Transiently catecholaminergic (TC) cells in the bowel of the fetal rat: precursors of noncatecholaminergic enteric neurons

Experiments were done to study the fate of transient catecholaminergic (TC) cells that develop in the rodent gut during ontogeny. When they are first detected, at Day E11 in rats, TC cells are distributed along the vagal pathway, in advance of the descending fibers of the vagus nerves, and in the foregut. The early TC cells coexpress the immunoreactivities of several neural markers, including 150-kDa neurofilament protein, peripherin, microtubule associated protein (MAP) 5, and growth-associated protein (GAP)-43, with those of the catecholamine biosynthetic enzymes tyrosine hydroxylase (TH) and dopamine-beta-hydroxylase (DBH). All cells in the fetal rat bowel at Day E11 that express neural markers also express TH immunoreactivity. The primitive TC cells also express the immunoreactivities of neural cell adhesion molecule (N-CAM), neuropeptide Y (NPY), and nerve growth factor (NGF) receptor (and NGF receptor mRNA). By Day E12 TC cells are found along the vagal pathway and throughout the entire preumbilical bowel. At this age TC cells acquire additional characteristics, including MAP 2 and synaptophysin immunoreactivities and acetylcholinesterase activity, which indicate that they continue to mature as neurons. In addition, TC cells of the rat are immunostained at Day E12 by the NC-1 monoclonal antibody, which in rats labels multiple cell types including migrating cells of neural crest origin. Despite their neural properties, at least some TC cells divide and therefore are neural precursors and not terminally differentiated neurons. At Day E10 TH mRNA-containing cells were not detected by in situ hybridization; however, by Day E11 TH mRNA was detected in sympathetic ganglia and in scattered cells in the mesenchyme of the foregut and vagal pathway. At this age, the number of enteric and vagal cells containing TH mRNA is about 30% less than the number of cells containing TH immunoreactivity in adjacent sections. The ratio of TH mRNA-containing cells to TH-immunoreactive vagal and enteric cells is even less at Day E12, especially in more caudal regions of the preumbilical bowel. A similar decline in the ratio of TH mRNA-containing to TH-immunoreactive cells was not observed in sympathetic ganglia. After Day E12 TH mRNA cannot be detected in enteric or vagal cells by in situ hybridization; nevertheless, TH immunoreactivity continues to be present through Day E14. DBH, NPY, and NGF receptor immunoreactivities are expressed by TH-immunoreactive transitional cells in the fetal rat gut after TH mRNA is no longer detectable.(ABSTRACT TRUNCATED AT 400 WORDS)     

Transient catecholaminergic (TC) cells in the vagus nerves and bowel of fetal mice: relationship to the development of enteric neurons

Catecholaminergic cells are transiently present during development of the fetal murine bowel. These transient catecholaminergic (TC) cells appear at Day E10, but by Day E13 can no longer be detected. In order to evaluate the hypothesis that these cells are the precursors of enteric neurons, we investigated the possibilities that TC cells coexpress neuronal and catecholaminergic markers, that they can be found along the presumed path followed by crest-derived cells migrating to the gut, and that they are proliferating. TC cells were identified immunocytochemically using polyclonal or monoclonal antibodies to tyrosine hydroxylase (TH). At Day E9.5, TH-immunoreactive cells were observed to be present along the wall of the primordial esophagus in lines that extended from the developing nodose ganglia down to the boundary of the stomach. At Day E9.5, TC cells were absent from the remaining foregut. These lines of esophageal TH-immunoreactive cells became continuous with similar cells in the wall of the stomach and duodenum on Day E10. Coincident expression of neurofilament immunoreactivity was seen in all of the esophageal TH-immunoreactive cells present at Day E9.5, as well as in the entire set of esophageal and lower enteric TH-immunoreactive cells present at Day E10 (or later); moreover, at Days E9.5 and E10, all of the neurofilament-immunoreactive cells in the esophagus, stomach, or duodenum were also TH-immunoreactive. In contrast, neurofilament immunoreactivity was not expressed by the endodermally derived pancreatic duct and islet cells, which were also TH-immunoreactive; nor could expression of neurofilament immunoreactivity be detected in the TH-immunoreactive cells of the nodose ganglia. It was not until Day E11 that neurofilament-immunoreactive cells, which did not coexpress TH immunoreactivity (the definitive phenotype of enteric neurons) began to appear in the gut. Vagal axons reached as far distally as the nodose ganglion on Day E9.5, the esophagogastric junction on Day E10, and did not enter the stomach until Day E11. When the vagus nerves reached their level, the TH-immunoreactive cells in the wall of the esophagus came to lie among the nerve fibers. TH-immunoreactive cells are thus present on the pathway ultimately followed by the vagus nerves, but they develop before vagal fibers reach their level. The vagal TH-immunoreactive cells, therefore, are probably not initially migrating on vagal fibers, but appear instead to be overtaken by the descending vagus nerves.(ABSTRACT TRUNCATED AT 400 WORDS)     

Neural and glial phenotypic expression by neural crest cells in culture: effects of control and presumptive aganglionic bowel from ls/ls mice

The enteric nervous system is formed by cells that migrate to the bowel from the neural crest. Previous experiments have established that avian crest cells in vitro will colonize explants of murine bowel and there give rise to neurons. It has been proposed that phenotypic expression by the crest-derived precursors of enteric neurons and glia is critically influenced by the microenvironment these cells encounter within the gut. To test this hypothesis, quail crest cells were cocultured with explants of control or presumptive aganglionic bowel from the ls/ls mutant mouse, and the effects of the enteric tissue on five phenotypic markers of crest cell development were followed. Aganglionosis develops in the terminal region of the colon of the ls/ls mouse because viable crest-derived neural and glial precursors fail to colonize this tissue. Expression of the phenotypic markers in the cocultures was compared with that in cultures of crest alone, crest plus neural tube, and gut grown alone. The markers examined were melanogenesis and immunostaining with antisera to 5-hydroxytryptamine (5-HT) and tyrosine hydroxylase (TH) and the monoclonal antibodies, NC-1 and GlN1. Explants of control, but not presumptive aganglionic ls/ls gut were found to increase the incidence of the expression of 5-HT and NC-1 immunoreactivities; moreover, especially near the gut, the assumption of a neuronal morphology by 5-HT-, NC-1-, and GlN1-immunoreactive cells was also increased. Coincidence of expression of 5-HT with NC-1 and GlN1 immunoreactivities was observed. The effect of the bowel was selective in that the expression of TH immunoreactivity, which is not a marker of mature enteric neurons, was reduced rather than enhanced. The effect of enteric explants on crest cell development was specific in that it was not mimicked by explants of metanephros, which inhibited expression of 5-HT immunoreactivity and the acquisition of a neuritic form by NC-1-immunoreactive cells. It is concluded that the enteric microenvironment affects the phenotypic expression of subsets of crest cells and that this action of the bowel is manifested in vitro. The inability of presumptive aganglionic gut from ls/ls mice to influence neural phenotypic expression may be due to the failure of this tissue to produce putative factor(s) required for the effect or to the inability of the crest-derived precursor cells to migrate into the abnormal enteric tissue.     

Colonization of the avian hindgut by cells derived from the sacral neural crest

Studies were done to test the hypothesis that the chick hindgut is colonized by emigrés from the sacral region of the neural crest. Crest-derived cells were identified immunocytochemically with the monoclonal antibody, NC-1, and by their ability to give rise to neurons or glia in the bowel. Neurons were recognized by demonstrating acetylcholinesterase activity, neurofilament immunoreactivity, or the immunoreactivity of a neurofilament-associated protein, NAPA-73, with a monoclonal antibody, E/C8. The visualization of glial fibrillary acidic protein immunoreactivity was employed to detect enteric glia. Separate rostral and caudal populations of NC-1-immunoreactive cells were detected in stage 21 embryos (Day E3.5) that extended in continuous streams from the sacral crest to the hindgut. The rostral group, coexpressed neural markers, while the caudal population did not. The rostral, dually labeled cells appeared to become embedded in the mesenchyme of the dorsal bowel by Day E4 and then to enter the mesentery by Day E5 to give rise to the ganglion of Remak. The caudal NC-1-immunoreactive group, which did not express neural markers, appeared to ascend within the colorectum and, in contrast to the rostral cells, fully encircled the gut. NC-1-immunoreactive neurons and glia developed in organotypic tissue cultures and chorioallantoic membrane grafts of both dorsal and ventral halves of the postumbilical bowel explanted at Days E4 and 5, ages known to precede the colonization of the hindgut by cells from the vagal crest. These observations are consistent with the view that NC-1-immunoreactive cells, which do not express neural markers, migrate from the sacral crest to the hindgut. A subset of these cells appears to be capable of giving rise to neurons in vitro, explaining the development of neurons in the explants of the ventral halves of the gut; however, the fate of the sacral crest-derived cells in situ remains to be established.     

From neural crest to bowel: Development of the enteric nervous system

The ENS resembles the brain and differs both physiologically and structurally from any other region of the PNS. Recent experiments in which crest cell migration has been studied with DiI, a replication-deficient retrovirus, or antibodies that label cells of neural crest origin, have confirmed that both the avian and mammalian bowel are colonized by émigrés from the sacral as well as the vagal level of the neural crest. Components of the extracellular matrix, such as laminin, may play roles in enteric neural and glial development. The observation that an overabundance of laminin develops in the presumptive aganglionic region of the gut in Is/Is mutant mice and is associated with the inability of crest-derived cells to colonize this region of the bowel has led to the hypothesis that laminin promotes the development of crest-derived cells as enteric neurons. Premature expression of a neuronal phenotype would cause crest-derived cells to cease migrating before they complete the colonization of the gut. The acquisition by crest-derived cells of a nonintegrin, nervespecific, 110 kD laminin-binding protein when they enter the bowel may enable these cells to respond to laminin differently from their pre-enteric migrating predecessors. Crest-derived cells migrating along the vagal pathway to the mammalian gut are transiently catecholaminergic (TC). This phenotype appears to be lost rapidly as the cells enter the bowel and begin to follow their program of terminal differentiation. The appearance and disappearance of TC cells may thus be an example of the effects of the enteric microenvironment on the differentiation of crest-derived cells in situ. Crest-derived cells can be isolated from the enteric microenvironment by immunoselection, a method that takes advantage of the selective expression on the surfaces of crest-derived cells of certain antigens. One neurotrophin, NT-3, promotes the development of enteric neurons and glia in vitro. Because trkC is expressed in the developing and mature gut, it seems likely that NT-3 plays a critical role in the development of the ENS in situ. Although the factors that are responsible for the development of the unique properties of the ENS remain unknown, progress made in understanding enteric neuronal development has recently accelerated. The application of new techniques and recently developed probes suggest that the accelerated pace of discovery in this area can be expected to continue. © 1993 John Wiley & Sons, Inc.     

The sympathoadrenal lineage in avian embryos. II. Effects of glucocorticoids on cultured neural crest cells

The neural crest-derived precursors of the sympathoadrenal lineage depend on environmental cues to differentiate as sympathetic neurons and pheochromocytes. We have used the monoclonal antibody A2B5 as a marker for neuronal differentiation and antisera against catecholamine synthesis enzymes to investigate the differentiation of catecholaminergic cells in cultures of quail neural crest cells. Cells corresponding phenotypically to sympathetic neurons and pheochromocytes can be identified in neural crest cell cultures after 5-6 days in vitro. Expression of the A2B5 antigen precedes expression of immunocytochemically detectable levels of tyrosine hydroxylase in cultured neural crest cells. Glucocorticoid treatment decreases the proportion of TH+ neural crest cells that express neuronal traits. We conclude that environmental cues normally encountered by sympathoadrenal precursors in vivo can influence the differentiation of a subpopulation of cultured neural crest cells in the sympathoadrenal lineage.     

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.     

Tissue effects on the expression of serotonin, tyrosine hydroxylase and GABA in cultures of neurogenic cells from the neuraxis and branchial arches

The phenotypically diverse neurones of the enteric nervous system are developmentally derived from precursors that migrate to the bowel from the vagal and sacral regions of the neuraxis. In order to gain insight into the generation of enteric neuronal diversity, we examined the expression of serotonin (5-HT), tyrosine hydroxylase and GABA in vitro. In the mature avian intestine, intrinsic neurones contain 5-HT or GABA but not tyrosine hydroxylase. These markers were demonstrated immunocytochemically, singly or simultaneously. All three phenotypic markers developed in cultures of cranial, vagal or truncal neural crest when the cultures were grown in enriched medium, containing horse serum and chick embryo extract; however, 5-HT and GABA, but not tyrosine hydroxylase-immunoreactive cells, also developed in cultures that were grown in partially defined medium. Tyrosine hydroxylase immunoreactivity was seen when partially defined medium was supplemented with nerve growth factor (NGF). Cultures of branchial arches (III and IV) contained cells that displayed tyrosine hydroxylase immunoreactivity, but not that of 5-HT- or GABA-; however, 5-HT immunoreactivity was seen when branchial arches were cocultured with aneuronal hindgut (from 4-day chick embryos). Cultures of cells from chick gut dissociated at 7 days contained tyrosine hydroxylase as well as 5-HT and GABA immunoreactivities; however, no cultures of bowel dissociated at 8 days or later expressed tyrosine hydroxylase immunoreactivity. When neuraxial cells were cocultured with branchial arches or heart instead of gut, no 5-HT-immunoreactive cells were seen; nevertheless, the further addition of explants of gut to the heart/crest cocultures did permit the expression of 5-HT immunoreactivity. These results are consistent with the hypotheses that precursors with the potential to give rise to cells that express 5-HT, GABA and tyrosine hydroxylase are found at several levels of the neuraxis; however, the ability to express these phenotypes may be suppressed either while the crest cells are migrating (for example, 5-HT and GABA expression by crest cells passing through the branchial arches) or in their final destination (for example, tyrosine hydroxylase in the gut). This suppression may be transient and reversed by the microenvironment of the target organs.     

Phenotypes of neural-crest-derived cells in vagal and sacral pathways

Enteric neurons arise from vagal and sacral level neural crest cells. To examine the phenotype of neural-crest-derived cells in vagal and sacral pathways, we used antisera to Sox10, p75, Phox2b, and Hu, and transgenic mice in which the expression of green fluorescent protein was under the control of the Ret promoter. Sox10 was expressed prior to the emigration of vagal cells, whereas p75 was expressed shortly after their emigration. Most crest-derived cells that emigrated adjacent to somites 1–4 migrated along a pathway that was later followed by the vagus nerve. A sub-population of these vagal cells coalesced to form vagal ganglia, whereas others continued their migration towards the heart and gut. Cells that coalesced into vagal ganglia showed a different phenotype from cells in the migratory streams proximal and distal to the ganglia. Only a sub-population of the vagal cells that first entered the foregut expressed Phox2b or Ret. Sacral neural crest cells gave rise to pelvic ganglia and some neurons in the hindgut. The pathways of sacral neural crest cells were examined by using DβH-nlacZ mice. Sacral cells appeared to enter the distal hindgut around embryonic day 14.5. Very few of the previously demonstrated, but rare, neurons that were present in the large intestine of Ret null mutants and that presumably arose from the sacral neural crest expressed nitric oxide synthase, unlike their counterparts in Ret heterozygous mice. This study was supported by the National Health and Medical Research Council of Australia (project grants nos. 145628 and 350311, C.J. Martin Fellowship no. 007144, and Senior Research Fellowship no. 170224).     

Effects of different regions of the developing gut on the migration of enteric neural crest-derived cells: a role for Sema3A, but not Sema3F

The enteric nervous system arises from vagal (caudal hindbrain) and sacral level neural crest-derived cells that migrate into and along the developing gut. Data from previous studies have suggested that (i) there may be gradients along the gut that induce the caudally directed migration of vagal enteric neural precursors (ENPs), (ii) exposure to the caecum might alter the migratory ability of vagal ENPs and (iii) Sema3A might regulate the entry into the hindgut of ENPs derived from sacral neural crest. Using co-cultures we show that there is no detectable gradient of chemoattractive molecules along the pre-caecal gut that specifically promotes the caudally directed migration of vagal ENPs, although vagal ENPs migrate faster caudally than rostrally along explants of hindgut. Exposure to the caecum did not alter the rate at which ENPs colonized explants of hindgut, but it did alter the ability of ENPs to colonize the midgut. The co-cultures also revealed that there is localized expression of a repulsive cue in the distal hindgut, which might delay the entry of sacral ENPs. We show that Sema3A is expressed by the hindgut mesenchyme and its receptor, neuropilin-1, is expressed by migrating ENPs. Furthermore, there is premature entry of sacral ENPs and extrinsic axons into the distal hindgut of fetal mice lacking Sema3A. These data show that Sema3A expressed by the distal hindgut regulates the entry of sacral ENPs and extrinsic axons into the hindgut. ENPs did not express neuropilin-2 and there was no detectable change in the timetable by which ENPs colonize the gut in mice lacking neuropilin-2.     

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.     

Dual function of Slit2 in repulsion and enhanced migration of trunk,but not vagal,neural crest cells

Neural crest precursors to the autonomic nervous system form different derivatives depending upon their axial level of origin; for example, vagal, but not trunk, neural crest cells form the enteric ganglia of the gut. Here, we show that Slit2 is expressed at the entrance of the gut, which is selectively invaded by vagal, but not trunk, neural crest. Accordingly, only trunk neural crest cells express Robo receptors. In vivo and in vitro experiments demonstrate that trunk, not vagal, crest cells avoid cells or cell membranes expressing Slit2, thereby contributing to the differential ability of neural crest populations to invade and innervate the gut. Conversely, exposure to soluble Slit2 significantly increases the distance traversed by trunk neural crest cells. These results suggest that Slit2 can act bifunctionally, both repulsing and stimulating the motility of trunk neural crest cells.     

Catecholaminergic Expression in 2N27 Immortal Neural Cell Line Is Enhanced by Glial-Derived Factors

The goal of this study was to examine the responsiveness of an immortalized catecholaminergic neuronal line, 2N27, to various growth factors and identify those which promote catecholaminergic expression. 2N27 is a newly established neural cell line derived from fetal rat mesencephalic tissue and, thus, contains tyrosine hydroxylase (TH), a reliable marker for catecholaminergic neurons. Using TH activity as a biochemical index, we examined the responsiveness to both recognized trophic factors (NGF, TGF- and basic- and acidic-FGF) as well as novel, glia-derived factors present in conditioned media from several glial sources. The glial cells included MACH, a normal cell line derived from aged mouse cerebral hemispheres NBCC, normal glia derived from newborn mouse cerebral hemispheres; and C-6 glioma cells, 2B clone, passage 72, predominately astrocytes. Cells were cultured in the presence of added factors from 0 to 3 days in vitro (DIV) and were harvested on day 4. We found that 2N27 neural cells responded differentially to growth factors. No change was observed in TH activity in response to NGF, TH activity even decreased in response to b-FGF ad TGF- addition to the culture medium. However, a dose dependent increase in TH activity was observed following treatment with a-FGF and the increase to a-FGF was associated to an increase in cell proliferation as compared to TH increase by cAMP associated to differentiation. However, the 2N27 cells responded with a marked increase in TH when cultured in the glial cell conditioned media. We conclude that immortal cells require a variety of microenvironmental signals to maintain their phenotype.     

Trigeminal ganglion cells cocultured with gut express vasoactive intestinal peptide

The plasticity of neural crest cells for the expression of adrenergic and cholinergic transmitter phenotypes has been well studied. The object of this study was to determine if cells of a sensory ganglion are capable of neuropeptide transmitter plasticity. We studied whether cells of the trigeminal ganglion, which do not express the neuropeptide vasoactive intestinal peptide (VIP) in vivo, would express this peptide when grown with a tissue the gut, that contains large numbers of VIP neurons. Embryonic aneural chick rectum was explanted with the embryonic quail trigeminal ganglion on the chorioallantoic membrane of chick hosts for 7-8 days. The explants were fixed, sectioned, and stained for VIP immunoreactivity (IR), for neurofilament protein immunoreactivity, and for the quail nucleolar marker. In sections of the explants we observed two populations of quail neurons: small (10-13 microns) VIP-IR cells and large (25-32 microns) cells lacking VIP-IR and resembling native trigeminal neurons. Trigeminal ganglia explanted with embryonic heart or trigeminal ganglia explanted alone lacked small VIP-IR cells but contained large VIP-negative neurons. These results show that cells of the trigeminal ganglion grown with the gut can express a neuropeptide they do not express in the absence of the gut or in vivo. Thus the embryonic trigeminal ganglion contains cells that are plastic with respect to neuropeptide expression.     

Proliferation and distribution of cells that transiently express a catecholaminergic phenotype during development in mice and rats

Cells that transiently express a catecholaminergic phenotype have previously been shown to appear in the rat gut during development. In the present study the immunocytochemical demonstration of the enzymes, tyrosine hydroxylase (TH) and dopamine-β-hydroxylase (DBH), were used as markers to examine tissues of rats and mice for catecholaminergic cells. The simultaneous radioautographic demonstration of labeling of identified catecholaminergic cells by tritiated thymidine was used to assess their ability to proliferate. Transient catecholaminergic cells were not limited to rat gut. They were also found in the gut of the mouse where they were present by 10 days' gestation and disappeared before Day 13. Similar cells were found in the mouse kidney, the mantle layer of the sacral spinal cord, and the dorsal mesentery. In mice, transient catecholaminergic cells contained TH but did not react with antiserum to DBH. Transient catecholaminergic cells in the rat gut and other locations synthesized DNA. We conclude that transient catecholaminergic cells (1) occur in both rat and mouse embryos, although the cells of mice may not contain DBH; (2) appear in other organs as well as the gut; (3) are able to proliferate. The ultimate fate of these cells remains to be demonstrated.     

Netrins and DCC in the guidance of migrating neural crest-derived cells in the developing bowel and pancreas

Vagal neural crest-derived precursors of the enteric nervous system colonize the bowel by descending within the enteric mesenchyme. Perpendicular secondary migration, toward the mucosa and into the pancreas, result, respectively, in the formation of submucosal and pancreatic ganglia. We tested the hypothesis that netrins guide these secondary migrations. Studies using RT-PCR, in situ hybridization, and immunocytochemistry indicated that netrins (netrins-1 and -3 mice and netrin-2 in chicks) and netrin receptors [deleted in colorectal cancer (DCC), neogenin, and the adenosine A2b receptor] are expressed by the fetal mucosal epithelium and pancreas. Crest-derived cells expressed DCC, which was developmentally regulated. Crest-derived cells migrated out of explants of gut toward cocultured cells expressing netrin-1 or toward cocultured explants of pancreas. Crest-derived cells also migrated inwardly toward the mucosa of cultured rings of bowel. These migrations were specifically blocked by antibodies to DCC and by inhibition of protein kinase A, which interferes with DCC signaling. Submucosal and pancreatic ganglia were absent at E12.5, E15, and P0 in transgenic mice lacking DCC. Netrins also promoted the survival/development of enteric crest-derived cells. The formation of submucosal and pancreatic ganglia thus involves the attraction of DCC-expressing crest-derived cells by netrins.     

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