Trunk Neural Crest Cells Research Articles

Role of retinoic acid in mouse neural crest cell development in vitro.

This study examines the role of retinoic acid (RA) in mouse neural crest cell development in culture. We have compared the effects of RA on the developmental behavior of mouse neural crest cells from different axial levels, that is the mesencephalic (cranial) and sympathoadrenal (trunk) levels by colony assay using antibodies against cell-type-specific markers. In control cultures in the absence of RA, the efficiency of colony formation by cranial neural crest cells was considerably lower than in colony cultures of trunk neural crest cells. Pulse-labelling experiments using 5-bromo-2'-deoxyuridine (BrdU) showed that the proportion of neural crest cells that incorporated BrdU was significantly smaller in day 5 cultures of cranial neural crest cells in the absence of RA compared to cultures from the trunk level. However, BrdU-incorporation matched the labelling observed in trunk crest cell cultures when RA was added to the culture medium. The efficiency of colony formation and the proportion of BrdU-incorporated cells in trunk crest cell cultures was similar in the presence and absence of RA. The results suggest that in the early stages of in vitro development, RA has a larger impact on proliferation and/or survival of cranial neural crest cells than of trunk neural crest cells. Moreover, the data indicate that RA significantly affects melanogenesis and the differentiation of serotonergic neurons in mouse neural crest cell cultures from both axial levels. Whereas melanogenesis was suppressed by RA treatment, serotonergic neurogenesis was promoted. Double-labelling experiments with antibodies against BrdU and serotonin (5-HT) indicated that RA selectively promoted proliferation of these neurons at a later stage of in vitro development. Furthermore, RA acted upon both committed cells and multipotent cells, Based on the results, we conclude that RA plays multiple critical roles in mouse neural crest cell development.

The relationship between migration and chondrogenic potential of trunk neural crest cells in Ambystoma mexicanum.

Based on results of transplantation experiments, it has long been believed that trunk neural crest cells are incapable of chondrogenesis. When pigmented trunk neural crest cells of Ambystoma mexicanum are transplanted to cranial levels of albino (a/a) embryos, the graft cells ultimately produce ectopic fins, but are incapable of following the chondrogenic cranial neural crest pathways. Therefore, heterotopic transplantation does not expose these cells to the same environment experienced by cranial neural crest cells, and is neither an adequate nor a sufficient test of chondrogenic potential. However, in vitro culture of trunk neural crest cells with pharyngeal endoderm does provide a direct test of chondrogenic ability. That cartilage does not form under these conditions demonstrates conclusively that trunk neural crest cells possess no chondrogenic potential.

Regulated expression of neurofibromin in migrating neural crest cells of avian embryos

Neurofibromatosis type 1 (NF1) is a common human genetic disease involving various neural crest (NC)-derived cell types, in particular, Schwann cells and melanocytes. The gene responsible for NF1 encodes the protein neurofibromin, which contains a domain with amino acid sequence homology to the ras-guanosine triphosphatase activating protein, suggesting that neurofibromin may play a role in intracellular signaling pathways regulating cellular proliferation or differentiation, or both. To determine whether neurofibromin plays a role in NC cell development, we used antibodies raised against human neurofibromin fusion proteins in western blot and immunocytochemical studies of early avian embryos. These antibodies specifically recognized the 235 kD chicken neurofibromin protein, which was expressed in migrating trunk and cranial NC cells of early embryos (E1.5 to E2), as well as in endothelial and smooth muscle cells of blood vessels and in a subpopulation of non-NC-derived cells in the dermamyotome. At slightly later stages (E3 to E5), neurofibromin immunostaining was observed in various NC derivatives, including dorsal root ganglia and peripheral nerves, as well as non-NC-derived cell types, including heart, skeletal muscle, and kidney. At still later stages (E7 to E9), neurofibromin immunoreactivity was found in almost all tissues in vivo. To determine whether the levels of neurofibromin changed during melanocyte and Schwann cell development, tissue culture experiments were performed. Cultured NC cells were found to express neurofibromin at early time points in culture, but the levels of immunoreactivity decreased as the cells underwent pigmentation. Schwann cells, on the other hand, continued to express neurofibromin in culture. These data suggest, therefore, that neurofibromin may play a role in the development of both NC cells and a variety of non-NC-derived tissues.

Role of the transforming growth factor-beta family in the expression of cranial neural crest-specific phenotypes.

Cranial and trunk neural crest cells produce different derivatives in vitro. Cranial neural crest cultures produce large numbers of cells expressing fibronectin (FN) and procollagen I (PCol I) immunoreactivities, two markers expressed by mesenchymal derivatives in vivo. Trunk neural crest cultures produce relatively few FN or PCol I immunoreactive cells, but they produce greater numbers of melanocytes than do cranial cultures. Treatment of trunk neural crest cultures with transforming growth factor-beta 1 (TGF-beta 1) stimulates them to express both FN and PCol I immunoreactivities at levels comparable to those normally seen in cranial cultures and simultaneously decreases their expression of melanin. These observations raised the possibility that endogenous TGF-beta is involved in specifying differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro. Consistent with this idea, we found that treatment of cranial cultures with a function-blocking TGF-beta antiserum inhibits the development of FN immunoreactive cells and stimulates the development of melanocytes. Cranial and trunk neural crest cells express approximately equal levels of TGF-beta mRNA. However, trunk neural crest cells are significantly less sensitive to the FN-inducing effect of TGF-beta 1 than are cranial neural crest cells. These results suggest that: (1) endogenous TGF-beta is required for the expression of mesenchymal phenotypes by cranial neural crest cells, and (2) differences in the phenotypes expressed by cranial and trunk neural crest cells in vitro result in part from differences in the sensitivities of these two cell populations to TGF-beta.

Cyclopamine, a steroidal alkaloid, disrupts development of cranial neural crest cells in Xenopus

Cyclopamine is a steroidal alkaloid which causes limb and craniofacial defects in many vertebrate species. We have used Xenopus laevis as a model system to characterize the defects caused by cyclopamine at the cellular level. The most dramatic consequence of cyclopamine treatment in the Xenopus embryo is a defect in formation of craniofacial cartilage. Much of this cartilage is absent in treated animals. As in avian and mammalian species, Xenopus craniofacial cartilage is derived primarily from cells of the cranial neural crest. Grafting experiments show that development of the cartilaginous derivatives of the cranial neural crest is impaired after cyclopamine treatment, and this is at least partially due to a direct effect on presumptive crest cells. A culture system was used to determine the cellular response to the drug. Cyclopamine did not block the initial emigration of cells from a neural plate explant. However, cell death is seen in treated cultures after 4 days. Trunk neural crest cells and transformed cell lines are resistant to cyclopamine. We therefore conclude that cyclopamine specifically causes death of cranial neural crest cells and that lethality is likely to account for the teratogenic effects of this compound.

Avian neural crest cells can migrate in the dorsolateral path only if they are specified as melanocytes.

Neural crest cells are conventionally believed to migrate arbitrarily into various pathways and to differentiate according to the environmental cues that they encounter. We present data consistent with the notion that melanocytes are directed, by virtue of their phenotype, into the dorsolateral path, whereas other neural crest derivatives are excluded. In the avian embryo, trunk neural crest cells that migrate ventrally differentiate largely into neurons and glial cells of the peripheral nervous system. Neural crest cells that migrate into the dorsolateral path become melanocytes, the pigment cells of the skin. Neural crest cells destined for the dorsolateral path are delayed in their migration until at least 24 hours after migration commences ventrally. Previous studies have suggested that invasion into the dorsolateral path is dependent upon a change in the migratory environment. A complementary possibility is that as neural crest cells differentiate into melanocytes they acquire the ability to take this pathway. When quail neural crest cells that have been grown in culture for 12 hours are labeled with Fluoro-gold and then grafted into the early migratory pathway at the thoracic level, they migrate only ventrally and are coincident with the host neural crest. When fully differentiated melanocytes (96 hours old) are back-grafted under identical conditions, however, they enter the dorsolateral path and invade the ectoderm at least one day prior to the host neural crest. Likewise, neural crest cells that have been cultured for at least 20 hours and are enriched in melanoblasts immediately migrate in the dorsolateral path, in addition to the ventral path, when back-grafted into the thoracic level. A population of neural crest cells depleted of melanoblasts--crest cells derived from the branchial arches--are not able to invade the dorsolateral path, suggesting that only pigment cells or their precursors are able to take this migratory route. These results suggest that as neural crest cells differentiate into melanocytes they can exploit the dorsolateral path immediately. Even when 12-hour crest cells are grafted into stage 19-21 embryos at an axial level where host crest are invading the dorsolateral path, these young neural crest cells do not migrate dorsolaterally. Conversely, melanoblasts or melanocytes grafted under the same circumstances are found in the ectoderm. These latter results suggest that during normal development neural crest cells must be specified, if not already beginning to differentiate, as melanocytes in order to take this path.(ABSTRACT TRUNCATED AT 400 WORDS)

Soluble and cell-bound forms of steel factor activity play distinct roles in melanocyte precursor dispersal and survival on the lateral neural crest migration pathway

Trunk neural crest cells segregate from the neuroepithelium and enter a 'migration staging area' lateral to the embryonic neural tube. After some crest cells in the migration staging area have begun to migrate on a medial pathway, a subpopulation of crest-derived cells remaining in the migration staging area expresses mRNAs for the receptor tyrosine kinase, c-kit, and tyrosinase-related protein-2, both of which are characteristic of melanocyte precursors. These putative melanocyte precursors are subsequently observed on the lateral crest migration pathway between the dermatome and overlying epithelium, and then dispersed in nascent dermal mesenchyme. Melanocyte precursors transiently require the c-kit ligand, Steel factor for survival. Although Steel factor mRNA is transiently expressed in the dorsal dermatome before the onset of trunk neural crest cell dispersal on the lateral pathway, it is no longer produced by dermatomal cells when melanocyte precursors have dispersed in the dermal mesenchyme. To assess the role of Steel factor in migration of melanocyte precursors on the lateral pathway, we analyzed melanocyte precursor dispersal and fate on the lateral pathway of two different Sl mutants, Sl, a null allele, and Sld, which lacks cell surface-associated Steel factor but produces a soluble form. No melanocyte precursors were detected in the dermatome of embryos homozygous for the Sl allele or in W mutants that lack functional c-kit. In contrast, in embryos homozygous for the Sld allele, melanocyte precursors appeared on the lateral pathway, but subsequently disappear from the dermis. These results suggest that soluble Steel factor is required for melanocyte precursor dispersal on the lateral pathway, or for their initial survival in the migration staging area. In contrast, membrane-bound Steel factor appears to promote melanocyte precursor survival in the dermis.

Changes in the fibronectin-specific integrin expression pattern modify the migratory behavior of sarcoma S180 cells in vitro and in the embryonic environment.

The molecules that mediate cell-matrix recognition, such as fibronectins (FN) and integrins, modulate cell behavior. We have previously demonstrated that FN and the beta 1-integrins are used during neural crest cell (NCC) migration in vitro as well as in vivo, and that the FN cell-binding domains I and II exhibit functional specificity in controlling either NCC attachment, spreading, or motility in vitro. In the present study, we have analyzed the effect of changes in the integrin expression patterns on migratory cell behavior in vivo. We have generated, after stable transfection, S180 cells expressing different levels of alpha 4 beta 1 or alpha 5 beta 1 integrins, two integrins that recognize distinct FN cell-binding domains. Murine S180 cells were chosen because they behave similarly to NCC after they are grafted into the NCC embryonic pathways in the chicken embryo. Thus, they provide a model system with which to investigate the mechanisms controlling in vitro and in vivo migratory cell behavior. We have observed that either the overexpression of alpha 5 beta 1 integrin or the induction of alpha 4 beta 1 expression in transfected S180 cells enhances their motility on FN in vitro. These genetically modified S180 cells also exhibit different migratory properties when grafted into the early trunk NCC migratory pathways. We observe that alpha 5 and low alpha 4 expressors migrate in both the ventral and dorsolateral paths simultaneously, in contrast to the parental S180 cells or the host NCC, which are delayed by 24 h in their invasion of the dorsolateral path. Moreover, the alpha 4 expressors exhibit different migratory properties according to their level of alpha 4 expression at the cell surface. Cells of the low alpha 4 expressor line invade both the ventral and dorsolateral pathways. In contrast, the high expressors remain as an aggregate at the graft site, possibly the result of alpha 4 beta 1-dependent homotypic aggregation. Thus, changes in the repertoire of FN-specific integrins enable the S180 cells to exploit different pathways in the embryo and regulate the speed with which they disperse in vivo and in culture. Our studies correlate well with known changes in integrin expression during neural crest morphogenesis and strongly suggest that neural crest cells that migrate into the dorsolateral path, i.e., melanoblasts, do so only after they have upregulated the expression of FN receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Chondrogenesis of neural crest cells: Effect of poly-l-lysine and bone extract

The mechanisms of chondrogenic differentiation are generally studied in vitro by analyzing the action of agents that promote or affect chondrogenesis in embryonic mesenchyme, such as cells of the embryonic limb bud. However, it is not known whether progenitor cells of the craniofacial skeleton, which are of a different embryonic origin and derived from the neural crest, are similarly responsive to such agents. To gain insight into the regulation of chondrogenic differentiation in cells derived from neural crest, we have treated chick embryonic neural crest explants in vitro with poly-L-lysine (PL, M(r) 380 kDa) or bovine bone extract (BBE), two agents known to enhance chondrogenesis of limb mesenchymal cells. Both cephalic (normally chondrogenic) and trunk (normally nonchondrogenic) neural crest cells were analyzed. Chondrogenic differentiation was determined by histological, immunohistochemical and autoradiographic methods. Our results indicate that both PL (380 kDa) and BBE significantly enhance chondrogenesis of cephalic neural crest cells, suggesting that the mechanism of chondrogenesis of these ectodermally derived cells is similar to that of mesodermally derived limb mesenchymal cells. However, trunk neural crest cells did not undergo chondrogenesis in response to PL or BBE. These data show that chondrogenesis can be enhanced in cranial ectodermal neural crest cells in a manner similar to that in the limb mesenchyme. However, since nonchondrogenic trunk neural crest cells are not responsive, an inherent potential for cartilaginous differentiation is necessary for exogenous stimulation of chondrogenesis.

Neural crest cell formation and migration in the developing embryo.

Neural crest cells arise from the neural tube shortly after its closure and migrate extensively through prescribed regions of the embryos, where they differentiate into most of the peripheral nervous system as well as the facial skeleton and pigment cells. Along the embryonic axis, several distinct neural crest populations differ both in their migratory pathways and range of derivatives. Whereas those cells arising from the midbrain migrate as a uniform sheet of cells, neural crest cells emerging from the hindbrain and trunk regions migrate in a segmented manner. For example, trunk neural crest cells move preferentially through the rostral, but not caudal, half of each somite. Interactions with tissues encountered during migration strongly influence this segmental migratory pattern. For example, the mesodermal somites dictate the segmental migration of trunk neural crest cells and the otic placode appears to attract hindbrain neural crest cells. Although little is known about the molecular basis underlying migration, patterns of gene expression in the hindbrain are thought to contribute to the segmental arrangement of neural crest cells. Furthermore, neural crest cells possess integrin receptors that may be important for interacting with extracellular matrix molecules in their surroundings.

Developmental potential of trunk neural crest cells in the mouse.

The availability of naturally occurring and engineered mutations in mice which affect the neural crest makes the mouse embryo an important experimental system for studying neural crest cell differentiation. Here, we determine the normal developmental potential of neural crest cells by performing in situ cell lineage analysis in the mouse by microinjecting lysinated rhodamine dextran (LRD) into individual dorsal neural tube cells in the trunk. Labeled progeny derived from single cells were found in the neural tube, dorsal root ganglia, sympathoadrenal derivatives, presumptive Schwann cells and/or pigment cells. Most embryos contained labeled cells both in the neural tube and at least one neural crest derivative, and numerous clones contributed to multiple neural crest derivatives. The time of injection influenced the derivatives populated by the labeled cells. Injections at early stages of migration yielded labeled progeny in both dorsal and ventral neural crest derivatives, whereas those performed at later stages had labeled cells only in more dorsal neural crest derivatives, such as dorsal root ganglion and presumptive pigment cells. The results suggest that in the mouse embryo: (1) there is a common precursor for neural crest and neural tube cells; (2) some neural crest cells are multipotent; and (3) the timing of emigration influences the range of possible neural crest derivatives.

Rostrocaudal differences in the expression of extracellular matrix proteins by avian neural crest cells in vitro

Cranial and trunk neural crest cells developing in vitro differed in their patterns of expression of two major extracellular matrix proteins, fibronectin and collagen I. Cranial neural crest cells showed two distinct phases of fibronectin expression: the first occurred during the initial migration of cells from explants onto the culture dish; the second was associated with the differentiative period of in vitro development. Fibronectin-immunoreactive cells eventually represented one of the most abundant cell types in cranial cultures. Large numbers of procollagen I-immunoreactive cells also developed in cranial cultures, and procollagen I was colocalized with fibronectin in individual cranial neural crest cells. Neither fibronectin nor procollagen I immunoreactivities were seen in either neurons or melanocytes, consistent with the idea that the fibronectin-immunoreactive cells in cranial neural crest cultures are committed to the mesenchymal lineage. In contrast to cranial neural crest, trunk neural crest produced very few fibronectin-immunoreactive cells at any time in vitro. Trunk neutral crest also produced smaller proportions of procollagen I-immunoreactive cells than did cranial explants. Mitotic labelling experiments showed that the differing proportions of fibronectin- and procollagen I-immunoreactive cells in cranial versus trunk cultures did not result from differences in rates of cell proliferation.

Trunk neural crest origin of caudal fin mesenchyme in the zebrafishBrachydanio rerio

The origin of fin mesenchyme and the cells forming the distal fin ray skeleton in fishes is unknown, but is often assumed to be the neural crest. To test this possibility, a series of experiments have been done using a vital cell marking technique to label candidate progenitor cell populations in early zebrafish embryos. Injections of the fluorescent membrane probe DiI were made in discrete focal points into the dorsal aspect of the caudal neural keel before trunk neural crest cell emigration at that axial level in embryos at the 15-20 somite stage. As controls, injections were made into both paraxial, unsegmented mesoderm, and somitic mesoderm before somite elevation, ventral and lateral to the dorsal keel injection site. Only after injection into the neural keel site were labelled cells subsequently found in the fin mesenchyme (along with labelling of other cells types traditionally regarded as crest derived). These data provide the first experimental evidence that early fin bud mesenchyme contains a contribution from the trunk neural crest, and support the hypothesis that the dermal skeleton depends upon a crest-derived mesenchyme for its formation. We suggest that a causal link between neural crest-derived mesenchyme and the development of the exoskeleton has been evolutionary conserved throughout the neural crest of teleosts but has been lost in the trunk crest of higher vertebrates.

Restriction of neural crest cell fate in the trunk of the embryonic zebrafish.

To learn when cell fate differences first arise in the zebrafish trunk neural crest, individual premigratory crest cells were labeled intracellularly with fluorescent vital dyes, followed in living embryos and complete lineages recorded. Although some of the earliest cells to migrate produced derivatives of multiple phenotypes, most zebrafish trunk neural crest cells appear to be lineage-restricted, generating type-restricted precursors that produce single kinds of derivatives. Further, cells that produce derivatives of multiple phenotypes appear to do so by first generating type-restricted precursors. Among the various types of derivatives, sensory and sympathetic cells arise only from early migrating crest cells. Some type-restricted precursors display cell-type-specific characteristics while still migrating. Taken together, these observations suggest that some trunk neural crest cells are specified before reaching their final locations.

Retinoic Acid Selectively Promotes the Survival and Proliferation of Neurogenic Precursors in Cultured Neural Crest Cell Populations

The neural crest produces both neuronal and nonneuronal derivatives in response to a variety of environmental cues. Here, we have analyzed the effects of retinoic acid on the differentiative behavior of quail trunk neural crest cells in vitro. We show that retinoic acid selectively increases neurogenesis in cultured neural crest cell populations, probably by promoting the survival and stimulating the proliferation of neuronal precursors. Our results suggest that neural crest-derived neurogenic precursors are specific targets for retinoic acid in vitro and raise the possibility that retinoic acid promotes the generation of peripheral neurons in vivo.

Three Populations of Migrating Amphibian Embryonic Cells Utilize Different Guidance Cues

Previous investigations designed to identify the molecule(s) governing the directed migration of the amphibian pronephric duct (PND) revealed a requirement for a glycosyl phosphatidylinositol (GPI)-linked cell surface molecule, possibly the ectoenzyme alkaline phosphatase (AP). Cranial neural crest cells (CNC) grafted to the flank migrate along the same pathways as the PND, suggesting that PND and CNC guidance systems might have a common molecular basis. Both PND and CNC migration pathways display AP. The present experiments demonstrate, however, that GPI-linked molecules on these pathways are not required for migration of either cell type. Because PND cells themselves express AP prominently but CNC cells do not, we asked whether the GPI-linked molecule required for PND migration resides on the PND cells themselves. Treatment of PND cells with phosphatidylinositol-specific phospholipase C, an enzyme that removes GPI-linked proteins, prevents their migration. Transplantation experiments show that although CNC cells are capable of following PND migration pathways, the converse is not the case. Extension of the transplantation experiments to include trunk neural crest (TNC) cells indicates that although CNC cells can follow PND guidance information on the flank, PND, CNC, and TNC cells all normally utilize different molecular cues to guide their migrations in situ.

Neural Crest Contributions to the Development of the Vertebrate Head

The neural crest is a major source of mesenchyme in the head, but not the body, of the vertebrate embryo. For some of these mesenchymal derivatives, the differences between the normal fates of cranial and trunk neural crest cells are not necessarily due to differences in the potentials of the cells for the various derivatives, but reflect a lack of interaction with appropriate inductive tissues. Chondrogenic potential, however, is restricted to those axial levels which normally give rise to cartilage. This chondrogenic subpopulation is not homogeneous, even prior to neural crest cell migration, cells from different axial levels display differences in migratory and morphogenetic abilities. While the events which give rise to these segregations have never been examined, some models have been proposed for the establishment of the neural crest itself.

Novel Markers of Melanocyte Differentiation in the Avian Embryo

In order to produce markers of melanoblasts and melanocytes, we immunized a mouse with trunk neural crest cells differentiated into melanocytes in culture. We obtained three monoclonal antibodies which react both with pigmented and unpigmented cells in epidermis and in neural crest cultures. These markers are characteristic of distinct differentiation steps. One of them, melanoblast/cyte early marker (MelEM) monoclonal antibody (Mab) detects melanoblasts as soon as they have reached the subectodermal mesenchyme, while the two others, Mel1 and Mel2 Mabs, detect antigens present in melanosomes and promelanosomes later in the differentiation process. Moreover, MelEM is a specific marker of melanoblasts/cytes deriving exclusively from neural crest and not of those from the neural retina. The antigen recognized by MelEM Mab is a protein of Mr 26,000. The epitopes recognized by Mel1 and Mel2 Mabs are carbohydrate moieties carried by glycoproteins of Mr 123,000 and 85,000, respectively.

Isolation and Analysis of Neural Crest Cell Subsets Using Fluorescence-Activated Cell Sorting and Cell Culture

The ability to analyze the developmental behavior of defined cell populations is an important tool for understanding several questions concerning how cell commitment and differentiation are established during the maturation of the nervous system. The application of fluorescence-activated cell sorting and cell culture to the study of the mechanisms of differentiation of avian trunk neural crest cells is described. Results from these experiments show that this method is useful for the isolation and analysis of subsets of precursors defined on the basis of their ability to bind specific antibodies. In particular, findings indicate that cell surface differences expressed on young neural crest cells are correlated with the later development of particular differentiated phenotypes. These results are consistent with a combinatorial hypothesis in which the generation of phenotypic diversity in the neural crest results from the interaction of distinct subsets of progenitor cells (defined by the display of distinct arrays of cell surface receptor molecules) with particular ligands (e.g., growth factors and extracellular matrix molecules) In the embryonic environment. As a consequence of different sets of receptors and ligands combining on discrete subpopulations of neural crest cells, different pathways of differentiation would be elicited in particular subpopulations of cells. In this model, the action of cues in the embryonic environment is superimposed on existing developmental biases in particular subsets of neural crest cells.

Regional differences between various axial segments of the avian neural crest regarding the formation of enteric ganglia

Abstract The vagal neural crest adjacent to somites 1–7 gives rise to the enteric ganglia along the entire digestive tract. It is generally assumed that formation of enteric ganglia in preumbilical gut is independent of the axial segment from which the neural crest originates. In postumbilical gut. however, there is evidence that the axial segment of origin of the neural crest might be relevant to neural differentiation. In this part of the gut, we previously identified a subpopulation of HNK-1-immuno-reactive cells within the enteric mesenchyme. This immunoreactivity disappeared upon formation of the enteric nervous system. We studied the interaction between various axial segments of quail neural crest and the microenvironment in aneural chicken hindgut using chorioallantoic membrane cocultures. We found that neural crest cells from various axial segments could migrate into the gut and home to the correct sites. However, whereas vagal neural crest cells differentiated into enteric neurons, neural crest cells from truncal segments mainly differentiated into melanocytes. The HNK-1-immunore-activity within the enteric mesenchyme only disappeared when neural crest cell colonization was followed by differentiation into enteric neurons and subsequent formation of enteric ganglia. To determine whether differentiation of neural crest cells in chorioallantoic membranc cocultures was influenced by the prolonged presence of the neural tube and notochord, we developed a new coculture system, using neural crest cells cultured in vitro. We found that the differentiation of vagal and trunk neural crest cells within the enteric mesenchyme was not influenced by the prolonged presence of the neural tube and notochord after 24 h. suggesting that there are intrinsic differences between these neural crest cell populations. Upon prolonged in vitro culturing. the properties of vagal neural crest cells changed, and concomitantly, they lost the ability to differentiate into enteric neurons and instead differentiated into melanocytes. We conclude that the disappearance of the HNK-1-immunore-activity within the enteric mesenchyme is correlated with formation of enteric ganglia. In our experimental system. cells from the vagal neural crest are more capable of neural differentiation in the hindgut than cells from other axial levels of the neural crest.