The development and distribution of the cranial neural crest in the rat embryo

Summary The head region of rat embryos was investigated by scanning electron microscopy after removal of the surface ectoderm with adhesive tape. Observations were made in embryos from 6-somite to 11-somite stages of development, in order to determine: (1) the sequence of emigration of neural crest cells from the different regions of the future brain; (2) the appearance of crest cells before, during, and after their conversion from an epithelial to a mesenchymal form; (3) the migration pathways.Emigration occurs first from the midbrain, and next from the rostral hindbrain; crest cells from these two regions migrate into the first visceral arch. Subsequently cells emigrate from the caudal hindbrain, but not in a rostrocaudal sequence. At the time of crest cell emigration, the neural fold morphology varies from a slightly convex, widely open plate (midbrain) to a closed tube (caudal hindbrain). Thus the timing of emigration is related neither to age (as reflected in rostrocaudal levels) nor to morphology of the neural epithelium.     

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.     

Cardiac neural crest of the mouse embryo: axial level of origin, migratory pathway and cell autonomy of the splotch (Sp2H) mutant effect

A sub-population of the neural crest is known to play a crucial role in development of the cardiac outflow tract. Studies in avians have mapped the complete migratory pathways taken by 'cardiac' neural crest cells en route from the neural tube to the developing heart. A cardiac neural crest lineage is also known to exist in mammals, although detailed information on its axial level of origin and migratory pattern are lacking. We used focal cell labelling and orthotopic grafting, followed by whole embryo culture, to determine the spatio-temporal migratory pattern of cardiac neural crest in mouse embryos. Axial levels between the post-otic hindbrain and somite 4 contributed neural crest cells to the heart, with the neural tube opposite somite 2 being the most prolific source. Emigration of cardiac neural crest from the neural tube began at the 7-somite stage, with cells migrating in pathways dorsolateral to the somite, medial to the somite, and between somites. Subsequently, cardiac neural crest cells migrated through the peri-aortic mesenchyme, lateral to the pharynx, through pharyngeal arches 3, 4 and 6, and into the aortic sac. Colonisation of the outflow tract mesenchyme was detected at the 32-somite stage. Embryos homozygous for the Sp2H mutation show delayed onset of cardiac neural crest emigration, although the pathways of subsequent migration resembled wild type. The number of neural crest cells along the cardiac migratory pathway was significantly reduced in Sp2H/Sp2H embryos. To resolve current controversy over the cell autonomy of the splotch cardiac neural crest defect, we performed reciprocal grafts of premigratory neural crest between wild type and splotch embryos. Sp2H/Sp2H cells migrated normally in the +/+ environment, and +/+ cells migrated normally in the Sp2H/Sp2H environment. In contrast, retarded migration along the cardiac route occurred when either Sp2H/+ or Sp2H/Sp2H neural crest cells were grafted into the Sp2H/Sp2H environment. We conclude that the retardation of cardiac neural crest migration in splotch mutant embryos requires the genetic defect in both neural crest cells and their migratory environment.     

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.     

Cell lineage in mammalian craniofacial mesenchyme

We have analysed the contributions of neural crest and mesoderm to mammalian craniofacial mesenchyme and its derivatives by cell lineage tracing experiments in mouse embryos, using the permanent genetic markers Wnt1-cre for neural crest and Mesp1-cre for mesoderm, combined with the Rosa26 reporter. At the end of neural crest cell migration (E9.5) the two patterns are reciprocal, with a mutual boundary just posterior to the eye. Mesodermal cells expressing endothelial markers (angioblasts) are found not to respect this boundary; they are associated with the migrating neural crest from the 5-somite stage, and by E9.5 they form a pre-endothelial meshwork throughout the cranial mesenchyme. Mesodermal cells of the myogenic lineage also migrate with neural crest cells, as the branchial arches form. By E17.5 the neural crest-mesoderm boundary in the subectodermal mesenchyme becomes out of register with that of the underlying skeletogenic layer, which is between the frontal and parietal bones. At E13.5 the primordia of these bones lie basolateral to the brain, extending towards the vertex of the skull during the following 4-5 days. We used DiI labelling of the bone primordia in ex-utero E13.5 embryos to distinguish between two possibilities for the origin of the frontal and parietal bones: (1) recruitment from adjacent connective tissue or (2) proliferation of the original primordia. The results clearly demonstrated that the bone primordia extend vertically by intrinsic growth, without detectable recruitment of adjacent mesenchymal cells.     

Defects in pathfinding by cranial neural crest cells in mice lacking the neuregulin receptor ErbB4

Mouse embryos with a loss-of-function mutation in the gene encoding the receptor tyrosine kinase ErbB4 exhibit misprojections of cranial sensory ganglion afferent axons. Here we analyse ErbB4-deficient mice, and find that morphological differences between wild-type and mutant cranial ganglia correlate with aberrant migration of a subpopulation of hindbrain-derived cranial neural crest cells within the paraxial mesenchyme environment. In transplantation experiments using new grafting techniques in cultured mouse embryos, we determine that this phenotype is non-cell-autonomous: wild-type and mutant neural crest cells both migrate in a pattern consistent with the host environment, deviating from their normal pathway only when transplanted into mutant embryos. ErbB4 signalling events within the hindbrain therefore provide patterning information to cranial paraxial mesenchyme that is essential for the proper migration of neural crest cells.     

Signalling between the hindbrain and paraxial tissues dictates neural crest migration pathways.

Cranial neural crest cells are a pluripotent population of cells derived from the neural tube that migrate into the branchial arches to generate the distinctive bone, connective tissue and peripheral nervous system components characteristic of the vertebrate head. The highly conserved segmental organisation of the vertebrate hindbrain plays an important role in patterning the pathways of neural crest cell migration and in generating the distinct or separate streams of crest cells that form unique structures in each arch. We have used focal injections of DiI into the developing mouse hindbrain in combination with in vitro whole embryo culture to map the patterns of cranial neural crest cell migration into the developing branchial arches. Our results show that mouse hindbrain-derived neural crest cells migrate in three segregated streams adjacent to the even-numbered rhombomeres into the branchial arches, and each stream contains contributions of cells from three rhombomeres in a pattern very similar to that observed in the chick embryo. There are clear neural crest-free zones adjacent to r3 and r5. Furthermore, using grafting and lineage-tracing techniques in cultured mouse embryos to investigate the differential ability of odd and even-numbered segments to generate neural crest cells, we find that odd and even segments have an intrinsic ability to produce equivalent numbers of neural crest cells. This implies that inter-rhombomeric signalling is less important than combinatorial interactions between the hindbrain and the adjacent arch environment in specific regions, in the process of restricting the generation and migration of neural crest cells. This creates crest-free territories and suggests that tissue interactions established during development and patterning of the branchial arches may set up signals that the neural plate is primed to interpret during the progressive events leading to the delamination and migration of neural crest cells. Using interspecies grafting experiments between mouse and chick embryos, we have shown that this process forms part of a conserved mechanism for generating neural crest-free zones and contributing to the separation of migrating crest populations with distinct Hox expression during vertebrate head development.     

Effect of hyaluronidase treatment on the distribution of cranial neural crest cells in the chick embryo

The cranial paraxial mesoblast is patterned into segmental units termed somitomeres. Recently we demonstrated the morphological relationship between the migratory pathways of cranial neural crest cells and the patterned primary mesenchyme of chick embryos (Anderson and Meier, '81). Since extracellular matrix, particularly hyaluronate, is also distributed in cranial crest pathways, embryos were given sub-blastodisc injections of hyaluronidase just prior to neural tube fusion and neural crest migration to remove matrix. Histological sections of enzyme-treated embryos showed that Alcian blue staining of hyaluronate was significantly reduced. Surface ectoderm appeared collapsed on the subjacent mesoderm as well. Examination of embryos with the scanning electron microscope (SEM) revealed that paraxial mesoderm remained segmentally patterned even though it appeared more condensed because of a reduction in intercellular space between mesenchymal cells. In enzyme-treated embryos, the rostral crest cells spread over the dorsal surfaces of the first four somitomeres, as they would do normally. This distribution of neural crest cells occurs even when enzyme treatment interferes with neural tube fusion at that level. We conclude that 1) neural tube fusion is not a prerequisite for the timely release of cranial crest in the chick embryo and 2) that much of the organized hyaluronate-rich matrix that lies in the path of cranial crest is not essential for crest emigration or patterned distribution.     

A monoclonal antibody against a laminin-heparan sulfate proteoglycan complex perturbs cranial neural crest migration in vivo

INO (inhibitor of neurite outgrowth) is a monoclonal antibody that blocks axon outgrowth, presumably by functionally blocking a laminin-heparan sulfate proteoglycan complex (Chiu, A. Y., W. D. Matthew, and P. H. Patterson. 1986. J. Cell Biol. 103: 1382-1398). Here the effect of this antibody on avian neural crest cells was examined by microinjecting INO onto the pathways of cranial neural crest migration. After injection lateral to the mesencephalic neural tube, the antibody had a primarily unilateral distribution. INO binding was observed in the basal laminae surrounding the neural tube, ectoderm, and endoderm, as well as within the cranial mesenchyme on the injected side of the embryo. This staining pattern was indistinguishable from those observed with antibodies against laminin or heparan sulfate proteoglycan. The injected antibody remained detectable for 18 h after injection, with the intensity of immuno-reactivity decreasing with time. Embryos ranging from the neural fold stage to the 9-somite stage were injected with INO and subsequently allowed to survive for up to 1 d after injection. These embryos demonstrated severe abnormalities in cranial neural crest migration. The predominant defects were ectopic neural crest cells external to the neural tube, neural crest cells within the lumen of the neural tube, and neural tube deformities. In contrast, embryos injected with antibodies against laminin or heparan sulfate proteoglycan were unaffected. When embryos with ten or more somites were injected with INO, no effects were noted, suggesting that embryos are sensitive for only a limited time during their development. Immunoprecipitation of the INO antigen from 2-d chicken embryos revealed a 200-kD band characteristic of laminin and two broad smears between 180 and 85 kD, which were resolved into several bands at lower molecular mass after heparinase digestion. These results indicate that INO precipitates both laminin and proteoglycans bearing heparan sulfate residues. Thus, microinjection of INO causes functional blockage of a laminin-heparan sulfate proteoglycan complex, resulting in abnormal cranial neural crest migration. This is the first evidence that a laminin-heparan sulfate proteoglycan complex is involved in aspects of neural crest migration in vivo.     

The IFT-A complex regulates Shh signaling through cilia structure and membrane protein trafficking

Hectd1 mutant mouse embryos exhibit the neural tube defect exencephaly associated with abnormal cranial mesenchyme. Cellular rearrangements in cranial mesenchyme are essential during neurulation for elevation of the neural folds. Here we investigate the molecular basis of the abnormal behavior of Hectd1 mutant cranial mesenchyme. We demonstrate that Hectd1 is a functional ubiquitin ligase and that one of its substrates is Hsp90, a chaperone protein with both intra- and extracellular clients. Extracellular Hsp90 enhances migration of multiple cell types. In mutant cranial mesenchyme cells, both secretion of Hsp90 and emigration of cells from cranial mesenchyme explants were enhanced. Importantly, we show that this enhanced emigration was highly dependent on the excess Hsp90 secreted from mutant cells. Together, our data set forth a model whereby increased secretion of Hsp90 in the cranial mesenchyme of Hectd1 mutants is responsible, at least in part, for the altered organization and behavior of these cells and provides a potential molecular mechanism underlying the neural tube defect.     

Pathogenesis of methanol-induced craniofacial defects in C57BL/6J mice

BACKGROUND: Methanol administered to C57BL/6J mice during gastrulation causes severe craniofacial dysmorphology. We describe dysmorphogenesis, cell death, cell cycle assessment, and effects on development of cranial ganglia and nerves observed following administration of methanol to pregnant C57BL/6J mice on gestation day (GD) 7. METHODS: Mice were injected (i.p.) on GD 7 with 0, 2.3, 3.4, or 4.9 gm/kg methanol, split into two doses. In embryos of mice treated with 0 or 4.9 gm/kg methanol, we used histology and LysoTracker red staining on GD 8 0 hr through GD 8 18 hr to examine cell death and dysmorphogenesis, and we also evaluated cell-cycle distribution and proliferation using flow cytometry (FCM) and BrdU immunohistochemistry. On GD 10, we evaluated the effect of GD 7 exposure to 0, 2.3, 3.4, or 4.9 gm/kg methanol on cranial ganglia and nerve development using neurofilament immunohistochemistry. RESULTS: Methanol treatment on GD 7 resulted in reduced mesenchyme surrounding the fore- and midbrain, and in the first branchial arches, by GD 8 12 hr. There were disruptions in the forebrain neuroepithelium and optic pit. Neural crest cell emigration from the mid- and hindbrain region was reduced in methanol-exposed embryos. Methanol had no apparent effect on BrdU incorporation or cell-cycle distribution on GD 8. Cell death was observed in the hindbrain region along the path of neural crest migration and in the trigeminal ganglion on GD 8 18 hr. Development of the cranial ganglia and nerves was adversely affected by methanol. Development of ganglia V, VIII, and IX was decreased at all dosage levels; ganglion VII was reduced at 3.4 and 4.9 gm/kg, and ganglion X was reduced at 4.9 gm/kg. CONCLUSIONS: These results suggest that gastrulation-stage methanol exposure affects neural crest cells and the anterior mesoderm and neuroepithelium. Cell death was evident in areas of migrating neural crest cells, but only at time points after methanol was cleared from the embryo, suggesting an indirect effect on these cells. Birth Defects Research (Part A), 2004. Published 2004 Wiley-Liss, Inc.     

Cranial neural crest emergence and migration in the Mexican axolotl (Ambystoma mexicanum)

The timing and pattern of cranial neural crest cell emergence and migration in the Mexican axolotl, Ambystoma mexicanum, are assessed using scanning electron microscopy (SEM). Cranial neural crest cells emerge and begin to migrate at the time of neural fold closure and soon form three distinct streams. The most anterior (mandibular) stream emerges first, at the level of the mesencephalon. Cells in this stream migrate rostroventrally around the optic vesicle. The second (hyoid) and third (branchial) streams emerge in close succession at the level of the rhombencephalon and extend ventrolaterally. Cells forming the hyoid stream migrate rostral to the otic vesicle, whereas the branchial stream divides into two parallel streams, which migrate caudal to the otic vesicle. At later stages (stage 26 onwards) the cranial neural crest cells disperse into the adjacent mesoderm and can no longer be followed by dissection and SEM. The pattern of cranial neural crest emergence and migration, and division into migratory streams is similar to that in other amphibians and in the Australian lungfish (Neoceratodus forsteri). Emergence of crest cells from the neural tube, relative to the time of neural tube closure, occurs relatively late in comparison to anurans, but much earlier than in the Australian lungfish. These results establish a morphological foundation for studies in progress on the further development and fate of cranial neural crest cells in the Mexican axolotl, as well as for studies of the role of cranial neural crest in cranial patterning.     

Cell surface beta 1,4-galactosyltransferase functions during neural crest cell migration and neurulation in vivo

Mesenchymal cell migration and neurite outgrowth are mediated in part by binding of cell surface beta 1,4-galactosyltransferase (GalTase) to N-linked oligosaccharides within the E8 domain of laminin. In this study, we determined whether cell surface GalTase functions during neural crest cell migration and neural development in vivo using antibodies raised against affinity-purified chicken serum GalTase. The antibodies specifically recognized two embryonic proteins of 77 and 67 kD, both of which express GalTase activity. The antibodies also immunoprecipitated and inhibited chick embryo GalTase activity, and inhibited neural crest cell migration on laminin matrices in vitro. Anti-GalTase antibodies were microinjected into the head mesenchyme of stage 7-9 chick embryos or cranial to Henson's node of stage 6 embryos. Anti-avian GalTase IgG decreased cranial neural crest cell migration on the injected side but did not cross the embryonic midline and did not affect neural crest cell migration on the uninjected side. Anti-avian GalTase Fab crossed the embryonic midline and perturbed cranial neural crest cell migration throughout the head. Neural fold elevation and neural tube closure were also disrupted by Fab fragments. Cell surface GalTase was localized to migrating neural crest cells and to the basal surfaces of neural epithelia by indirect immunofluorescence, whereas GalTase was undetectable on neural crest cells prior to migration. These results suggest that, during early embryogenesis, cell surface GalTase participates during neural crest cell migration, perhaps by interacting with laminin, a major component of the basal lamina. Cell surface GalTase also appears to play a role in neural tube formation, possibly by mediating neural epithelial adhesion to the underlying basal lamina.     

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Annexin A6 Modulates Chick Cranial Neural Crest Cell Emigration

The vertebrate neural crest is a population of migratory cells that originates in the dorsal aspect of the embryonic neural tube. These cells undergo an epithelial-to-mesencyhmal transition (EMT), delaminate from the neural tube and migrate extensively to generate an array of differentiated cell types. Elucidating the gene regulatory networks involved in neural crest cell induction, migration and differentiation are thus crucial to understanding vertebrate development. To this end, we have identified Annexin A6 as an important regulator of chick midbrain neural crest cell emigration. Annexin proteins comprise a family of calcium-dependent, membrane-binding molecules that mediate a variety of cellular and physiological processes including cell adhesion, migration and invasion. Our data indicate that Annexin A6 is expressed in the proper spatio-temporal pattern in the chick midbrain to play a potential role in neural crest cell ontogeny. To investigate Annexin A6 function, we have depleted or overexpressed Annexin A6 in the developing midbrain neural crest cell population. Our results show that knock-down or overexpression of Annexin A6 reduces or expands the migratory neural crest cell domain, respectively. Importantly, this phenotype is not due to any change in cell proliferation or cell death but can be correlated with changes in the size of the premigratory neural crest cell population and with markers associated with EMT. Taken together, our data indicate that Annexin A6 plays a pivotal role in modulating the formation of cranial migratory neural crest cells during vertebrate development.     

Analysis of neural crest cell lineage and migration.

In this review, we describe the results of recent experiments designed to investigate various aspects of neural crest cell lineage and migration. We have analyzed the lineage of individual premigratory neural crest cells by injecting a fluorescent lineage tracer dye, lysinated fluorescein dextran, into cells within the dorsal neural tube. Individual clones contained cells that were located in very diverse sites consistent with their being sensory neurons, prepigment cells, Schwann cells, adrenergic cells, and neural tube cells. These results suggest that some neural crest cells in the trunk and cranial regions are multipotent prior to their emigration from the neural tube. The environment through which neural crest cells move influences both the pattern and direction of their migration. We have shown that the sclerotomal portion of the somites are responsible for the rostrocaudal pattern of trunk neural crest cell movement, whereas the neural tube appears to govern the dorsoventral position of neural crest-derived ganglia. In addition, the notochord inhibits the movement of neural crest cells. In order to understand necessary cell-matrix interactions in neural crest migration, we have performed perturbation experiments, in which antibodies directed against cell surface or extracellular matrix molecules were introduced along neural crest pathways. We find that integrins, fibronectin, laminin, and tenascin all play some role in cranial neural crest emigration. Thus, multiple factors may be involved in controlling neural crest cell migration, and different factors may be important for migration in different regions of the embryo.     

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.