Sacral neural crest cells colonise aganglionic hindgut in vivo but fail to compensate for lack of enteric ganglia

The vagal neural crest is the origin of majority of neurons and glia that constitute the enteric nervous system, the intrinsic innervation of the gut. We have recently confirmed that a second region of the neuraxis, the sacral neural crest, also contributes to the enteric neuronal and glial populations of both the myenteric and the submucosal plexuses in the chick, caudal to the level of the umbilicus. Results from this previous study showed that sacral neural crest-derived precursors colonised the gut in significant numbers only 4 days after vagal-derived cells had completed their migration along the entire length of the gut. This observation suggested that in order to migrate into the hindgut and differentiate into enteric neurons and glia, sacral neural crest cells may require an interaction with vagal-derived cells or with factors or signalling molecules released by them or their progeny. This interdependence may also explain the inability of sacral neural crest cells to compensate for the lack of ganglia in the terminal hindgut of Hirschsprung's disease in humans or aganglionic megacolon in animals. To investigate the possible interrelationship between sacral and vagal-derived neural crest cells within the hindgut, we mapped the contribution of various vagal neural crest regions to the gut and then ablated appropriate sections of chick vagal neural crest to interrupt the migration of enteric nervous system precursor cells and thus create an aganglionic hindgut model in vivo. In these same ablated animals, the sacral level neural axis was removed and replaced with the equivalent tissue from quail embryos, thus enabling us to document, using cell-specific antibodies, the migration and differentiation of sacral crest-derived cells. Results showed that the vagal neural crest contributed precursors to the enteric nervous system in a regionalised manner. When quail-chick grafts of the neural tube adjacent to somites 1-2 were performed, neural crest cells were found in enteric ganglia throughout the preumbilical gut. These cells were most numerous in the esophagus, sparse in the preumbilical intestine, and absent in the postumbilical gut. When similar grafts adjacent to somites 3-5 or 3-6 were carried out, crest cells were found within enteric ganglia along the entire gut, from the proximal esophagus to the distal colon. Vagal neural crest grafts adjacent to somites 6-7 showed that crest cells from this region were distributed along a caudal-rostral gradient, being most numerous in the hindgut, less so in the intestine, and absent in the proximal foregut. In order to generate aneural hindgut in vivo, it was necessary to ablate the vagal neural crest adjacent to somites 3-6, prior to the 13-somite stage of development. When such ablations were performed, the hindgut, and in some cases also the cecal region, lacked enteric ganglionated plexuses. Sacral neural crest grafting in these vagal neural crest ablated chicks showed that sacral cells migrated along normal, previously described hindgut pathways and formed isolated ganglia containing neurons and glia at the levels of the presumptive myenteric and submucosal plexuses. Comparison between vagal neural crest-ablated and nonablated control animals demonstrated that sacral-derived cells migrated into the gut and differentiated into neurons in higher numbers in the ablated animals than in controls. However, the increase in numbers of sacral neural crest-derived neurons within the hindgut did not appear to be sufficiently high to compensate for the lack of vagal-derived enteric plexuses, as ganglia containing sacral neural crest-derived neurons and glia were small and infrequent. Our findings suggest that the neuronal fate of a relatively fixed subpopulation of sacral neural crest cells may be predetermined as these cells neither require the presence of vagal-derived enteric precursors in order to colonise the hindgut, nor are capable of dramatically altering their proliferation or d     

In ovo transplantation of enteric nervous system precursors from vagal to sacral neural crest results in extensive hindgut colonisation

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC). Within the embryonic avian gut, vagal NCC migrate in a rostrocaudal direction to form the majority of neurons and glia along the entire length of the gastrointestinal tract, whereas sacral NCC migrate in an opposing caudorostral direction, initially forming the nerve of Remak, and contribute a smaller number of ENS cells primarily to the distal hindgut. In this study, we have investigated the ability of vagal NCC, transplanted to the sacral region of the neuraxis, to colonise the chick hindgut and form the ENS in an experimentally generated hypoganglionic hindgut in ovo model. Results showed that when the vagal NC was transplanted into the sacral region of the neuraxis, vagal-derived ENS precursors immediately migrated away from the neural tube along characteristic pathways, with numerous cells colonising the gut mesenchyme by embryonic day (E) 4. By E7, the colorectum was extensively colonised by transplanted vagal NCC and the migration front had advanced caudorostrally to the level of the umbilicus. By E10, the stage at which sacral NCC begin to colonise the hindgut in large numbers, myenteric and submucosal plexuses in the hindgut almost entirely composed of transplanted vagal NCC, while the migration front had progressed into the pre-umbilical intestine, midway between the stomach and umbilicus. Immunohistochemical staining with the pan-neuronal marker, ANNA-1, revealed that the transplanted vagal NCC differentiated into enteric neurons, and whole-mount staining with NADPH-diaphorase showed that myenteric and submucosal ganglia formed interconnecting plexuses, similar to control animals. Furthermore, using an anti-RET antibody, widespread immunostaining was observed throughout the ENS, within a subpopulation of sacral NC-derived ENS precursors, and in the majority of transplanted vagal-to-sacral NCC. Our results demonstrate that: (1) a cell autonomous difference exists between the migration/signalling mechanisms used by sacral and vagal NCC, as transplanted vagal cells migrated along pathways normally followed by sacral cells, but did so in much larger numbers, earlier in development; (2) vagal NCC transplanted into the sacral neuraxis extensively colonised the hindgut, migrated in a caudorostral direction, differentiated into neuronal phenotypes, and formed enteric plexuses; (3) RET immunostaining occurred in vagal crest-derived ENS cells, the nerve of Remak and a subpopulation of sacral NCC within hindgut enteric ganglia.     

The receptor tyrosine kinase RET regulates hindgut colonization by sacral neural crest cells

The enteric nervous system (ENS) is formed from vagal and sacral neural crest cells (NCC). Vagal NCC give rise to most of the ENS along the entire gut, whereas the contribution of sacral NCC is mainly limited to the hindgut. This, and data from heterotopic quail-chick grafting studies, suggests that vagal and sacral NCC have intrinsic differences in their ability to colonize the gut, and/or to respond to signalling cues within the gut environment. To better understand the molecular basis of these differences, we studied the expression of genes known to be essential for ENS formation, in sacral NCC within the chick hindgut. Our results demonstrate that, as in vagal NCC, Sox10, EdnrB, and Ret are expressed in sacral NCC within the gut. Since we did not detect a qualitative difference in expression of these ENS genes we performed DNA microarray analysis of vagal and sacral NCC. Of 11 key ENS genes examined from the total data set, Ret was the only gene identified as being highly differentially expressed, with a fourfold increase in expression in vagal versus sacral NCC. We also found that over-expression of RET in sacral NCC increased their ENS developmental potential such that larger numbers of cells entered the gut earlier in development, thus promoting the fate of sacral NCC towards that of vagal NCC.     

Experimental analysis of the migration and differentiation of neuroblasts of the autonomic nervous system and of neurectodermal mesenchymal derivatives, using a biological cell marking technique

By isotopic and isochronic transplantations of fragments of quail neural tube into chick, it has been previously shown that enteric ganglion cells arise from the “vagal” (somites 1–7) and the “lumbo-sacral” (behind somite 28) levels of the neural crest, while the trunk region (somites 8–28) gives rise to orthosympathetic ganglion chain and adrenomedullary cells. The latter originate precisely from the neural crest corresponding to somites 18–24 (i.e., “adrenomedullary” level of the crest). Heterotopic transplantations of fragments of quail neural tube into chick have been carried out in the present work. When the “adrenomedullary” level of the quail neural tube is grafted into the “vagal” region of a chick, the crest cells colonize the gut and differentiate into enteric ganglia of Auerbach's and Meissner's plexi. If quail cephalic neural crest is transplanted in the “adrenomedullary” level of a chick, quail cells migrate into the suprarenal glands and differentiate into adrenomedullary cells. Mesectodermal cells migrate laterally, and differentiate into cartilage, dermis and connective tissues. Thus it appears that preferential pathways located at precise levels of the embryo lead crest cells to their definitive sites. On the other hand the differentiation of the autonomic neuroblasts is controlled by the environment in which crest cells are localized at the end of their migration. On the contrary, mesenchymal derivatives of the cephalic neural crest appear to be early determined since they differentiate according to their presumptive fate when transplanted into the trunk.     

Sacral neural crest cell migration to the gut is dependent upon the migratory environment and not cell-autonomous migratory properties

Avian neural crest cells from the vagal (somite level 1-7) and the sacral (somite level 28 and posterior) axial levels migrate into the gut and differentiate into the neurons and glial cells of the enteric nervous system. Neural crest cells that emigrate from the cervical and thoracic levels stop short of the dorsal mesentery and do not enter the gut. In this study we tested the hypothesis that neural crest cells derived from the sacral level have cell-autonomous migratory properties that allow them to reach and invade the gut mesenchyme. We heterotopically grafted neural crest cells from the sacral axial level to the thoracic level and vice versa and observed that the neural crest cells behaved according to their new position, rather than their site of origin. Our results show that the environment at the sacral level is sufficient to allow neural crest cells from other axial levels to enter the mesentery and gut mesenchyme. Our study further suggests that at least two environmental conditions at the sacral level enhance ventral migration. First, sacral neural crest cells take a ventral rather than a medial-to-lateral path through the somites and consequently arrive near the gut mesenchyme many hours earlier than their counterparts at the thoracic level. Our experimental evidence reveals only a narrow window of opportunity to invade the mesenchyme of the mesentery and the gut, so that earlier arrival assures the sacral neural crest of gaining access to the gut. Second, the gut endoderm is more dorsally situated at the sacral level than at the thoracic level. Thus, sacral neural crest cells take a more direct path to the gut than the thoracic neural crest, and also their target is closer to the site from which they initiate migration. In addition, there appears to be a barrier to migration at the thoracic level that prevents neural crest cells at that axial level from migrating ventral to the dorsal aorta and into the mesentery, which is the portal to the gut.     

Lack of organ specific commitment of vagal neural crest cell derivatives as shown by back-transplantation of GFP chicken tissues

Neural crest cells (NCC) are multipotent progenitors that migrate extensively throughout the developing embryo and generate a diverse range of cell types. Vagal NCC migrate from the hindbrain into the foregut and from there along the gastrointestinal tract to form the enteric nervous system (ENS), the intrinsic innervation of the gut, and into the developing lung buds to form the intrinsic innervation of the lungs. The aim of this study was to determine the developmental potential of vagal NCC that had already colonised the gut or the lungs. We used transgenic chicken embryos that ubiquitously express green fluorescent protein (GFP) to permanently mark and fate-map vagal NCC using intraspecies grafting. This was combined with back-transplantation of gut and lung segments, containing GFP-positive NCC, into the vagal region of a second recipient embryo to determine, using immunohistochemical staining, whether gut or lung NCC are competent of re-colonising both these organs, or whether their fate is restricted. Chick(GFP)-chick intraspecies grafting efficiently labelled NCC within the gut and lung of chick embryos. When segments of embryonic day (E)5.5 pre-umbilical midgut containing GFP-positive NCC were back-transplanted into the vagal region of E1.5 host embryos, the GFP-positive NCC remigrated to colonise both the gut and lungs and differentiated into neurons in stereotypical locations. However, GFP-positive lung NCC did not remigrate when back-transplanted. Our studies suggest that gut NCC are not restricted to colonising only this organ, since upon back-transplantation GFP-positive gut NCC colonised both the gut and the lung.     

Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.     

The effect of back-transplants of the embryonic gut wall on growth of the neural tube

Experiments in which the developing gut of avian embryos was back-transplanted to permit the bowel to interact with the developing neural tube were undertaken. Segments of intestine from 4-day quail embryos were implanted between the somites and neural tubes of chick embryos of 7 to 24 somites. The spinal cord responded to the presence of the bowel by enlarging unilaterally on the side of the graft. This effect encompassed both gray and white matter and was accompanied by the extension of neuritic projections from the spinal cord into the enteric grafts. The growth-promoting effect of enteric transplants was manifest at all levels of the neural tube where the grafts were made and led to enlargement of the brain as well as the spinal cord; however, truncal neural crest derivatives in the region of the grafts, such as developing sympathetic and spinal ganglia, were unaffected. Neither sham operations nor grafts of ciliary ganglion, lung, pancreas, mesonephros, or rudiment of the eye mimicked the action of the gut. The effect of the bowel was manifest as early as 24 hr following back-transplantation and was found to be due to an increase in the number of cells in the neuroepithelium. The cell responsible for the ability of the gut wall to enhance neuroepithelial proliferation was not identified, but the effect lacked species specificity and could be elicited in the absence of endoderm or neural crest derivatives in the explant. We propose that the musculoconnective tissue of the gut produces a short-range diffusible factor that induces mitogenic activity in the neuroepithelial cells of the neural tube, but not in the crest cells that form sympathetic or sensory ganglia. Since the gut is not normally in apposition to the neural tube, we suggest that the physiological targets of this factor are the specialized crest cells that colonize the bowel and give rise to the enteric nervous system.     

Redefining the head-trunk interface for the neural crest

The head-trunk interface lies at the occipito-cervical boundary, which corresponds to the somite 5/6 level. Previous studies have demonstrated that neural crest cells also behave differently either side of this boundary and that this may be due to intrinsic differences between cranial and trunk crest. However, it is also possible that some of the observed differences between cranial and trunk crest are assigned by environmental cues. We have therefore scrutinised the behaviour of the neural crest cells generated either side of the occipito-cervical boundary in chick and, interestingly, find that both behave in a truncal fashion by traversing the anterior half of their adjacent somites. Furthermore, although not previously described, we find that transient DRGs form opposite somites 4 and 5. Crest cells produced anterior of the somite 3/4 boundary avoid the somites and behave in a non-truncal fashion; these cells populate the pharyngeal arches, and thus contribute to the developing head. We have further shown, via somite transplantations, that differential behaviour of the posterior versus anterior occipital crest is assigned by the somites. If somites 1 to 3 are replaced by trunk somites, then the anterior occipital crest will behave in a truncal fashion by invading the somites. Correspondingly, if these anterior occipital somites are transplanted in place of trunk somites, they perturb the migration of trunk crest. Thus, for the neural crest, the head-trunk interface does not lie at the occipito-cervical boundary, but rather lies at the somite 3/4 level and is defined by the somites. The fact that this boundary lies at the somite 3/4 level in chick is significant as it reflects the more ancient posterior occipital boundary; in fish, only the first three somites contribute to the occipital bone.     

Enteric neural crest-derived cells promote their migration by modifying their microenvironment through tenascin-C production

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells that migrate, proliferate, and differentiate into enteric neurons and glia within the gut wall. The mechanisms regulating enteric neural crest-derived cell (ENCC) migration are poorly characterized despite the importance of this process in gut formation and function. Characterization of genes involved in ENCC migration is essential to understand ENS development and could provide targets for treatment of human ENS disorders. We identified the extracellular matrix glycoprotein tenascin-C (TNC) as an important regulator of ENCC development. We find TNC dynamically expressed during avian gut development. It is absent from the cecal region just prior to ENCC arrival, but becomes strongly expressed around ENCCs as they enter the ceca and hindgut. In aganglionic hindguts, TNC expression is strong throughout the outer mesenchyme, but is absent from the submucosal region, supporting the presence of both ENCC-dependent and independent expression within the gut wall. Using rat–chick coelomic grafts, neural tube cultures, and gut explants, we show that ENCCs produce TNC and that this ECM protein promotes their migration. Interestingly, only vagal neural crest-derived ENCCs express TNC, whereas sacral neural crest-derived cells do not. These results demonstrate that vagal crest-derived ENCCs actively modify their microenvironment through TNC expression and thereby help to regulate their own migration.     

Slow muscle regulates the pattern of trunk neural crest migration in zebrafish

In avians and mice, trunk neural crest migration is restricted to the anterior half of each somite. Sclerotome has been shown to play an essential role in this restriction; the potential role of other somite components in specifying neural crest migration is currently unclear. By contrast, in zebrafish trunk neural crest, migration on the medial pathway is restricted to the middle of the medial surface of each somite. Sclerotome comprises only a minor part of zebrafish somites, and the pattern of neural crest migration is established before crest cells contact sclerotome cells, suggesting other somite components regulate the pattern of zebrafish neural crest migration. Here, we use mutants to investigate which components regulate the pattern of zebrafish trunk neural crest migration on the medial pathway. The pattern of trunk neural crest migration is aberrant in spadetail mutants that have very reduced somitic mesoderm, in no tail mutants injected with spadetail morpholino antisense oligonucleotides that entirely lack somitic mesoderm and in somite segmentation mutants that have normal somite components but disrupted segment borders. Fast muscle cells appear dispensable for patterning trunk neural crest migration. However, migration is abnormal in Hedgehog signaling mutants that lack slow muscle cells, providing evidence that slow muscle cells regulate the pattern of trunk neural crest migration. Consistent with this idea, surgical removal of adaxial cells, which are slow muscle precursors, results in abnormal patterning of neural crest migration; normal patterning can be restored by replacing the ablated adaxial cells with ones transplanted from wild-type embryos.     

A Quantitative Cell Migration Assay for Murine Enteric Neural Progenitors

Neural crest cells (NCC) are a transient and multipotent cell population that originates from the dorsal neural tube and migrates extensively throughout the developing vertebrate embryo. In addition to providing peripheral glia and neurons, NCC generate melanocytes as well as most of the cranio-facial skeleton. NCC migration and differentiation is controlled by a combination of their axial origin along the neural tube and their exposure to regionally distinct extracellular cues. Such contribution of extracellular ligands is especially evident during the formation of the enteric nervous system (ENS), a complex interconnected network of neural ganglia that locally controls (among other things) gut muscle movement and intestinal motility. Most of the ENS is derived from a small initial pool of NCC that undertake a long journey in order to colonize - in a rostral to caudal fashion - the entire length of the prospective gut. Among several signaling pathways known to influence enteric NCC colonization, GDNF/RET signaling is recognized as the most important. Indeed, spatiotemporally controlled secretion of the RET ligand GDNF by the gut mesenchyme is chiefly responsible for the attraction and guidance of RET-expressing enteric NCC to and within the embryonic gut. Here, we describe an ex vivo cell migration assay, making use of a transgenic mouse line possessing fluorescently labeled NCC, which allows precise quantification of enteric NCC migration potential in the presence of various growth factors, including GDNF.     

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.     

Vital dye labelling demonstrates a sacral neural crest contribution to the enteric nervous system of chick and mouse embryos

We have used the vital dye, DiI, to analyze the contribution of sacral neural crest cells to the enteric nervous system in chick and mouse embryos. In order to label premigratory sacral neural crest cells selectively, DiI was injected into the lumen of the neural tube at the level of the hindlimb. In chick embryos, DiI injections made prior to stage 19 resulted in labelled cells in the gut, which had emerged from the neural tube adjacent to somites 29-37. In mouse embryos, neural crest cells emigrated from the sacral neural tube between E9 and E9.5. In both chick and mouse embryos, DiI-labelled cells were observed in the rostral half of the somitic sclerotome, around the dorsal aorta, in the mesentery surrounding the gut, as well as within the epithelium of the gut. Mouse embryos, however, contained consistently fewer labelled cells than chick embryos. DiI-labelled cells first were observed in the rostral and dorsal portion of the gut. Paralleling the maturation of the embryo, there was a rostral-to-caudal sequence in which neural crest cells populated the gut at the sacral level. In addition, neural crest cells appeared within the gut in a dorsal-to-ventral sequence, suggesting that the cells entered the gut dorsally and moved progressively ventrally. The present results resolve a long-standing discrepancy in the literature by demonstrating that sacral neural crest cells in both the chick and mouse contribute to the enteric nervous system in the postumbilical gut.     

T-cadherin expression alternates with migrating neural crest cells in the trunk of the avian embryo.

Trunk neural crest cells and motor axons move in a segmental fashion through the rostral (anterior) half of each somitic sclerotome, avoiding the caudal (posterior) half. This metameric migration pattern is thought to be caused by molecular differences between the rostral and caudal portions of the somite. Here, we describe the distribution of T-cadherin (truncated-cadherin) during trunk neural crest cell migration. T-cadherin, a novel member of the cadherin family of cell adhesion molecules was selectively expressed in the caudal half of each sclerotome at all times examined. T-cadherin immunostaining appeared graded along the rostrocaudal axis, with increasing levels of reactivity in the caudal halves of progressively more mature (rostral) somites. The earliest T-cadherin expression was detected in a small population of cells in the caudal portion of the somite three segments rostral to last-formed somite. This initial T-cadherin expression was observed concomitant with the invasion of the first neural crest cells into the rostral portion of the same somite in stage 16 embryos. When neural crest cells were ablated surgically prior to their emigration from the neural tube, the pattern of T-cadherin immunoreactivity was unchanged compared to unoperated embryos, suggesting that the metameric T-cadherin distribution occurs independent of neural crest cell signals. This expression pattern is consistent with the possibility that T-cadherin plays a role in influencing the pattern of neural crest cell migration and in maintaining somite polarity.     

Developmental potential of neural crest-derived cells migrating from segments of developing quail bowel back-grafted into younger chick host embryos

The technique of back-transplantation was used to investigate the developmental potential of neural crest-derived cells that have migrated to and colonized the avian bowel. Segments of quail bowel (removed at E4) were grafted between the somites and neural tube of younger (E2) chick host embryos. Grafts were placed at a truncal level, adjacent to somites 14-24. Initial experiments, done in vitro, confirmed that crest-derived cells are capable of migrating out of segments of foregut explanted at E4. The foregut, which at E4 has been colonized by cells derived from the vagal crest, served as the donor tissue. Comparative observations were made following grafts of control tissues, which included hindgut, lung primordia, mesonephros and limb bud. Additional experiments were done with chimeric bowel in which only the crest-derived cells were of quail origin. Targets in the host embryos colonized by crest-derived cells from the foregut grafts included the neural tube, spinal roots and ganglia, peripheral nerves, sympathetic ganglia and the adrenals, but not the gut. Donor cells in these target organs were immunostained by the monoclonal antibody, NC-1, indicating that they were crest-derived and developing along neural or glial lineages. Some of the crest-derived cells (NC-1-immunoreactive) that left the bowel and reached sympathetic ganglia, but not peripheral nerves or dorsal root ganglia, co-expressed tyrosine hydroxylase immunoreactivity, a neural characteristic never expressed by crest-derived cells in the avian gut. None of the cells leaving enteric back-grafts produced pigment. Cells of mesodermal origin were also found to leave donor explants and aggregate in dermis and feather germs near the grafts. These observations indicate that crest-derived cells, having previously migrated to the bowel, retain the ability to migrate to distant sites in a younger embryo. The routes taken by these cells appear to reflect, not their previous migratory experience, but the level of the host embryo into which the graft is placed. Some of the population of crest-derived cells that leave the back-transplanted gut remain capable of expressing phenotypes that they do not express within the bowel in situ, but which are appropriate for the site in the host embryo to which they migrate.