Sacral Neural Crest Research Articles

20-P002 Pre-enteric migration of sacral neural crest cells is affected in Dominant megacolon embryos

Our previous study showed that mouse neural crest cells (NCCs) from the sacral level were able to migrate from the dorsal neural tube over long distances to enter the hindgut. In the present study, we sought to determine whether Sox10 mutation affected the migration of sacral NCCs prior to their entry to the hindgut. Dominant megacolon (Dom) mice with a spontaneous mutation of Sox10, and experimental techniques including cell labelling, whole embryo culture and multiple immunohistochemical staining were used. We found that in wild type embryos, sacral NCCs caudal to somite 24 began their migration from the neural tube at E9.5, started to aggregate on two sides of the hindgut to form pelvic ganglia at E11.5 and by around E14.0 entered the hindgut at S2 and S3 vertebral levels. In Sox10Dom heterozygous mutants, sacral NCCs migrated along the same spatio-temporal pathway, although the number of migrating sacral NCCs was significantly reduced. In Sox10Dom homozygotes, the number of migrating sacral NCCs was further reduced and no NCCs reached the pelvic ganglia region. TUNEL assay did not reveal any significant increase in sacral NCC death in either Sox10Dom/+ or Sox10Dom/Dom mutants. Our observations indicate that the Sox10 mutation in Dom embryos affects the migration of sacral NCC before they enter the hindgut by reducing the number of migrating sacral NCCs.

Inhibition of cell death results in hyperganglionosis: implications for enteric nervous system development

The enteric nervous system (ENS) is derived from vagal and sacral neural crest cells (NCC) that delaminate from the neural tube and undergo extensive migration and proliferation in order to colonize the entire length of the gut and differentiate into many millions of neurons and glial cells. Although apoptotic programmed cell death is an essential physiological process during development of the majority of the vertebrate nervous system, apoptosis within early ENS development has not been comprehensively investigated. The aim of this study was to determine the presence and extent of apoptosis within the vagal NCC population that gives rise to most of the ENS in the chick embryo. We demonstrated that apoptotic cells, as shown by terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling and active caspase-3 immunoreactivity, are present within an electroporated green fluorescent protein (GFP) and human natural killer-1 (HNK-1) immunopositive NCC population migrating from the vagal region of the neural tube to the developing foregut. Inhibition of caspase activity in vagal NCC, by electroporation with a dominant-negative form of caspase-9, increased the number of vagal NCC available for ENS formation, as shown by 3-dimensional reconstruction of serial GFP or HNK-1 labelled sections, and resulted in hyperganglionosis within the proximal foregut, as shown by NADPH-diaphorase whole gut staining. These findings suggest that apoptotic cell death may be a normal process within the precursor pool of pre-enteric NCC that migrates to the gut, and as such it may play a role in the control of ENS formation.

Effects of NGF, NT-3 and GDNF family members on neurite outgrowth and migration from pelvic ganglia from embryonic and newborn mice

BackgroundPelvic ganglia are derived from the sacral neural crest and contain both sympathetic and parasympathetic neurons. Various members of the neurotrophin and GDNF families of neurotrophic factors have been shown to play important roles in the development of a variety of peripheral sympathetic and parasympathetic neurons; however, to date, the role of these factors in the development of pelvic ganglia has been limited to postnatal and older ages. We examined the effects of NGF, NT-3, GDNF, neurturin and artemin on cell migration and neurite outgrowth from explants of the pelvic ganglia from embryonic and newborn mice grown on collagen gels, and correlated the responses with the immunohistochemical localization of the relevant receptors in fixed tissue.ResultsCell migration assays showed that GDNF strongly stimulated migration of tyrosine hydroxylase (TH) cells of pelvic ganglia from E11.5, E14.5 and P0 mice. Other factors also promoted TH cell migration, although to a lesser extent and only at discrete developmental stages. The cells and neurites of the pelvic ganglia were responsive to each of the GDNF family ligands – GDNF, neurturin and artemin – from E11.5 onwards. In contrast, NGF and NT-3 did not elicit a significant neurite outgrowth effect until E14.5 onwards. Artemin and NGF promoted significant outgrowth of sympathetic (TH+) neurites only, whereas neurturin affected primarily parasympathetic (TH-negative) neurite outgrowth, and GDNF and NT-3 enhanced both sympathetic and parasympathetic neurite outgrowth. In comparison, collagen gel assays using gut explants from E11.5 and E14.5 mice showed neurite outgrowth only in response to GDNF at E11.5 and to neurturin only in E14.5 mice.ConclusionOur data show that there are both age-dependent and neuron type-dependent differences in the responsiveness of embryonic and neo-natal pelvic ganglion neurons to growth factors.

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

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

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.

Colonisation of the gut by neural crest‐derived enteric nervous system precursors: migration pathways and developmental potentials

The neurons and glia of the enteric nervous system (ENS) are derived from vagal and sacral neural crest cells (NCC). Vagal (hindbrain) NCC migrate from the dorsal neural tube, accumulate in the caudal branchial arches, enter the foregut and colonise the entire gut in a rostro-caudal direction, reaching the terminal hindgut of the chick at embryonic day 7.5. Sacral NCC, which arise caudal to the 28th pair of somites, initially form the nerve of Remak in the chick, then enter the gut after it has been colonised by vagal cells. The contribution of sacral cells to the ENS is restricted to the post-umbilical gut, and mainly the hindgut. It therefore appears that vagal and sacral NCC have different ENS developmental potentials, since vagal cells form most of the ENS along the entire gut, whereas sacral NCC provide a limited number of cells to the hindgut. This idea is further supported by heterotopic grafting experiments: transplanting vagal NCC into the sacral region results in rapid and extensive gut colonisation, whereas transplanting sacral cells into the vagal region results in diminished colonisation, compared with vagal NCC. To better understand the differences in ENS developmental potentials between these two populations, we analysed the expression of ENS signalling molecules in vagal and sacral NCC, and show that increasing the expression of the receptor tyrosine kinase RET in sacral NCC leads to increased gut colonisation.

The early migratory pathways of mouse sacral neural crest cells

The early migratory pathways of mouse sacral neural crest cells

Vagal neural crest contribution to the chick embryo cloaca

Intrinsic innervation of the developing chick cloaca is provided by the enteric nervous system, a network of neurons and glia that lies within its walls. The enteric nervous system originates from neural crest cells that migrate from the vagal and sacral regions of the neural tube during the early stages of development. Abnormal cloacal development can cause a number of anorectal anomalies including persistent cloaca. Our study aimed to investigate the contribution of vagal neural crest cells to the total population of enteric neurons and glia within the chick embryo cloaca, using quail-chick chimeras. Chicken embryos were incubated until the 10-12 somite stage (ss). The vagal neural tube, corresponding to somites 1-7, was then microsurgically ablated in ovo and isochronic and isotopic quail grafts were performed. The eggs were then reincubated until embryos were harvested at E12. Whole embryos were fixed in Bouin's fluid, embedded in paraffin wax and sectioned. Immunohistochemistry was carried out using the HNK-1 antibody to label all neural crest cells, and the quail-specific antibody, QCPN, to label quail cells. QCPN-immunoreactive cells were seen to make up a large proportion of enteric neurons and glia within the walls of the embryonic cloaca. HNK-1 labelled all neural crest cells in the myenteric and submucosal plexuses as well as the sacral crest-derived nerve of Remak, while QCPN-positive cells were evident in both plexuses but mostly in the submucosal plexus, where they appeared to make up the majority of neurons. Results show that the chick embryo cloaca is primarily innervated by vagal neural crest cells. Further studies to investigate the contribution of sacral neural crest cells to the same region will give further insight into the development of the enteric nervous system within the embryonic cloaca.

Pelvic plexus contributes ganglion cells to the hindgut enteric nervous system

The hindgut enteric nervous system (ENS) contains cells originating from vagal and sacral neural crest. In avians, the sacral crest gives rise to the nerve of Remak (NoR) and pelvic plexus. Whereas the NoR has been suggested to serve as the source of sacral crest-derived cells to the gut, the contribution of the pelvic ganglia is unknown. The purpose of this study was to test the hypothesis that the pelvic ganglia contribute ganglion cells to the hindgut ENS. We observed that the quail pelvic plexus develops from neural crest-derived cells that aggregate around the cloaca at embryonic day 5. Using chick-quail tissue recombinations, we found that hindgut grafts did not contain enteric ganglia unless the pelvic plexus was included. Neurofibers extended from the NoR into the intestine, but no ganglion cell contribution from the NoR was identified. These results demonstrate that the pelvic plexus, and not the NoR, serves as the staging area for sacral crest-derived cells to enter the avian hindgut, confirming the evolutionary conservation of this important embryologic process.

Lack of β1 integrins in enteric neural crest cells leads to a Hirschsprung-like phenotype

The enteric nervous system arises mainly from vagal and sacral neural crest cells that colonise the gut between 9.5 and 14 days of development in mice. Using the Cre-LoxP system, we removed beta1 integrins in the neural crest cells when they emerge from the neural tube. beta1-null enteric neural crest cells fail to colonise the gut completely, leading to an aganglionosis of the descending colon, which resembles the human Hirschsprung's disease. Moreover, beta1-null enteric neural crest cells form abnormal aggregates in the gut wall, leading to a severe alteration of the ganglia network organisation. Organotypic cultures of gut explants reveal that beta1-null enteric neural crest cells show impaired adhesion on extracellular matrix and enhanced intercellular adhesion properties. They display migration defects in collagen gels and gut tissue environments. We also provide evidence that beta1 integrins are required for the villi innervation in the small intestine. Our findings highlight the crucial roles played by beta1 integrins at various steps of enteric nervous system development.

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 DbetaH-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.

The effect of vagal neural crest ablation on the chick embryo cloaca

The cloaca, the caudal limit of the avian gastrointestinal tract, acts as a collecting chamber into which the gastrointestinal, urinary, and genital tracts discharge. It is intrinsically innervated by the enteric nervous system, which is derived from neural crest emigres that migrate from the vagal and sacral regions of the neural tube. Abnormal cloacal development can cause a number of anorectal anomalies, including persistent cloaca. Ablation of the vagal neural crest has previously been shown to result in an aganglionic hindgut to the extent of the colorectum. The aim of our study was to investigate the effect of vagal neural crest ablation on the cloaca, the limit of the hindgut in the developing chick embryo. Chick embryos were incubated until the 10-12 somite stage. The vagal neural tube corresponding to the level of somites 3-6 was then ablated, and eggs were incubated until harvested on embryonic day 11 (E11). Whole chick embryos were fixed, embedded in paraffin, and sectioned. Immunohistochemistry was then carried out using the HNK-1 monoclonal antibody to label neural crest cells, and results were assessed by light microscopy. Vagal neural crest ablation resulted in a dramatic decrease in the number of neural crest cells colonizing the chick embryo cloaca compared with control embryos. Ablated embryos contained only a small number of HNK-1-positive neural crest cells, which were scattered within the myenteric plexus in a disorganised pattern. Hypoganglionosis was also evident in other regions of the hindgut in ablated embryos. Ablation of the vagal neural crest results in a hypoganglionic cloaca in addition to hypoganglionosis of the hindgut. These results suggest that the cloaca is largely innervated by vagal neural crest emigres. Further studies involving quail-chick chimeras to investigate the exact contribution provided by both vagal and sacral neural crest cells to the cloaca should increase our understanding of the pathophysiology of conditions like persistent cloaca.

Enteric neural crest-derived cells and neural stem cells: biology and therapeutic potential.

The enteric nervous system arises from two regions of the neural crest; the vagal neural crest which gives rise to the vast majority of enteric neurones throughout the gastrointestinal tract, and the sacral neural crest which contributes a smaller number of cells that are mainly distributed within the hindgut. The migration of vagal neural crest cells into, and along the gut is promoted by GDNF, which is expressed by the gut mesenchyme and is the ligand for the Ret/GFRalpha1 signalling complex present on migrating vagal-derived crest cells. Sacral neural crest cells enter the gut after it has been colonized by vagal neural crest cells, but the molecular control of sacral neural crest cell development has yet to be elucidated. Under the influence of both intrinsic and extrinsic cues, neural crest cells differentiate into glia and different types of enteric neurones at different developmental stages. Recently, the potential for neural stem cells to form an enteric nervous system has been examined, with the ultimate aim of using neural stem cells as a therapeutic strategy for some gut disorders where enteric neurones are reduced or absent.

Enteric nervous system patterning in the avian hindgut.

The enteric nervous system (ENS) is principally derived from vagal and sacral neural crest cells that migrate throughout the gastrointestinal tract before differentiating into neurons and glia. These cells form two concentric rings of ganglia and regulate intestinal motility, absorption, and secretion. Abnormalities of ENS development can lead to disorders of intestinal function, including Hirschsprung's disease. These disorders are generally limited to the distal hindgut, suggesting unique features to development of this region. This study characterized the normal spatiotemporal development of the ENS within the avian hindgut. Neural crest cells begin to populate the hindgut at E8, with patterning of both plexuses complete by embryonic day 9. Crest-derived cells arrive in the submucosal layer before the myenteric layer, as well as differentiate to a neuronal phenotype first. The cloaca demonstrates a unique pattern, characterized by a disorganized myenteric plexus and a flattened nerve of Remak. Detailed understanding of normal avian hindgut ENS development will allow better utilization of this model system to study abnormalities of the intestinal nervous system.

Dissimilar regulation of cell differentiation in mesencephalic (cranial) and sacral (trunk) neural crest cells in vitro.

During development neural crest cells give rise to a wide variety of specialized cell types in response to cytokines from surrounding tissues. Depending on the cranial-caudal level of their origin, different populations of neural crest cells exhibit differential competence to respond to these signals as exemplified by the unique ability of cranial neural crest to form skeletal cell types. We show that in addition to differences in whether they respond to particular signals, cranial neural crest cells differ dramatically from the trunk neural crest cells in how they respond to specific extracellular signals, such that under identical conditions the same signal induces dissimilar cell fate decisions in the two populations in vitro. Conversely, the same differentiated cell types are induced by different signals in the two populations. These in vitro differences in neural crest response are consistent with in vivo manipulations. We also provide evidence that these differences in responsiveness are modulated, at least in part, by differential expression of Hox genes within the neural crest.

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.

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Enteric neural crest‐derived cells: Origin, identification, migration, and differentiation

Enteric nervous system development: analysis of the selective developmental potentialities of vagal and sacral neural crest cells using quail-chick chimeras.

The majority of the enteric nervous system (ENS) is derived from vagal neural crest cells (NCC). For many years, the contribution from a second region of the neuraxis (the sacral neural crest) to the ENS has been less clear, with conflicting reports appearing in the literature. To resolve this longstanding issue, we documented the spatiotemporal migration and differentiation of vagal and sacral-derived NCC within the developing chick embryo using quail-chick grafting and antibody labelling. Results showed that vagal NCC colonised the entire length of the gut in a rostrocaudal direction. The hindgut, the region of the gastrointestinal tract most frequently affected in developmental disorders, was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before colonising the myenteric plexus region. In contrast, sacral NCC, which colonised the hindgut in a caudorostral direction, were primarily located in the myenteric plexus region from where they subsequently migrated to the submucosa. We also observed that sacral NCC migrated into the hindgut in significant numbers only after vagal-derived cells had colonised the entire length of the gut. This suggested that to participate in ENS formation, sacral cells may require an interaction with vagal-derived cells, or with factors or signalling molecules released by them or their progeny. To investigate this possible inter-relationship, we ablated sections of vagal neural crest (NC) to prevent the rostrocaudal migration of ENS precursors and, thus, create an aganglionic hindgut model. In the same NC ablated animals, quail-chick sacral NC grafts were performed. In the absence of vagal-derived ganglia, sacral NCC migrated and differentiated in an apparently normal manner. Although the numbers of sacral cells within the hindgut was slightly higher in the absence of vagal-derived cells, the increase was not sufficient to compensate for the lack of enteric ganglia. As vagal NCC appear to be more invasive than sacral NCC, since they colonise the entire length of the gut, we investigated the ability of transplanted vagal cells to colonise the hindgut by grafting the vagal NC into the sacral region. We found that when transplanted, vagal cells retained their invasive capacity and migrated into the hindgut in large numbers. Although sacral-derived cells normally contribute a relatively small number of precursors to the post-umbilical gut, many heterotopic vagal cells were found within the hindgut enteric plexuses at much earlier stages of development than normal. Heterotopic grafting of invasive vagal NCC into the sacral neuraxis may, therefore, be a means of rescuing an aganglionic hindgut phenotype.

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 DβH–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.

Transplantation of enteric nervous system precursors from the vagal to the sacral neural crest region results in extensive hindgut colonisation

Transplantation of enteric nervous system precursors from the vagal to the sacral neural crest region results in extensive hindgut colonisation