interactions also are required for proper emigration of cranial neural crest cells, the results suggest that the balance between cell-cell and cell-matrix adhesion may be critical for this process.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: (1) what establishes the pathways of migration? And (2), what controls the final destination and differentiation of various neural crest subpopulations? These questions will be addressed in this Review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk.The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: (1) the vagal-level neural crest cells exhibit modest developmental bias; (2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively and (3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but take the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.Key words: neural crest, morphogenesis, cell migration, chicken embryo, fate restriction, vagal neural crest, pathways     

Immunohistochemical localisation of chondroitin sulphate proteoglycans and the effects of chondroitinase ABC in 9- to 11-day rat embryos

Studies on cell behaviour in vitro have indicated that the chondroitin sulphate proteoglycan (CSPG) family of molecules can participate in the control of cell proliferation, differentiation and adhesion, but its morphogenetic functions had not been investigated in intact embryos. Chondroitin/chondroitin sulphates have been identified in rat embryos at low levels at the start of neurulation (day 9) and at much higher levels on day 10. In this study we have sought evidence for the morphogenetic functions of CSPGs in rat embryos during the period of neurulation and neural crest cell migration by a combination of two approaches: immunocytochemical localization of CSPG by means of an antibody, CS-56, to the chondroitin sulphate component of CSPG, and exposure of embryos to the enzyme chondroitinase ABC. Staining of the CS-56 epitope was poor at the beginning of cranial neurulation; bright staining was at first confined to the primary mesenchyme under the convex neural folds late on day 9. In day 10 embryos, all mesenchyme cells were stained, but at different levels of intensity, so that primary mesenchyme, neural crest and sclerotomal cells could be distinguished from each other. Basement membranes were also stained, particularly bright staining being present where two epithelial were basally apposed, e.g., neural/surface ectoderms, dorsal aorta/neural tube, prior to migration of a population of cells between them. Staining within the neural epithelium was first confined to the dorsolateral edge region, and associated with the onset of neural crest cell emigration; after neural tube closure, neuroepithelial staining was more general. Neural crest cells were stained during migration, but the reaction was absent in areas associated with migration end-points (trigeminal ganglion anlagen, frontonasal mesenchyme). Embryos exposed to chondroitinase ABC in culture showed no abnormalities until early day 10, when cranial neural crest cell emigration from the neural epithelium was inhibited and neural tube closure was retarded. Sclerotomal cells failed to take their normal pathway between the dorsal aorta and neural tube. Correlation of the results of these two methods suggests: (1) that by decreasing adhesiveness within the neural epithelium at specific stages, CSPG facilitates the emigration of neural crest cells and the migratory movement of neuroblasts, and may also provide increased flexibility during the generation of epithelial curvatures; (2) that by decreasing the adhesiveness of fibronectin-containing extracellular matrices, CSPG facilitates the migration of neural crest and sclerotomal cells. This second function is particularly important when migrating cells take pathways between previously apposed tissues.     

Regional differences in neural crest morphogenesis

Neural crest cells are pluripotent cells that emerge from the neural epithelium, migrate extensively, and differentiate into numerous derivatives, including neurons, glial cells, pigment cells and connective tissue. Major questions concerning their morphogenesis include: 1) what establishes the pathways of migration and 2) what controls the final destination and differentiation of various neural crest subpopulations. These questions will be addressed in this review. Neural crest cells from the trunk level have been explored most extensively. Studies show that melanoblasts are specified shortly after they depart from the neural tube, and this specification directs their migration into the dorsolateral pathway. We also consider other reports that present strong evidence for ventrally migrating neural crest cells being similarly fate restricted. Cranial neural crest cells have been less analyzed in this regard but the preponderance of evidence indicates that either the cranial neural crest cells are not fate-restricted, or are extremely plastic in their developmental capability and that specification does not control pathfinding. Thus, the guidance mechanisms that control cranial neural crest migration and their behavior vary significantly from the trunk. The vagal neural crest arises at the axial level between the cranial and trunk neural crest and represents a transitional cell population between the head and trunk neural crest. We summarize new data to support this claim. In particular, we show that: 1) the vagal-level neural crest cells exhibit modest developmental bias; 2) there are differences in the migratory behavior between the anterior and the posterior vagal neural crest cells reminiscent of the cranial and the trunk neural crest, respectively; 3) the vagal neural crest cells take the dorsolateral pathway to the pharyngeal arches and the heart, but the ventral pathway to the peripheral nervous system and the gut. However, these pathways are not rigidly specified because of prior fate restriction. Understanding the molecular, cellular and behavioral differences between these three populations of neural crest cells will be of enormous assistance when trying to understand the evolution of the neck.     

Anterior/Posterior Influences on Neural Crest-Derived Pigment Cell Differentiation

The neural crest of vertebrate embryos has been used to elucidate steps involved in early embryonic cellular processes such as differentiation and migration. Neural crest cells form a ridge along the dorsal midline and subsequently they migrate throughout the embryo and differentiate into a wide variety of cell types. Intrinsic factors and environmental cues distributed along the neural tube, along the migratory pathways, and/or at the location of arrest influence the fate of neural crest cells. Although premigratory cells of the cranial and trunk neural crest exhibit differences in their differentiation potentials, premigratory trunk neural crest cells are generally assumed to have equivalent developmental potentials. Axolotl neural crest cells from different regions of origin, different stages of development, and challenged with different culture media have been analyzed for differentiation preferences pertaining to the pigment cell lineages. We report region-dependent differentiation of chromatophores from trunk neural crest at two developmental stages. Also, dosage with guanosine produces region-specific influences on the production of xanthophores from wild-type embryos. Our results support the hypothesis that spatial and temporal differences among premigratory trunk neural crest cells found along the anteroposterior axis influence developmental potentials and diminish the equivalency of axolotl neural crest cells.     

Origins and Developmental Potential of the Neural Crest

Neural crest cells are a migratory population that forms most of the peripheral nervous system, facial skeleton, and numerous other derivatives. These cells arise from the neural ectoderm and are first recognizable as discrete cells after neural tube closure. In this review, I summarize the results of studies from our laboratory on neural crest cell lineage and origin. Our recent experiments demonstrate that interactions between the presumptive neural plate and the nonneural ectoderm are likely to be instrumental in the induction of the avian neural crest. Juxtaposition of these tissues at early stages results in the formation of neural crest cells at the interface. However, neural crest cells do not appear to be segregated from other neuroepithelial cells; cell lineage studies have demonstrated that individual precursor cells within the neural tube can give rise to both neural crest and neural tube derivatives as diverse as sensory, commissural, and motor neurons. This suggests that individual neuroectodermal cells are multipotent, such that a precursor within the neural tube has the ability to form both neural tube (central nervous system) and neural crest (peripheral nervous system and other) derivatives. Further support for flexibility in the developmental program of neuroepithelial cells comes from experiments in which the cranial neural folds are ablated; this results in regulation by the remaining ventral neural tube cells to form neural crest cells after the endogenous neural crest is removed. At later stage of development, this regulative capacity is lost. Following their emigration from the neural tube, neural crest cells become progressively restricted to defined embryonic states. Taken together, these experiments demonstrate that: (1) the neural crest is an induced population that arises by interactions within the ectoderm; (2) initially, progenitor cells are multipotent, having the potential to form multiple neural crest and neural tube derivatives; and (3) with time, the precursors become progressively restricted to form neural crest derivatives and eventually to individual phenotypes.     

Cranial neural crest migration: New rules for an old road

The neural crest serve as an excellent model to better understand mechanisms of embryonic cell migration. Cell tracing studies have shown that cranial neural crest cells (CNCCs) emerge from the dorsal neural tube in a rostrocaudal manner and are spatially distributed along stereotypical, long distance migratory routes to precise targets in the head and branchial arches. Although the CNCC migratory pattern is a beautifully choreographed and programmed invasion, the underlying orchestration of molecular events is not well known. For example, it is still unclear how single CNCCs react to signals that direct their choice of direction and how groups of CNCCs coordinate their interactions to arrive at a target in an ordered manner. In this review, we discuss recent cellular and molecular discoveries of the CNCC migratory pattern. We focus on events from the time when CNCCs encounter the tissue adjacent to the neural tube and their travel through different microenvironments and into the branchial arches. We describe the patterning of discrete cell migratory streams that emerge from the hindbrain, rhombomere (r) segments r1-r7, and the signals that coordinate directed migration. We propose a model that attempts to unify many complex events that establish the CNCC migratory pattern, and based on this model we integrate information between cranial and trunk neural crest development.     

Wnt and BMP signaling govern lineage segregation of melanocytes in the avian embryo

Recent studies show that specification of some neural crest lineages occurs prior to or at the time of migration from the neural tube. We investigated what signaling events establish the melanocyte lineage, which has been shown to migrate from the trunk neural tube after the neuronal and glial lineages. Using in situ hybridization, we find that, although Wnts are expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, the Wnt inhibitor cfrzb-1 is expressed in the neuronal and glial precursors and not in melanoblasts. This expression pattern suggests that Wnt signaling may be involved in specifying the melanocyte lineage. We further report that Wnt-3a-conditioned medium dramatically increases the number of pigment cells in quail neural crest cultures while decreasing the number of neurons and glial cells, without affecting proliferation. Conversely, BMP-4 is expressed in the dorsal neural tube throughout the time when neural crest cells are migrating, but is decreased coincident with the timing of melanoblast migration. This expression pattern suggests that BMP signaling may be involved in neural and glial cell differentiation or repression of melanogenesis. Purified BMP-4 reduces the number of pigment cells in culture while increasing the number of neurons and glial cells, also without affecting proliferation. Our data suggest that Wnt signaling specifies melanocytes at the expense of the neuronal and glial lineages, and further, that Wnt and BMP signaling have antagonistic functions in the specification of the trunk neural crest.     

Division of labor during trunk neural crest development

Neural crest cells, the migratory precursors of numerous cell types including the vertebrate peripheral nervous system, arise in the dorsal neural tube and follow prescribed routes into the embryonic periphery. While the timing and location of neural crest migratory pathways has been well documented in the trunk, a comprehensive collection of signals that guides neural crest migration along these paths has only recently been established. In this review, we outline the molecular cascade of events during trunk neural crest development. After describing the sequential routes taken by trunk neural crest cells, we consider the guidance cues that pattern these neural crest trajectories. We pay particular attention to segmental neural crest development and the steps and signals that generate a metameric peripheral nervous system, attempting to reconcile conflicting observations in chick and mouse. Finally, we compare cranial and trunk neural crest development in order to highlight common themes.     

Neural crest cell migration in the developing embryo

In vertebrate embryos, neural crest cells migrate extensively to defined sites where they differentiate into a complex array of derivatives, ranging from neurons to pigment cells. Neural crest cells emerge uniformly from the neural tube but their subsequent migratory pattern is segmented along much of the body axis. What factors control this segmental migration? At trunk levels, it is imposed by the intrinsic segmentation of the neighbouring somitic mesoderm, while in the head, intrinsic information within the neural tube as well as extrinsic influences from the ectoderm are involved. A variety of cell-cell and cell-extracellular matrix interactions are thought to influence initiation and movement of neural crest cells. This review summarizes recent progress from both experimental embryology and cell biology approaches in uncovering the mechanisms underlying neural crest cell migration.     

Evidence for collapsin-1 functioning in the control of neural crest migration in both trunk and hindbrain regions

Collapsin-1 belongs to the Semaphorin family of molecules, several members of which have been implicated in the co-ordination of axon growth and guidance. Collapsin-1 can function as a selective chemorepellent for sensory neurons, however, its early expression within the somites and the cranial neural tube (Shepherd, I., Luo, Y. , Raper, J. A. and Chang, S. (1996) Dev. Biol. 173, 185-199) suggest that it might contribute to the control of additional developmental processes in the chick. We now report a detailed study on the expression of collapsin-1 as well as on the distribution of collapsin-1-binding sites in regions where neural crest cell migration occurs. collapsin-1 expression is detected in regions bordering neural crest migration pathways in both the trunk and hindbrain regions and a receptor for collapsin-1, neuropilin-1, is expressed by migrating crest cells derived from both regions. When added to crest cells in vitro, a collapsin-1-Fc chimeric protein induces morphological changes similar to those seen in neuronal growth cones. In order to test the function of collapsin-1 on the migration of neural crest cells, an in vitro assay was used in which collapsin-1-Fc was immobilised in alternating stripes consisting of collapsin-Fc/fibronectin versus fibronectin alone. Explanted neural crest cells derived from both trunk and hindbrain regions avoided the collapsin-Fc-containing substratum. These results suggest that collapsin-1 signalling can contribute to the patterning of neural crest cell migration in the developing chick.     

Alterations in neural crest migration by a monoclonal antibody that affects cell adhesion

The possible role of a 140-kD cell surface complex in neural crest adhesion and migration was examined using a monoclonal antibody JG22, first described by Greve and Gottlieb (1982, J. Cell. Biochem. 18:221-229). The addition of JG22 to neural crest cells in vitro caused a rapid change in morphology of cells plated on either fibronectin or laminin substrates. The cells became round and phase bright, often detaching from the dish or forming aggregates of rounded cells. Other tissues such as somites, notochords, and neural tubes were unaffected by the antibody in vitro even though the JG22 antigen is detectable in embryonic tissue sections on the surface of the myotome, neural tube, and notochord. The effects of the JG22 on neural crest migration in vivo were examined by a new perturbation approach in which both the antibody and the hybridoma cells were microinjected onto neural crest pathways. Hybridoma cells were labeled with a fluorescent cell marker that is nondeleterious and that is preserved after fixation and tissue sectioning. The JG22 antibody and hybridoma cells caused a marked reduction in cranial neural crest migration, a build-up of neural crest cells within the lumen of the neural tube, and some migration along aberrant pathways. Neural crest migration in the trunk was affected to a much lesser extent. In both cranial and trunk regions, a cell free zone of one or more cell diameters was generally observed between neural crest cells and the JG22 hybridoma cells. Two other monoclonal antibodies, 1-B and 1-N, were used as controls. Both 1-B and 1-N bind to bands of the 140-kD complex precipitated by JG22. Neither control antibody affected neural crest adhesion in vitro or neural crest migration in situ. This suggests that the observed alterations in neural crest migration are due to a functional block of the 140-kD complex.     

Environmental influences on neural crest cell migration

Neural crest cells migrate extensively and interact with numerous tissues and extracellular matrix components during their movement. Cell marking techniques have shown that neural crest cells in the trunk of the avian embryo migrate through the anterior, but not posterior, half of each sclerotome and avoid the region around the notochord. A possible mechanism to account for this migratory pattern is that neural crest cells may be inhibited from entering the posterior sclerotome and the perinotochordal space. Thus, interactions with other tissue may prescribe the pattern of neural crest cell migration in the trunk. In contrast, interactions between neural crest cells and the extracellular matrix may mediate the primary interactions controlling neural crest cells migration in the head region. © 1993 John Wiley & Sons, Inc.     

Transforming growth factor-beta control of cell-substratum adhesion during avian neural crest cell emigration in vitro.

It has been proposed that, in higher vertebrates, the onset of neural crest cell migration from the neural tube involves spatially and temporally coordinated changes in cellular adhesiveness that are under the control of external signals released in the extracellular milieu by neighboring tissues. In the present study, we have analyzed the dynamics of changes in cell-substratum adhesiveness during crest cell emigration and searched for regulatory cues using an in vitro model system. This model is based on the fact that, in vivo, crest cell dispersion occurs gradually along a rostrocaudal wave, allowing us to explant portions of the neural axis, termed migratory and premigratory levels, that differ in the time in culture at which neural crest cells initiate migration and in the locomotory behavior of the cells. We found that neural crest cell emigration is not triggered by the main extracellular matrix molecules present in the migratory pathways, as none of these molecules could abolish the intrinsic difference in the timing of emigration between the different axial levels. Using an in vitro adhesion assay, we found that presumptive neural crest cells from premigratory level explants gradually acquired the ability to respond to extracellular matrix material with time in culture, suggesting that acquisition of appropriate, functional integrin receptors was a necessary step for migration. Finally, we showed that members of the transforming growth factor-beta family reduced in a dose-dependent manner the delay of neural crest cell emigration from premigratory level explants and were able to increase significantly the substratum-adhesion properties of crest cells. Our results suggest that acquisition of substratum adhesion by presumptive neural crest cells is a key event during their dispersion from the neural tube in vitro, and that members of the transforming growth factor-beta family may act as potent inducers of crest cell emigration, possibly by increasing the substratum adhesion of the cells.     

Integrin alpha5beta1 supports the migration of Xenopus cranial neural crest on fibronectin

During early embryonic development, cranial neural crest cells emerge from the developing mid- and hindbrain. While numerous studies have focused on integrin involvement in trunk neural crest cell migration, comparatively little is known about mechanisms of cranial neural crest cell migration. We show that fibronectin, but not laminin, vitronectin, or type I collagen can support cranial neural crest cell migration and segmentation in vitro. These behaviors require both the RGD and "synergy" sites located within the central cell-binding domain of fibronectin. While these two sites are sufficient for cranial neural crest cell migration, we find that the second Heparin-binding domain of fibronectin can provide additional support for cranial neural crest cell migration in vitro. Finally, using a function blocking monoclonal antibody, we show that cranial neural crest cell migration on fibronectin requires the integrin alpha5beta1.     

Abnormalities in neural crest cell migration in laminin alpha5 mutant mice

Although numerous in vitro experiments suggest that extracellular matrix molecules like laminin can influence neural crest migration, little is known about their function in the embryo. Here, we show that laminin alpha5, a gene up-regulated during neural crest induction, is localized in regions of newly formed cranial and trunk neural folds and adjacent neural crest migratory pathways in a manner largely conserved between chick and mouse. In laminin alpha5 mutant mice, neural crest migratory streams appear expanded in width compared to wild type. Conversely, neural folds exposed to laminin alpha5 in vitro show a reduction by half in the number of migratory neural crest cells. During gangliogenesis, laminin alpha5 mutants exhibit defects in condensing cranial sensory and trunk sympathetic ganglia. However, ganglia apparently recover at later stages. These data suggest that the laminin alpha5 subunit functions as a cue that restricts neural crest cells, focusing their migratory pathways and condensation into ganglia. Thus, it is required for proper migration and timely differentiation of some neural crest populations.     

Patterning of the cranial nerves in the chick embryo is dependent on cranial mesoderm and rhombomeric metamerism

The vertebrate peripheral nervous system (PNS) consists of two groups of nerves that have a metamerical series of proximal roots along the body axis: the branchial and spinal nerves. Spinal nerve metamerism is brought about by the presence of somites, while that of the branchial nerves is, in part, intrinsic to rhombomeres, the segmental compartments of the hind-brain. As the distribution pattern of neural crest cells prefigures the morphology of the PNS, we constructed tissue-recombinant chick embryos in order to determine factors that might regulate the crest cell distribution pattern. When the segmental plate was transplanted between the hind-brain and the head mesoderm before crest cell emigration, it developed into ectopic somites that inhibited the dorsolateral migration of crest cells such that formation of the cranial nerve trunks was disturbed. Even so, proximal portions of the nerve roots were intact. An ectopic graft of lateral mesoderm did not inhibit the directional migration of the crest cells, but allowed their ectopic distribution, resulting in the fusion of cranial nerve trunks. When spinal neurectoderm was transplanted into the hind-brain, the graft behaved like an even-numbered rhombomere and caused the fusion of cranial nerve roots. The identity of the spinal neurectoderm was preserved in the ectopic site analyzed by the immunolocalization of Hoxb-5 protein, a spinal cord marker. We conclude that the spatial distribution of cephalic crest cells is regulated by successive processes that act on their proximal and distal distribution. The migratory behavior of crest cells is achieved partly by an embryonic environment that is dependent upon the presence of somitomeres, which do not epithelialize as somites, in the trunk.