The Delayed Entry of Thoracic Neural Crest Cells into the Dorsolateral Path Is a Consequence of the Late Emigration of Melanogenic Neural Crest Cells from the Neural Tube

Neural crest cells migrate along two pathways in the trunk: the ventral path, between the neural tube and somite, and the dorsolateral path, between the somite and overlying ectoderm. In avian embryos, ventral migration precedes dorsolateral migration by nearly 24 h, and the onset of dorsolateral migration coincides with the cessation of ventral migration. Neural crest cells in the ventral path differentiate predominantly as neurons and glial cells of the peripheral nervous system, whereas those in the dorsolateral path give rise to the melanocytes of the skin. Thus, early- and late-migrating neural crest cells exhibit unique morphogenetic behaviors and give rise to different subsets of neural crest derivatives. Here we present evidence that these differences reflect the appearance of specified melanocyte precursors, or melanoblasts, from late- but not early-migrating neural crest cells. We demonstrate that serum from Smyth line (SL) chickens specifically immunolabels melanocyte precursors, or melanoblasts. Using SL serum as a marker, we first detect melanoblasts immediately dorsal and lateral to the neural tube beginning at stage 18, which is prior to the onset of dorsolateral migration. At later stages every neural crest cell in the dorsolateral path is SL-positive, demonstrating that only melanoblasts migrate dorsolaterally. Thus, melanoblast specification precedes dorsolateral migration, and only melanoblasts migrate dorsolaterally at the thoracic level. Together with previous work (Erickson, C. A., and Goins, T. L.,Development121, 915–924, 1995), these data argue that specification as a melanoblast is a prerequisite for dorsolateral migration. This conclusion suggested that the delay in dorsolateral migration (relative to ventral migration) may reflect a delay in the emigration of melanogenic neural crest cells from the neural tube. Several experiments support this hypothesis. There are no melanoblasts in the ventral path, as revealed by the absence of SL-positive cells in the ventral path, and neural crest cells isolated from the ventral path do not give rise to melanocytes when explanted in culture, suggesting that early, ventrally migrating neural crest cells are limited in their ability to differentiate as melanocytes. Similarly, neural crest cells that emigrate from the neural tubein vitroduring the first 6 h fail to give rise to any melanocytes or SL-positive melanoblasts, whereas neural crest cells that emigrate at progressively later times show a dramatic increase in melanogenesis under identical culture conditions. Thus, the timing of dorsolateral migration at the thoracic level is ultimately controlled by the late emigration of melanogenic neural crest cells from the neural tube.     

Control of neural crest cell dispersion in the trunk of the avian embryo

Many hypotheses have been advanced to explain the orientation and directional migration of neural crest cells. These include positive and negative chemotaxis, haptotaxis, galvanotaxis, and contact inhibition. To test directly the factors that may control the directional dispersion of the neural crest, I have employed a variety of grafting techniques in living embryos. In addition, time-lapse video microscopy has been used to study neural crest cells in tissue culture. Trunk neural crest cells normally disperse from their origin at the dorsal neural tube along two extracellular pathways. One pathway extends laterally between the ectoderm and somites. When either pigmented neural crest cells or neural crest cells isolated from 24-hr cultures are grafted into the space lateral to the somites, they migrate: (1) medially toward the neural tube in the space between the ectoderm and somites and (2) ventrally along intersomitic blood vessels. Once the grafted cells contact the posterior cardinal vein and dorsal aorta they migrate along both blood vessels for several somite lengths in the anterior-posterior axis. Neural crest cells grafted lateral to the somites do not immediately move laterally into the somatic mesoderm of the body wall or the limb. Dispersion of neural crest cells into the mesoderm occurs only after blood vessels and nerves have first invaded, which the grafted cells then follow. The other neural crest pathway extends ventrally alongside the neural tube in the intersomitic space. When neural crest cells were grafted to a ventral position, between the notochord and dorsal aorta, in this intersomitic pathway at the axial level of the last somite, the grafted cells migrate rapidly within 2 hr in two directions: (1) dorsally, in the intersomitic space, until the grafted cells contact the ventrally moving stream of the host neural crest and (2) laterally, along the dorsal aorta and endoderm. All of the above experiments indicate that neither a preestablished chemotactic nor adhesive (haptotactic) gradient exists in the embryo since the grafted neural crest cells will move in the reverse direction along these pathways toward the dorsal neural tube. For the same reason, these experiments also show that dispersal of the neural crest is not directed passively by other environmental controls, since the cells can clearly move counter to their usual pathway and against such putative passive mechanisms.(ABSTRACT TRUNCATED AT 400 WORDS)     

A vital dye analysis of the timing and pathways of avian trunk neural crest cell migration

To permit a more detailed analysis of neural crest cell migratory pathways in the chick embryo, neural crest cells were labelled with a nondeleterious membrane intercalating vital dye, DiI. All neural tube cells with endfeet in contact with the lumen, including premigratory neural crest cells, were labelled by pressure injecting a solution of DiI into the lumen of the neural tube. When assayed one to three days later, migrating neural crest cells, motor axons, and ventral root cells were the only cells types external to the neural tube labelled with DiI. During the neural crest cell migratory phase, distinctly labelled cells were found along: (1) a dorsolateral pathway, under the epidermis, as well adjacent to and intercalating through the dermamyotome; and (2) a ventral pathway, through the rostral portion of each sclerotome and around the dorsal aorta as described previously. In contrast to those cells migrating through the sclerotome, labelled cells on the dorsolateral pathway were not segmentally arranged along the rostrocaudal axis. DiI-labelled cells were observed in all truncal neural crest derivatives, including subepidermal presumptive pigment cells, dorsal root ganglia, and sympathetic ganglia. By varying the stage at which the injection was performed, neural crest cell emigration at the level of the wing bud was shown to occur from stage 13 through stage 22. In addition, neural crest cells were found to populate their derivatives in a ventral-to-dorsal order, with the latest emigrating cells migrating exclusively along the dorsolateral pathway.     

Molecular identification of distinct neurogenic and melanogenic neural crest sublineages

Clonal and lineage analyses have demonstrated that although some neural crest cells have the ability to generate multiple cell types and display self-renewal ability, other crest cells generate a single or limited repertoire of cell types. However, it is not yet clear when, and in what order, crest cells become specified to adopt a particular fate. We report that the receptor tyrosine kinases TrkC and C-Kit are expressed by distinct neural crest subpopulations in vitro. We then analyzed the lineages of individual receptor-expressing crest cells and found that TrkC-expressing cells that have just emerged from the neural tube give rise to clones containing neurons or glial cells, or both, but never produce melanocytes. A short time later, TrkC-expressing cells only generate pure neuronal clones. By contrast, from their earliest appearance in neural tube outgrowths, C-Kit-expressing cells invariably give rise to clones containing only melanocytes. Our results directly demonstrate that distinct neurogenic and melanogenic sublineages diverge before or soon after crest cells emerge from the neural tube, that fate-restricted precursors are present in nascent neural crest populations and that these sublineages can be distinguished by their cell type-specific expression of receptor tyrosine kinases.     

Fate map and morphogenesis of presumptive neural crest and dorsal neural tube

In contrast to the classical assumption that neural crest cells are induced in chick as the neural folds elevate, recent data suggest that they are already specified during gastrulation. This prompted us to map the origin of the neural crest and dorsal neural tube in the early avian embryo. Using a combination of focal dye injections and time-lapse imaging, we find that neural crest and dorsal neural tube precursors are present in a broad, crescent-shaped region of the gastrula. Surprisingly, static fate maps together with dynamic confocal imaging reveal that the neural plate border is considerably broader and extends more caudally than expected. Interestingly, we find that the position of the presumptive neural crest broadly correlates with the BMP4 expression domain from gastrula to neurula stages. Some degree of rostrocaudal patterning, albeit incomplete, is already evident in the gastrula. Time-lapse imaging studies show that the neural crest and dorsal neural tube precursors undergo choreographed movements that follow a spatiotemporal progression and include convergence and extension, reorientation, cell intermixing, and motility deep within the embryo. Through these rearrangement and reorganization movements, the neural crest and dorsal neural tube precursors become regionally segregated, coming to occupy predictable rostrocaudal positions along the embryonic axis. This regionalization occurs progressively and appears to be complete in the neurula by stage 7 at levels rostral to Hensen's node.     

Mapping of neural crest pathways in Xenopus laevis using inter- and intra-specific cell markers

This study examines the pathways of migration followed by neural crest cells in Xenopus embryos using two recently described cell marking techniques. The first is an interspecific chimera created by grafting Xenopus borealis cells into Xenopus laevis hosts. The cells of these closely related species can be distinguished by their nuclear dimorphism. The second type of marker is created by microinjection of lysinated dextrans into fertilized eggs which can then be used for intraspecific grafting. These recently developed fluorescent dyes are fixable and identifiable in both living and fixed embryos. After grafting labeled donor neural tubes into unlabeled host embryos, the distribution of neural crest cells at various stages after grafting was used to define the pathways of neural crest migration. To control for possible grafting artifacts, fluorescent lysinated dextran was injected into a single blastomere which gives rise to a large number of neural crest cells, thereby labeling the neural crest without grafting. By all three techniques, Xenopus neural crest cells were observed along two predominant pathways in the trunk. The majority of neural crest cells were observed along a "ventral" route, between the neural tube and somite, the notochord and somite, and along the dorsal mesentery. A second group of neural crest cells was observed "dorsally" where they populated the dorsal fin. A third minor "lateral" pathway was observed primarily in borealis/laevis chimerae and in blastomere-injected embryos; some neural crest cells were observed underneath the ectoderm lateral to the neural tube. Along the rostrocaudal axis, neural crest cells were not continuously distributed but were primarily located across from the caudal two-thirds of the somite. Fewer than 3% of the neural crest cells were observed across from the rostral third of each somite. When grafted to ventral locations, neural crest cells were not able to migrate dorsally but migrated laterally along the dorsal mesentery. Labeled neural crest cells gave rise to cells of the spinal, sympathetic, and enteric ganglia as well as to adrenal chromaffin cells, Schwann cells, pigment cells, mesenchymal cells of the dorsal fin, and some cells in the integuments and in the region of the pronephros. These results show that the neural crest migratory pathways in Xenopus differ from those in the avian embryo. In avians NC cells migrate as a closely associated sheet of cells while in Xenopus they migrate as individual cells. Both species exhibit a metamerism in the neural crest cell distribution pattern along the rostrocaudal axis.(ABSTRACT TRUNCATED AT 400 WORDS)     

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.     

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.     

The developmental potentials of the caudalmost part of the neural crest are restricted to melanocytes and glia

The avian spinal cord is characterized by an absence of motor nerves and sensory nerves and ganglia at its caudalmost part. Since peripheral sensory neurons derive from neural crest cells, three basic mechanisms could account for this feature: (i) the caudalmost neural tube does not generate any neural crest cells; (ii) neural crest cells originating from the caudal part of the neural tube cannot give rise to dorsal root ganglia or (iii) the caudal environment is not permissive for the formation of dorsal root ganglia. To solve this problem, we have first studied the pattern of expression of ventral (HNF3beta) and dorsal (slug) marker genes in the caudal region of the neural tube; in a second approach, we have recorded the emergence of neural crest cells using the HNK1 monoclonal antibody; and finally, we have analyzed the developmental potentials of neural crest cells arising from the caudalmost part of the neural tube in avian embryo in in vitro culture and by means of heterotopic transplantations in vivo. We show here that neural crest cells arising from the neural tube located at the level of somites 47-53 can differentiate both in vitro and in vivo into melanocytes and Schwann cells but not into neurons. Furthermore, the neural tube located caudally to the last pair of somites (i.e. the 53rd pair) does not give rise to neural crest cells in any of the situations tested. The specific anatomical aspect of the avian spinal cord can thus be accounted for by limited developmental potentials of neural crest cells arising from the most caudal part of the neural tube.     

The dorsal neural tube: A dynamic setting for cell fate decisions

The dorsal neural tube first generates neural crest cells that exit the neural primordium following an epithelial‐to‐mesenchymal conversion to become sympathetic ganglia, Schwann cells, dorsal root sensory ganglia, and melanocytes of the skin. Following the end of crest emigration, the dorsal midline of the neural tube becomes the roof plate, a signaling center for the organization of dorsal neuronal cell types. Recent lineage analysis performed before the onset of crest delamination revealed that the dorsal tube is a highly dynamic region sequentially traversed by fate‐restricted crest progenitors. Furthermore, prospective roof plate cells were shown to originate ventral to presumptive crest and to progressively relocate dorsalward to occupy their definitive midline position following crest delamination. These data raise important questions regarding the mechanisms of cell emigration in relation to fate acquisition, and suggest the possibility that spatial and/or temporal information in the dorsal neural tube determines initial segregation of neural crest cells into their derivatives. In addition, they emphasize the need to address what controls the end of neural crest production and consequent roof plate formation, a fundamental issue for understanding the separation between central and peripheral lineages during development of the nervous system. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 796–812, 2010.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Dual origin of melanocytes defined by Sox1 expression and their region‐specific distribution in mammalian skin

Melanocytes are pigment‐producing cells generated from neural crest cells (NCCs) that delaminate from the dorsal neural tube. The widely accepted premise that NCCs migrating along the dorsolateral pathway are the main source of melanocytes in the skin was recently challenged by the finding that Schwann cell precursors are the major cellular source of melanocytes in the skin. Still, in a wide variety of vertebrate embryos, melanocytes are exclusively derived from NCCs. In this study, we show that a NCC population that is not derived from Sox1⁺ dorsal neuroepithelial cells but are derived from Sox1^? cells differentiate into a significant population of melanocytes in the skin of mice. Later, these Sox1^? cells clearly segregate from cells that originated from Sox1⁺ dorsal neuroepithelial cell‐derived NCCs. The possible derivation of Sox1^? cells from epidermal cells also strengthens their non‐neuroepithelial origin.     

Formation of the dorsal root ganglia in the avian embryo: Segmental origin and migratory behavior of neural crest progenitor cells

The segmental origin and migratory pattern of neural crest cells at the trunk level of avian embryos was studied, with special emphasis on the formation of the dorsal root ganglia (DRG) which organize in the anterior half of each somite. Neural crest cells were visualized using the quail-chick marker and HNK-1 immunofluorescence. The migratory process turned out to be closely correlated with somitic development: when the somites are epithelial in structure few labeled cells were found in a dorsolateral position on the neural tube, uniformly distributed along the craniocaudal axis. Following somitic dissociation into dermomyotome and sclerotome labeled cells follow defined migratory pathways restricted to each anterior somitic half. In contrast, opposite the posterior half of the somites, cells remain grouped in a dorsolateral position on the neural tube. The fate of crest cells originating at the level of the posterior somitic half was investigated by grafting into chick hosts short segments of quail neural primordium, which ended at mid-somitic or at intersomitic levels. It was found that neural crest cells arising opposite the posterior somitic half participate in the formation of the DRG and Schwann cells lining the dorsal and ventral root fibers of the same somitic level as well as of the subsequent one, whereas those cells originating from levels facing the anterior half of a somite participate in the formation of the corresponding DRG. Moreover, crest cells from both segmental halves segregate within each ganglion in a distinct topographical arrangement which reflects their segmental origin on the neural primordium. Labeled cells which relocate from posterior into anterior somitic regions migrate longitudinally along the neural tube. Longitudinal migration of neural crest cells was first observed when the somites are epithelial in structure and is completed after the disappearance of the last cells from the posterior somitic region at a stage corresponding to the organogenesis of the DRG.     

The influence of the metameric pattern in the mesoderm on migration of cranial neural crest cells in the chick embryo

In recent studies of chick embryos, the cranial paraxial mesoblast was found to be patterned into segmental units termed somitomeres. Anterior to the first segmental cleft, seven contiguous segments are aligned, with somitomeric interfaces forming grooves at right angles to the midline. In this study, the morphological relationship between the migratory pathways of cranial neural crest cells and patterned primary mesenchyme was analyzed with the scanning electron microscope, utilizing stereo imaging. In addition, the development of neuromeres in the adjacent neural tube was monitored. It was found that cranial neural crest first appears along the dorsal midline as a ridge of cells which loosens from the wall of the neural tube and migrates laterally as discrete populations. The mesencephalic crest appears first, immediately following neural tube fusion at that level, and migrates over the dorsal surface of the adjacent third somitomere and into the grooves formed by its juncture with the second and fourth somitomeres. Later, the addition of prosencephalic and rostral rhombencephalic crest extends the mesencephalic population to form a shelf of crest which spreads over the dorsal surface of the first four somitomeres. Component cells of this most cranial crest shelf become oriented and mimic the metameric pattern of the subjacent somitomeres. Crest cells adjacent to the fifth somitomeres appear along the midline, but do not migrate, creating a gap anterior to the otic crest. By stage 9, a narrow finger-like segment of the otic crest migrates from a specific neuromere into the grooved interface between the fifth and sixth somitomeres, delimiting the rostral border of the otic placode in the ectoderm above. By the end of stage 9, crest cells delimiting the caudal border of the placode have migrated along the interface of the seventh and eighth somitomeres. The crest cells adjacent to the sixth and seventh somitomeres, between the rostral and caudal otic populations, appear but do not migrate, remaining condensed along the midline. Thus, otic crest cells form a ring which circumscribes the invaginating otic placode. This study suggests that the precise distribution of cranial neural crest cells may result from their introduction at specific times, as specific populations from specific brain regions (neuromeres), onto a patterned mesodermal layer.     

Behavior of neural crest cells on embryonic basal laminae

Neural crest cells separate from the neural epithelium in a region devoid of a basal lamina and migrate along pathways bordered by intact basal laminae. The distribution of basal laminae suggests that they might have an important role in the morphogenesis of the neural crest by acting as a barrier to migration. The experiments reported here have tested directly whether neural crest cells can penetrate a basal lamina. Isolated neural tubes, neural crest cells cultured for 24 hr, or pigmented neural crest cells were explanted onto human placental amnions from which the epithelium had been removed to expose the basal lamina. In no case did neural crest cells or crest derivatives penetrate the basal lamina to invade the underlying stroma. If crest cells were grown on the stromal side of the amnion, they invaded the connective tissue. Pigmented neural crest derivative and [3H]thymidine-labeled nonpigmented crest cells were also confronted with chick embryonic basal laminae by grafting the cells into the lumen of the neural tube at the axial levels where host crest migration had commenced. Most of the grafted cells invaded the neural epithelium and accumulated after 24 hr at the basal surface of the neural tube. A few crest cells escaped through the dorsal surface of the neural tube and entered the overlying ectoderm, presumably through the wound created during the grafting procedure. Some of these grafted cells, located initially by light microscopy, were examined at the higher magnification and resolution offered by the transmission electron microscope to determine the relationship of the grafted cells to the basal lamina. In 50% (14 total) of the cases, the crest cells never reached the basal lamina of the neural tube, but were trapped by cell junctions between the neural epithelial cells. Of the remaining grafted cells that were relocated in the TEM (50%, total 15) all were spread on the basal lamina, but were not seen penetrating it. Likewise, in the three cases where crest cells were found in the epidermal ectoderm, all were in contact with the basal lamina of the ectoderm but did not have any processes extending through it. In three cases, at the level of the light microscope, crest cells were found to extend through the basal surface of the neural tube. In all these instances, the cells followed the dorsal root nerve exiting through a region of the neural tube that is devoid of a basal lamina.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular origin and developmental mechanisms during the formation of skin melanocytes

Melanocytes are derived from the neural crest (NC), which are transient multipotent cells arising by delamination from the developing dorsal neural tube. During recent years, signaling systems and molecular mechanisms of melanocyte development have been studied in detail, but the exact diversification of the NC into melanocytes and how they migrate, expand and disperse in the skin have not been fully understood. The recent finding that Schwann cell precursors (SCPs) of the growing nerve represents a stem cell niche from which various cell types, including Schwann cells, endoneural fibroblasts and melanocytes arise has exposed new knowledge on the cellular basis for melanocyte development. This opens for the identification of new factors and reinterpretation of old data on cell fate instructive, proliferative, survival and cell homing factors participating in melanocyte development.     

The early migration of neural crest cells in the trunk region of the avian embryo: an electron microscopic study

Four phases of neural crest migration characteristic of early avian trunk regions are described: (a) appearance, during which crest cells reside in the dorsal neural tube, but are separated from each other dorsally by large spaces; (b) condensation, during which large spaces between the crest cells become reduced, the cells elongate, flatten upon the surface of the neural tube, and become oriented tangentially (i.e., with their long axes perpendicular to the longitudinal axes of the neural tube); (c) early migration, during which the crest population expands uniformly to meet the dorsal apex of the somites; and (d) advanced migration, during which crest cells appear in the extracellular space dorsal to the somites. At the most advanced phases, the crest population at the dorsal midline decreased in number, with a concomitant loss of tangential orientation and the appearance of spaces between the cells. Extracellular components of the acellular spaces through which crest cells migrate are also described. The observations are discussed in terms of (1) those morphological changes undergone by crest cells during migration, and (2) possible factors that might delimit crest pathways. It is suggested that the operation of contact inhibition of movement within the crest population is sufficient to determine the direction of crest migration.     

Regulation of the onset of neural crest migration by coordinated activity of BMP4 and Noggin in the dorsal neural tube.

For neural crest cells to engage in migration, it is necessary that epithelial premigratory crest cells convert into mesenchyme. The mechanisms that trigger cell delamination from the dorsal neural tube remain poorly understood. We find that, in 15- to 40-somite-stage avian embryos, BMP4 mRNA is homogeneously distributed along the longitudinal extent of the dorsal neural tube, whereas its specific inhibitor noggin exists in a gradient of expression that decreases caudorostrally. This rostralward reduction in signal intensity coincides with the onset of emigration of neural crest cells. Hence, we hypothesized that an interplay between Noggin and BMP4 in the dorsal tube generates graded concentrations of the latter that in turn triggers the delamination of neural crest progenitors. Consistent with this suggestion, disruption of the gradient by grafting Noggin-producing cells dorsal to the neural tube at levels opposite the segmental plate or newly formed somites, inhibited emigration of HNK-1-positive crest cells, which instead accumulated within the dorsal tube. Similar results were obtained with explanted neural tubes from the same somitic levels exposed to Noggin. Exposure to Follistatin, however, had no effect. The Noggin-dependent inhibition was overcome by concomitant treatment with BMP4, which when added alone, also accelerated cell emigration compared to untreated controls. Furthermore, the observed inhibition of neural crest emigration in vivo was preceded by a partial or total reduction in the expression of cadherin-6B and rhoB but not in the expression of slug mRNA or protein. Altogether, these results suggest that a coordinated activity of Noggin and BMP4 in the dorsal neural tube triggers delamination of specified, slug-expressing neural crest cells. Thus, BMPs play multiple and discernible roles at sequential stages of neural crest ontogeny, from specification through delamination and later differentiation of specific neural crest derivatives.