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.     

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).     

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.     

Critical numbers of neural crest cells are required in the pathways from the neural tube to the foregut to ensure complete enteric nervous system formation

The enteric nervous system (ENS) is mainly derived from vagal neural crest cells (NCC) that arise at the level of somites 1-7. To understand how the size and composition of the NCC progenitor pool affects ENS development, we reduced the number of NCC by ablating the neural tube adjacent to somites 3-6 to produce aganglionic gut. We then back-transplanted various somite lengths of quail neural tube into the ablated region to determine the 'tipping point', whereby sufficient progenitors were available for complete ENS formation. The addition of one somite length of either vagal, sacral or trunk neural tube into embryos that had the neural tube ablated adjacent to somites 3-6, resulted in ENS formation along the entire gut. Although these additional cells contributed to the progenitor pool, the quail NCC from different axial levels retained their intrinsic identities with respect to their ability to form the ENS; vagal NCC formed most of the ENS, sacral NCC contributed a limited number of ENS cells, and trunk NCC did not contribute to the ENS. As one somite length of vagal NCC was found to comprise almost the entire ENS, we ablated all of the vagal neural crest and back-transplanted one somite length of vagal neural tube from the level of somite 1 or somite 3 into the vagal region at the position of somite 3. NCC from somite 3 formed the ENS along the entire gut, whereas NCC from somite 1 did not. Intrinsic differences, such as an increased capacity for proliferation, as demonstrated in vitro and in vivo, appear to underlie the ability of somite 3 NCC to form the entire ENS.     

Functional Loss of Semaphorin 3C and/or Semaphorin 3D and Their Epistatic Interaction with Ret Are Critical to Hirschsprung Disease Liability

Innervation of the gut is segmentally lost in Hirschsprung disease (HSCR), a consequence of cell-autonomous and non-autonomous defects in enteric neuronal cell differentiation, proliferation, migration, or survival. Rare, high-penetrance coding variants and common, low-penetrance non-coding variants in 13 genes are known to underlie HSCR risk, with the most frequent variants in the ret proto-oncogene (RET). We used a genome-wide association (220 trios) and replication (429 trios) study to reveal a second non-coding variant distal to RET and a non-coding allele on chromosome 7 within the class 3 Semaphorin gene cluster. Analysis in Ret wild-type and Ret-null mice demonstrates specific expression of Sema3a, Sema3c, and Sema3d in the enteric nervous system (ENS). In zebrafish embryos, sema3 knockdowns show reduction of migratory ENS precursors with complete ablation under conjoint ret loss of function. Seven candidate receptors of Sema3 proteins are also expressed within the mouse ENS and their expression is also lost in the ENS of Ret-null embryos. Sequencing of SEMA3A, SEMA3C, and SEMA3D in 254 HSCR-affected subjects followed by in silico protein structure modeling and functional analyses identified five disease-associated alleles with loss-of-function defects in semaphorin dimerization and binding to their cognate neuropilin and plexin receptors. Thus, semaphorin 3C/3D signaling is an evolutionarily conserved regulator of ENS development whose dys-regulation is a cause of enteric aganglionosis.     

Endoderm-derived Sonic hedgehog and mesoderm Hand2 expression are required for enteric nervous system development in zebrafish

The zebrafish enteric nervous system (ENS), like those of all other vertebrate species, is principally derived from the vagal neural crest cells (NCC). The developmental controls that govern the migration, proliferation and patterning of the ENS precursors are not well understood. We have investigated the roles of endoderm and Sonic hedgehog (SHH) in the development of the ENS. We show that endoderm is required for the migration of ENS NCC from the vagal region to the anterior end of the intestine. We show that the expression of shh and its receptor ptc-1 correlate with the development of the ENS and demonstrate that hedgehog (HH) signaling is required in two phases, a pre-enteric and an enteric phase, for normal ENS development. We show that HH signaling regulates the proliferation of vagal NCC and ENS precursors in vivo. We also show the zebrafish hand2 is required for the normal development of the intestinal smooth muscle and the ENS. Furthermore we show that endoderm and HH signaling, but not hand2, regulate gdnf expression in the intestine, highlighting a central role of endoderm and SHH in patterning the intestine and the ENS.     

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.     

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.     

Perturbation of Hoxb5 signaling in vagal and trunk neural crest cells causes apoptosis and neurocristopathies in mice

Neural crest cells (NCCs) migrate from different regions along the anterior–posterior axis of the neural tube (NT) to form different structures. Defective NCC development causes congenital neurocristopathies affecting multiple NCC-derived tissues in human. Perturbed Hoxb5 signaling in vagal NCC causes enteric nervous system (ENS) defects. This study aims to further investigate if perturbed Hoxb5 signaling in trunk NCC contributes to defects of other NCC-derived tissues besides the ENS. We perturbed Hoxb5 signaling in NCC from the entire NT, and investigated its impact in the development of tissues derived from these cells in mice. Perturbation of Hoxb5 signaling in these NCC resulted in Sox9 downregulation, NCC apoptosis, hypoplastic sympathetic and dorsal root ganglia, hypopigmentation and ENS defects. Mutant mice with NCC-specific Sox9 deletion also displayed some of these phenotypes. In vitro and in vivo assays indicated that the Sox9 promoter was bound and trans-activated by Hoxb5. In ovo studies further revealed that Sox9 alleviated apoptosis induced by perturbed Hoxb5 signaling, and Hoxb5 induced ectopic Sox9 expression in chick NT. This study demonstrates that Hoxb5 regulates Sox9 expression in NCC and disruption of this signaling causes Sox9 downregulation, NCC apoptosis and multiple NCC-developmental defects. Phenotypes such as ENS deficiency, hypopigmentation and some of the neurological defects are reported in patients with Hirschsprung disease (HSCR). Whether dysregulation of Hoxb5 signaling and early depletion of NCC contribute to ENS defect and other neurocristopathies in HSCR patients deserves further investigation.     

Vascularisation is not necessary for gut colonisation by enteric neural crest cells

The vasculature and nervous system share striking similarities in their networked, tree-like architecture and in the way they are super-imposed in mature organs. It has previously been suggested that the intestinal microvasculature network directs the migration of enteric neural crest cells (ENCC) along the gut to promote the formation of the enteric nervous system (ENS). To investigate the inter-relationship of migrating ENCC, ENS formation and gut vascular development we combined fate-mapping of ENCC with immunolabelling and intravascular dye injection to visualise nascent blood vessel networks. We found that the enteric and vascular networks initially had very distinct patterns of development. In the foregut, ENCC migrated through areas devoid of established vascular networks. In vessel-rich areas, such as the midgut and hindgut, the distribution of migrating ENCC did not support the idea that these cells followed a pre-established vascular network. Moreover, when gut vascular development was impaired, either genetically in Vegfa^120/120 or Tie2-Cre;Nrp1^fl/− mice or using an in vitro Wnt1-Cre;Rosa26^Yfp/+ mouse model of ENS development, ENCC still colonised the entire length of the gut, including the terminal hindgut. These results demonstrate that blood vessel networks are not necessary to guide migrating ENCC during ENS development. Conversely, in miRet⁵¹ mice, which lack ENS in the hindgut, the vascular network in this region appeared to be normal suggesting that in early development both networks form independently of each other.     

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.     

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     

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.     

Neurofilament expression in vagal neural crest-derived precursors of enteric neurons

In order to gain insight into the potential role of the enteric microenvironment in the neuronal determination of the neural crest-derived precursor cells of enteric neurons, an attempt was made to ascertain when and where along the migratory route of these cells that they first express neuronal properties. The immunocytochemical detection of the 160-kDa component of the triplet of the chick neurofilament peptides served as a neuronal marker. In addition, neurogenic potential was assessed by growing explants of tissue suspected of containing presumptive neuroblasts in culture or as grafts on the chorioallantoic membrane of chick embryonic hosts. Neurofilament immunoreactivity was first detected in the foregut by Day 4 of development and spread to the hindgut by Day 7. Within the hindgut, development was more advanced within the colorectum than within the more proximal terminal ileum and caecal appendages. This probably reflects the distal-proximal migration of sacral neural crest cells in the postumbilical bowel. The ability of enteric explants to show neuronal development in vitro correlated with whether or not cells containing neurofilament immunoreactivity had reached that segment of gut at the age of explantation. These data suggest that enteric neuronal precursors have already begun to differentiate as neurons by the time they colonize the gut. Prior to the appearance of fibrillar neurofilament immunoreactivity in the foregut, cells that express this marker were found transiently within the mesenchyme of branchial arches 3, 4, and 5. These cells had disappeared from this region by developmental Day 6. The neurogenic potential of branchial arches 3 and 4 was demonstrated by the correlation that was found between the ability of explants of these arches to show neuronal development in vitro and the presence within them of cells that display neurofilament immunoreactivity. No similar neurogenic potential was found in the more rostral branchial arches which lacked the masses of neurofilament-immunoreactive cells. The location of the caudal branchial arches below the migrating vagal neural crest, the transience of the neurofilament immunoreactivity in them, and the coincident transience of their neurogenic potential in vitro, suggested that the masses of neurofilament immunoreactive cells in the caudal branchial arches might be vagal neural crest-derived neuronal precursor cells en route to the pharynx and the rest of the gut. This possibility was supported by the observation of neurofilament immunoreactivity in a subset of cells of the premigratory and early migratory neural crest in the vagal, but not other, regions of the neuraxis prior to the appearance of neurofilament immunoreactivity in the branchial arches. Proliferative expansion of cells with neurofilament immunoreactivity was indicated by the observation of mitotic figures in them. It is suggested that the vagal neural crest cells that populate the ENS are already committed to the neuronal lineage while still in the vagal region of the neuraxis. It is therefore not likely that the enteric microenvironment plays a role in this process.     

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut, Gdnf expression was upregulated in a more posterior region - the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k- and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.     

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.     

Colonization of the murine hindgut by sacral crest-derived neural precursors: experimental support for an evolutionarily conserved model

Enteric ganglia in the hindgut are derived from separate vagal and sacral neural crest populations. Two conflicting models, based primarily on avian data, have been proposed to describe the contribution of sacral neural crest cells. One hypothesizes early colonization of the hindgut shortly after neurulation, and the other states that sacral crest cells reside transiently in the extraenteric ganglion of Remak and colonize the hindgut much later, after vagal crest-derived neural precursors arrive. In this study, I show that Wnt1-lacZ-transgene expression, an "early" marker of murine neural crest cells, is inconsistent with the "early-colonization" model. Although Wnt1-lacZ-positive sacral crest cells populate pelvic ganglia in the mesenchyme surrounding the hindgut, they are not found in the gut prior to the arrival of vagal crest cells. Similarly, segments of murine hindgut harvested prior to the arrival of vagal crest cells and grafted under the renal capsule fail to develop enteric neurons, unless adjacent pelvic mesenchyme is included in the graft. When pelvic mesenchyme from DbetaH-nlacZ transgenic embryos is apposed with nontransgenic hindgut, neural precursors from the mesenchyme colonize the hindgut and form intramural ganglion cells that express the transgenic marker. Contribution of sacral crest-derived cells to the enteric nervous system is not affected by cocolonization of grafts by vagal crest-derived neuroglial precursors. The findings complement recent studies of avian chimeras and support an evolutionarily conserved model in which sacral crest cells first colonize the extramural ganglion and secondarily enter the hindgut mesenchyme.