Cell-intrinsic differences between stem cells from different regions of the peripheral nervous system regulate the generation of neural diversity

Stem cells in different regions of the nervous system give rise to different types of mature cells. This diversity is assumed to arise in response to local environmental differences, but the contribution of cell-intrinsic differences between stem cells has been unclear. At embryonic day (E)14, neural crest stem cells (NCSCs) undergo primarily neurogenesis in the gut but gliogenesis in nerves. Yet gliogenic and neurogenic factors are expressed in both locations. NCSCs isolated by flow-cytometry from E14 sciatic nerve and gut exhibited heritable, cell-intrinsic differences in their responsiveness to lineage determination factors. Gut NCSCs were more responsive to neurogenic factors, while sciatic nerve NCSCs were more responsive to gliogenic factors. Upon transplantation of uncultured NCSCs into developing peripheral nerves in vivo, sciatic nerve NCSCs gave rise only to glia, while gut NCSCs gave rise primarily to neurons. Thus, cell fate in the nerve was stem cell determined.     

Neural crest stem cells persist in the adult gut but undergo changes in self-renewal,neuronal subtype potential,and factor responsiveness

We found neural crest stem cells (NCSCs) in the adult gut. Postnatal gut NCSCs were isolated by flow-cytometry and compared to fetal gut NCSCs. They self-renewed extensively in culture but less than fetal gut NCSCs. Postnatal gut NCSCs made neurons that expressed a variety of neurotransmitters but lost the ability to make certain subtypes of neurons that are generated during fetal development. Postnatal gut NCSCs also differed in their responsiveness to lineage determination factors, affecting cell fate determination in vivo and possibly explaining their reduced neuronal subtype potential. These perinatal changes in gut NCSCs parallel perinatal changes in hematopoietic stem cells, suggesting that stem cells in different tissues undergo similar developmental transitions. The persistence of NCSCs in the adult PNS opens up new possibilities for regeneration after injury or disease.     

Prospective identification, isolation by flow cytometry, and in vivo self-renewal of multipotent mammalian neural crest stem cells

Multipotent and self-renewing neural stem cells have been isolated in culture, but equivalent cells have not yet been prospectively identified in neural tissue. Using cell surface markers and flow cytometry, we have isolated neural crest stem cells (NCSCs) from mammalian fetal peripheral nerve. These cells are phenotypically and functionally indistinguishable from NCSCs previously isolated by culturing embryonic neural tube explants. Moreover, in vivo BrdU labeling indicates that these stem cells self-renew in vivo. NCSCs freshly isolated from nerve tissue can be directly transplanted in vivo, where they generate both neurons and glia. These data indicate that neural stem cells persist in peripheral nerve into late gestation by undergoing self-renewal. Such persistence may explain the origins of some PNS tumors in humans.     

Neural crest stem cells undergo cell-intrinsic developmental changes in sensitivity to instructive differentiation signals

Rat neural crest stem cells (NCSCs) prospectively isolated from uncultured E14.5 sciatic nerve and transplanted into chick embryos generate fewer neurons than do NCSCs isolated from E10.5 neural tube explants. In addition, they differentiate primarily to cholinergic parasympathetic neurons, although in culture they can also generate noradrenergic sympathetic neurons. This in vivo behavior can be explained, at least in part, by a reduced sensitivity of sciatic nerve-derived NCSCs to the neurogenic signal BMP2 and by the observation that cholinergic neurons differentiate at a lower BMP2 concentration than do noradrenergic neurons in vitro. These results demonstrate that neural stem cells can undergo cell-intrinsic changes in their sensitivity to instructive signals, while maintaining multipotency and self-renewal capacity. They also suggest that the choice between sympathetic and parasympathetic fates may be determined by the local concentration of BMP2.     

Temporally distinct requirements for endothelin receptor B in the generation and migration of gut neural crest stem cells

Loss of Endothelin-3/Endothelin receptor B (EDNRB) signaling leads to aganglionosis of the distal gut (Hirschsprung's disease), but it is unclear whether it is required primarily for neural crest progenitor maintenance or migration. Ednrb-deficient gut neural crest stem cells (NCSCs) were reduced to 40% of wild-type levels by embryonic day 12.5 (E12.5), but no further depletion of NCSCs was subsequently observed. Undifferentiated NCSCs persisted in the proximal guts of Ednrb-deficient rats throughout fetal and postnatal development but exhibited migration defects after E12.5 that prevented distal gut colonization. EDNRB signaling may be required to modulate the response of neural crest progenitors to migratory cues, such as glial cell line-derived neurotrophic factor (GDNF). This migratory defect could be bypassed by transplanting wild-type NCSCs directly into the aganglionic region of the Ednrb(sl/sl) gut, where they engrafted and formed neurons as efficiently as in the wild-type gut.     

Isolation of Enteric Nervous System Progenitor Cells from the Aganglionic Gut of Patients with Hirschsprung’s Disease

Enteric nervous system progenitor cells isolated from postnatal human gut and cultured as neurospheres can then be transplanted into aganglionic gut to restore normal patterns of contractility. These progenitor cells may be of future use to treat patients with Hirschprung’s disease, a congenital condition characterized by hindgut dysmotility due to the lack of enteric nervous system ganglia. Here we demonstrate that progenitor cells can also be isolated from aganglionic gut removed during corrective surgery for Hirschsprung’s disease. Although the enteric nervous system marker calretinin is not expressed in the aganglionic gut region, de novo expression is initiated in cultured neurosphere cells isolated from aganglionic Hirschsprung bowel. Furthermore, expression of the neural markers NOS, VIP and GFAP also increased during culture of aganglionic gut neurospheres which we show can be transplantation into cultured embryonic mouse gut explants to restore a normal frequency of contractility. To determine the origin of the progenitor cells in aganglionic region, we used fluorescence-activated cell sorting to demonstrate that only p75-positive neural crest-derived cells present in the thickened nerve trunks characteristic of the aganglionic region of Hirschsprung gut gave rise to neurons in culture. The derivation of enteric nervous system progenitors in the aganglionic gut region of Hirschprung’s patients not only means that this tissue is a potential source of cells for future autologous transplantation, but it also raises the possibility of inducing the differentiation of these endogenous cells in situ to compensate for the aganglionosis.     

Ontogeny and multipotency of neural crest-derived stem cells in mouse bone marrow, dorsal root ganglia, and whisker pad

Although recent reports have described multipotent, self-renewing, neural crest-derived stem cells (NCSCs), the NCSCs in various adult rodent tissues have not been well characterized or compared. Here we identified NCSCs in the bone marrow (BM), dorsal root ganglia, and whisker pad and prospectively isolated them from adult transgenic mice encoding neural crest-specific P0-Cre/Floxed-EGFP and Wnt1-Cre/Floxed-EGFP. Cultured EGFP-positive cells formed neurosphere-like structures that expressed NCSC genes and could differentiate into neurons, glial cells, and myofibroblasts, but the frequency of the cell types was tissue source dependent. Interestingly, we observed NCSCs in the aorta-gonad-mesonephros region, circulating blood, and liver at the embryonic stage, suggesting that NCSCs migrate through the bloodstream to the BM and providing an explanation for how neural cells are generated from the BM. The identification of NCSCs in accessible adult tissue provides a new potential source for autologous cell therapy after nerve injury or disease.     

Identification of Sox8 as a modifier gene in a mouse model of Hirschsprung disease reveals underlying molecular defect

Mice carrying heterozygous mutations in the Sox10 gene display aganglionosis of the colon and represent a model for human Hirschsprung disease. Here, we show that the closely related Sox8 functions as a modifier gene for Sox10-dependent enteric nervous system defects as it increases both penetrance and severity of the defect in Sox10 heterozygous mice despite having no detectable influence on enteric nervous system development on its own. Sox8 exhibits an expression pattern very similar to Sox10 with occurrence in vagal and enteric neural crest cells and later confinement to enteric glia. Loss of Sox8 alleles in Sox10 heterozygous mice impaired colonization of the gut by enteric neural crest cells already at early times. Whereas proliferation, apoptosis, and neuronal differentiation were normal for enteric neural crest cells in the gut of mutant mice, apoptosis was dramatically increased in vagal neural crest cells outside the gut. The defects in enteric nervous system development of mice with Sox10 and Sox8 mutations are therefore likely caused by a reduction of the pool of undifferentiated vagal neural crest cells. Our study suggests that Sox8 and Sox10 are jointly required for the maintenance of these vagal neural crest stem cells.     

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 migratory behavior of immature enteric neurons

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.     

The boundary cap: a source of neural crest stem cells that generate multiple sensory neuron subtypes

The boundary cap (BC) is a transient neural crest-derived group of cells located at the dorsal root entry zone (DREZ) that have been shown to differentiate into sensory neurons and glia in vivo. We find that when placed in culture, BC cells self-renew, show multipotency in clonal cultures and express neural crest stem cell (NCSCs) markers. Unlike sciatic nerve NCSCs, the BC-NCSC (bNCSCs) generates sensory neurons upon differentiation. The bNCSCs constitute a common source of cells for functionally diverse types of neurons, as a single bNCSC can give rise to several types of nociceptive and thermoreceptive sensory neurons. Our data suggests that BC cells comprise a source of multipotent sensory specified stem cells that persist throughout embryogenesis.     

Migration of neural crest-derived enteric nervous system precursor cells to and within the gastrointestinal tract

The enteric nervous system, the intrinsic innervation of the gastrointestinal tract, consists of large numbers of phenotypically diverse neurons and glial cells, arranged in complex interconnecting plexuses situated between the smooth muscle layers of the gut wall. Recently, the enteric nervous system has attracted much attention from developmental biologists whose efforts have focused on analysing the cellular origins of enteric nervous system precursor cells, how these cells migrate to and within the gut and the identification of signalling mechanisms which cause migrating cells to differentiate into the appropriate phenotypes in the appropriate locations. This review summarises the state of knowledge concerning the early stages of enteric nervous system development and concentrates on: (i) the embryological origins of the neural crest cells which colonise the gastrointestinal tract, (ii) their spatiotemporal migration within the gut, (iii) the possible pre-specification of neural crest cells as enteric nervous system precursors and (iv) factors influencing their directional migration within the gut.     

The location and phenotype of proliferating neural-crest-derived cells in the developing mouse gut

Neural crest cells that originate in the caudal hindbrain migrate into and along the developing gastrointestinal tract to form the enteric nervous system. While they are migrating, neural-crest-derived cells are also proliferating. Previous studies have shown that the expression of glial-derived neurotrophic factor (GDNF) and endothelin-3 is highest in the embryonic caecum, and that GDNF alone or in combination with endothelin-3 promotes the proliferation of enteric neural-crest-derived cells in vitro. However, whether neural proliferative zones, like those in the central nervous system, are found along the developing gut is unknown. We used a fluorescent nucleic acid stain to identify dividing cells or BrdU labelling (2 h after administration of BrdU to the mother), combined with antibodies specific to neural crest cells to determine the percentage of proliferating crest-derived cells in various gut regions of embryonic day 11.5 (E11.5) and E12.5 mice. The rate of proliferation of crest-derived cells did not vary significantly in different regions of the gut (including the caecum) or at different distances from the migratory wavefront of vagal crest-derived cells. The phenotype of mitotic enteric crest-derived cells was also examined. Cells expressing the pan-neuronal markers, neurofilament-M and Hu, or the glial marker, S100b, were observed undergoing mitosis. However, no evidence was found for proliferation of cells expressing neuron-type-specific markers, such as nitric oxide synthase (at E12.5) or calcitonin gene-related peptide (at E18.5). Thus, for enteric neurons, exit from the cell cycle appears to occur after the expression of pan-neuronal proteins but prior to the expression of markers of terminally differentiated neurons.This work was supported by the Australian Research Council (DP0345298) and the National Health and Medical Research Council of Australia (Project grant 145628 and Senior Research Fellowship 170224).     

Phactr4: A new integrin modulator required for directional migration of enteric neural crest cells

The enteric nervous system (ENS) is critically important for many intestinal functions such as peristalsis and secretion. Defects in the embryonic formation of the ENS cause Hirschsprung disease (HSCR) or megacolon, a severe birth defect that affects approximately 1 in 5,000 newborns. One of the least understood aspects of ENS development are the cellular and molecular mechanisms that control chain migration of the ENS cells during their migration into and along the embryonic gut. We recently reported a mouse model of HSCR in which mutant embryos carrying a hypomorphic allele of the Phactr4 gene show an embryonic gastrointestinal defect due to loss of enteric neurons in the colon. We found that Phactr4 modulates integrin signaling and cofilin activity to coordinate the forces that drive enteric neural crest cell (ENCC) migration in the mammalian embryo. In this extra view, we briefly summarize the current knowledge on integrin signaling in ENCC migration and introduce the Phactr protein family. Employing the ENS as a model, we shed some light on the mechanisms by which Phactr4 regulates integrin signaling and controls the cell polarity required for directional ENCC migration in the mouse developing gut.     

Ret isoform function and marker gene expression in the enteric nervous system is conserved across diverse vertebrate species

The enteric nervous system (ENS) derives from migratory neural crest cells that colonize the developing gut tube, giving rise to an integrated network of neurons and glial cells, which together regulate important aspects of gut function, including coordinating the smooth muscle contractions of the gut wall. The absence of enteric neurons in portions of the gut (aganglionosis) is the defining feature of Hirschsprung’s disease (HSCR) and has been replicated in a number of mouse models. Mutations in the RET tyrosine kinase account for over half of familial cases of HSCR and mice mutant for Ret exhibit aganglionosis. RET exists in two main isoforms, RET9 and RET51 and studies in mouse have shown that RET9 is sufficient to allow normal development of the ENS. In the last several years, zebrafish has emerged as a model of vertebrate ENS development, having been supported by a number of demonstrations of conservation of gene function between zebrafish, mouse and human. In this study we further analyse the potential similarities and differences between ENS development in zebrafish, mouse and human. We demonstrate that zebrafish Ret is required in a dose-dependent manner to regulate colonization of the gut by neural crest derivatives, as in human. Additionally, we show that as in mouse and human, zebrafish ret is produced as two isoforms, ret9 and ret51. Moreover, we show that, as in mouse, the Ret9 isoform is sufficient to support colonization of the gut by enteric neurons. Finally, we identify zebrafish orthologues of genes previously identified to be expressed in the mouse ENS and demonstrate that these genes are expressed in the developing zebrafish ENS, thereby identifying useful ENS markers in this model organism. These studies reveal that the similarities between gene expression and gene function across vertebrate species is more extensive than previously appreciated, thus supporting the use of zebrafish as a general model for vertebrate ENS development and the use of zebrafish genetic screens as a way to identify candidate genes mutated in HSCR cases.     

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     

Development of the enteric nervous system

The mature enteric nervous system (ENS) is characterized by a degree of neuronal phenotypic diversity and independence of central nervous system control unequaled by any other region of the peripheral nervous system. Studies that have utilized the immunocytochemical demonstration of neurofilament protein and explanation of primordial gut with subsequent growth in culture have indicated that the neural crest precursors of enteric neurons are already committed to the neuronal lineage when they colonize the bowel; however, neuronal phenotypic expression occurs within the gut itself. It is likely that precursors able to give rise to each type of neuron found in the mature ENS are present among the earliest neural crest émigrés to reach the bowel. In mice a proximodistal wave of neuronal phenotypic expression occurs that does not appear to reflect the descent of neuronal precursors. This observation, the known plasticity of developing neural crest-derived neurons, and the demonstration of a persistent population of proliferating neuroblasts in the gut raise the possibility that enteric neuronal phenotypic expression is influenced by the enteric microenvironment.     

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.     

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

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

Localization of the netrin guidance receptor, DCC, in the developing peripheral and enteric nervous systems

Over recent years the secreted guidance cue, netrin-1, and its receptor, DCC, have been shown to be an essential guidance system driving axon pathfinding within the developing vertebrate central nervous system (CNS). Mice lacking DCC exhibit severe defects in commissural axon extension towards the floor plate demonstrating that the DCC-netrin guidance system is largely responsible for directing axonal projections toward the ventral midline in the developing spinal cord (Fazeli et al., Nature 386 (1997) 796). In addition, these mutants lack several major commissures within the forebrain, including the corpus callosum and the hippocampal commissure. In contrast to the CNS, the role of the DCC guidance receptor in the development of the mammalian peripheral and enteric nervous systems (PNS and ENS) has not been investigated. Here we demonstrate using immunohistochemical analysis that the DCC receptor is present in the developing mouse PNS where it is found on spinal, segmental, and sciatic nerves, and in developing sensory ganglia and their associated axonal projections. In addition, DCC is present in the ENS throughout the early developmental phase.