Sacral Neural Crest Research Articles

Single-cell profiling coupled with lineage analysis reveals vagal and sacral neural crest contributions to the developing enteric nervous system.

During development, much of the enteric nervous system (ENS) arises from the vagal neural crest that emerges from the caudal hindbrain and colonizes the entire gastrointestinal tract. However, a second ENS contribution comes from the sacral neural crest that arises in the caudal neural tube and populates the post-umbilical gut. By coupling single-cell transcriptomics with axial-level-specific lineage tracing in avian embryos, we compared the contributions of embryonic vagal and sacral neural crest cells to the chick ENS and the associated peripheral ganglia (Nerve of Remak and pelvic plexuses). At embryonic day (E) 10, the two neural crest populations form overlapping subsets of neuronal and glia cell types. Surprisingly, the post-umbilical vagal neural crest much more closely resembles the sacral neural crest than the pre-umbilical vagal neural crest. However, some differences in cluster types were noted between vagal and sacral derived cells. Notably, RNA trajectory analysis suggests that the vagal neural crest maintains a neuronal/glial progenitor pool, whereas this cluster is depleted in the E10 sacral neural crest which instead has numerous enteric glia. The present findings reveal sacral neural crest contributions to the hindgut and associated peripheral ganglia and highlight the potential influence of the local environment and/or developmental timing in differentiation of neural crest-derived cells in the developing ENS.

HPSC-derived sacral neural crest enables rescue in a severe model of Hirschsprung's disease.

The enteric nervous system (ENS) is derived from both the vagal and sacral component of the neural crest (NC). Here, we present the derivation of sacral ENS precursors from human PSCs via timed exposure to FGF, WNT, and GDF11, which enables posterior patterning and transition from posterior trunk to sacral NC identity, respectively. Using a SOX2::H2B-tdTomato/T::H2B-GFP dual reporter hPSC line, we demonstrate that both trunk and sacral NC emerge from a double-positive neuro-mesodermal progenitor (NMP). Vagal and sacral NC precursors yield distinct neuronal subtypes and migratory behaviors invitro and invivo. Remarkably, xenografting of both vagal and sacral NC lineages is required to rescue a mouse model of total aganglionosis, suggesting opportunities in the treatment of severe forms of Hirschsprung's disease.

The RET gene encodes RET protein, which triggers intracellular signaling pathways for enteric neurogenesis, and RET mutation results in Hirschsprung's disease.

Enteric neurons and ganglia are derived from vagal and sacral neural crest cells, which undergo migration from the neural tube to the gut wall. In the gut wall, they first undergo rostrocaudal migration followed by migration from the superficial to deep layers. After migration, they proliferate and differentiate into the enteric plexus. Expression of the Rearranged During Transfection (RET) gene and its protein RET plays a crucial role in the formation of enteric neurons. This review describes the molecular mechanism by which the RET gene and the RET protein influence the development of enteric neurons. Vagal neural crest cells give rise to enteric neurons and glia of the foregut and midgut while sacral neural crest cells give rise to neurons of the hindgut. Interaction of RET protein with its ligands (glial cell derived neurotrophic factor (GDNF), neurturin (NRTN), and artemin (ARTN)) and its co-receptors (GDNF receptor alpha proteins (GFRα1-4)) activates the Phosphoinositide-3-kinase-protein kinase B (PI3K-PKB/AKT), RAS mitogen-activated protein kinase (RAS/MAPK) and phospholipase Cγ (PLCγ) signaling pathways, which control the survival, migration, proliferation, differentiation, and maturation of the vagal and sacral neural crest cells into enteric neurons. Abnormalities of the RET gene result in Hirschsprung's disease.

Direct Interaction of Sox10 With Cadherin-19 Mediates Early Sacral Neural Crest Cell Migration: Implications for Enteric Nervous System Development Defects

The enteric nervous system, which regulates many gastrointestinal functions, is derived from neural crest cells (NCCs). Defective NCC migration during embryonic development may lead to enteric neuropathies such as Hirschsprung's disease (hindgut aganglionosis). Sox10 is known to be essential for cell migration but downstream molecular events regulating early NCC migration have not been fully elucidated. This study aimed to determine how Sox10 regulates migration of sacral NCCs toward the hindgut using Dominant megacolon mice, an animal model of Hirschsprung's disease with a Sox10 mutation. We used the following: time-lapse live cell imaging to determine the migration defects of mutant sacral NCCs; genome-wide microarrays, site-directed mutagenesis, and whole embryo culture to identify Sox10 targets; and liquid chromatography and tandem mass spectrometry to ascertain downstream effectors of Sox10. Sacral NCCs exhibited retarded migration to the distal hindgut in Sox10-null embryos with simultaneous down-regulated expression of cadherin-19 (Cdh19). Sox10 was found to bind directly to the Cdh19 promoter. Cdh19 knockdown resulted in retarded sacral NCC migration invitro and exvivo, whereas re-expression of Cdh19 partially rescued the retarded migration of mutant sacral NCCs invitro. Cdh19 formed cadherin-catenin complexes, which then bound to filamentous actin of the cytoskeleton during cell migration. Cdh19 is a direct target of Sox10 during early sacral NCC migration toward the hindgut and forms cadherin-catenin complexes which interact with the cytoskeleton in migrating cells. Elucidation of this novel molecular pathway helps to provide insights into the pathogenesis of enteric nervous system developmental defects.

5-HT3 Signaling Alters Development of Sacral Neural Crest Derivatives That Innervate the Lower Urinary Tract.

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.

Altered sacral neural crest development in Pax3 spina bifida mutants underlies deficits of bladder innervation and function

Mouse models of Spina bifida (SB) have been instrumental for identifying genes, developmental processes, and environmental factors that influence neurulation and neural tube closure. Beyond the prominent neural tube defects, other aspects of the nervous system can be affected in SB with significant changes in essential bodily functions such as urination. SB patients frequently experience bladder dysfunction and SB fetuses exhibit reduced density of bladder nerves and smooth muscle although the developmental origins of these deficits have not been determined. The Pax3 Splotch-delayed (Pax3Sp-d) mouse model of SB is one of a very few mouse SB models that survives to late stages of gestation. Through analysis of Pax3Sp-d mutants we sought to define how altered bladder innervation in SB might arise by tracing sacral neural crest (NC) development, pelvic ganglia neuronal differentiation, and assessing bladder nerve fiber density. In Pax3Sp-d/Sp-d fetal mice we observed delayed migration of Sox10+ NC-derived progenitors (NCPs), deficient pelvic ganglia neurogenesis, and reduced density of bladder wall innervation. We further combined NC-specific deletion of Pax3 with the constitutive Pax3Sp-d allele in an effort to generate viable Pax3 mutants to examine later stages of bladder innervation and postnatal bladder function. Neural crest specific deletion of a Pax3 flox allele, using a Sox10-cre driver, in combination with a constitutive Pax3Sp-d mutation produced postnatal viable offspring that exhibited altered bladder function as well as reduced bladder wall innervation and altered connectivity between accessory ganglia at the bladder neck. Combined, the results show that Pax3 plays critical roles within sacral NC that are essential for initiation of neurogenesis and differentiation of autonomic neurons within pelvic ganglia.

Neuron-Glia Interaction in the Developing and Adult Enteric Nervous System.

The enteric nervous system (ENS) constitutes the largest part of the peripheral nervous system. In recent years, ENS development and its neurogenetic capacity in homeostasis and allostasishave gained increasing attention. Developmentally, the neural precursors of the ENS are mainly derived from vagal and sacral neural crest cell portions. Furthermore, Schwann cell precursors, as well as endodermal pancreatic progenitors, participate in ENS formation. Neural precursors enherite three subpopulations: a bipotent neuron-glia, a neuronal-fated and a glial-fated subpopulation. Typically, enteric neural precursors migrate along the entire bowel to the anal end, chemoattracted by glial cell-derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) molecules. During migration, a fraction undergoes differentiation into neurons and glial cells. Differentiation is regulated by bone morphogenetic proteins (BMP), Hedgehog and Notch signalling. The fully formed adult ENS may react to injury and damage with neurogenesis and gliogenesis. Nevertheless, the origin of differentiating cells is currently under debate. Putative candidates are an embryonic-like enteric neural progenitor population, Schwann cell precursors and transdifferentiating glial cells. These cells can be isolated and propagated in culture as adult ENS progenitors and may be used for cell transplantation therapies for treating enteric aganglionosis in Chagas and Hirschsprung’s diseases.

PROX1 is a specific and dynamic marker of sacral neural crest cells in the chicken intestine.

The enteric nervous system (ENS) is a complex network constituted of neurons and glial cells that ensures the intrinsic innervation of the gastrointestinal tract. ENS cells originate from vagal and sacral neural crest cells that are initially located at the border of the neural tube. In birds, sacral neural crest cells (sNCCs) first give rise to an extramural ganglionated structure (the so-called Nerve of Remak [NoR]) and to the pelvic plexus. Later, sNCCs enter the colon mesenchyme to colonize and contribute to the intrinsic innervation of the caudal part of the gut. However, no specific sNCC marker has been described. Here, we report the expression pattern of prospero-related homeobox 1 (PROX1) in the developing chick colon. PROX1 is a homeobox domain transcription factor that plays a role in cell type specification in various tissues. Using in situ hybridization and immunofluorescence techniques, we showed that PROX1 is expressed in sNCCs localized in the NoR and in the pelvic plexus. Then, using real-time quantitative PCR we found that PROX1 displays a strong and highly dynamic expression pattern during NoR development. Moreover, we demonstrated using in vivo cell tracing, that sNCCs are the source of the PROX1-positive cells within the NoR. Our results indicate that PROX1 is the first marker that specifically identifies sNCCs. This might help to better identify the role of the different neural crest cell populations in distal gut innervation, and consequently to improve the diagnosis of diseases linked to incomplete ENS formation, such as Hirschsprung's disease.

Migration pathways of sacral neural crest during development of lower urogenital tract innervation

The migration and fate of cranial and vagal neural crest-derived progenitor cells (NCPCs) have been extensively studied; however, much less is known about sacral NCPCs particularly in regard to their distribution in the urogenital system. To construct a spatiotemporal map of NCPC migration pathways into the developing lower urinary tract, we utilized the Sox10-H2BVenus transgene to visualize NCPCs expressing Sox10. Our aim was to define the relationship of Sox10-expressing NCPCs relative to bladder innervation, smooth muscle differentiation, and vascularization through fetal development into adulthood. Sacral NCPC migration is a highly regimented, specifically timed process, with several potential regulatory mileposts. Neuronal differentiation occurs concomitantly with sacral NCPC migration, and neuronal cell bodies are present even before the pelvic ganglia coalesce. Sacral NCPCs reside within the pelvic ganglia anlagen through 13.5 days post coitum (dpc), after which they begin streaming into the bladder body in progressive waves. Smooth muscle differentiation and vascularization of the bladder initiate prior to innervation and appear to be independent processes. In adult bladder, the majority of Sox10+ cells express the glial marker S100β, consistent with Sox10 being a glial marker in other tissues. However, rare Sox10+ NCPCs are seen in close proximity to blood vessels and not all are S100β+, suggesting either glial heterogeneity or a potential nonglial role for Sox10+ cells along vasculature. Taken together, the developmental atlas of Sox10+ NCPC migration and distribution profile of these cells in adult bladder provided here will serve as a roadmap for future investigation in mouse models of lower urinary tract dysfunction.

The Role of Rho/ROCK Pathway on the Sacral Neural Crest Cell Migration in the Mouse Gut

The enteric nervous system (ENS) originates from neural crest cells (NCCs) at two different levels along the neuraxis: vagal (somites 1–7) and sacral (caudal to somite 24 in the mouse) levels. While the development of vagal NCCs has been widely studied, little has been known about sacral NCCs, particularly in mammals. Our objective was to test whether inhibition of small GTPases such as ROCK (an effector of Rho) affects sacral NCC migration in the mouse gut.First, cultured sacral NCCs from pelvic ganglia were treated with different concentrations of Y‐27632 to suppress active ROCK in vitro, and results showed that inhibition of ROCK significantly decreased neurite outgrowths and migration distances of sacral NCCs. Immunofluorescent localization demonstrated that the expression of cofilin which is a downstream protein of Rho/ROCK pathway remained unchanged while its phosphorylated form p‐cofilin (inactive state) was increased significantly. Expression of phalloidin which is a marker of stress fibers decreased after Y‐27632 treatment. Time‐lapse live cell imaging also showed that the speed and persistence of sacral NCC migration decreased, and the directional migration was disrupted after Y‐27632 treatment. Interestingly, expression of both phalloidin and p‐cofilin in sacral NCCs was restored to normal levels by Rho activator treatment.Next, we established an embryonic gut explant culture, where a pelvic ganglion (serving as a source of sacral NCCs) was recombined with a recipient hindgut segment ex vivo. Whole‐mount preparation and cryosection staining showed that the migration and differentiation of endogenous vagal NCCs in the hindgut explant cultured ex vivo was similar to those in the hindgut developed from E12.5 to E16.5 in vivo. Hence the gut culture explant system could support the normal development of the hindgut explant ex vivo. The recipient hindgut segment into which sacral NCCs from the pelvic ganglion had migrated 2 day after cultured ex vivo was then treated with different concentrations of Y‐27632. It was found that Y‐27632 reduced the migration distance, cell number and axonal extension of sacral NCCs within the hindgut segment. Moreover, time‐lapse live cell imaging also suggested that the speed and persistence of axonal extension of sacral NCCs decreased, and directional migration was disrupted after Y‐27632 treatment.In summary, these observations provide evidence that Rho/ROCK pathway regulates stress fiber formation and directional migration during sacral NCC migration.Support or Funding InformationThe work was supported by grants from RGC General Research Fund (Project Reference: 14102214) and RGC Theme‐based Research Scheme (Project Reference: T12C‐714/14‐R).

Molecular changes leading to the abnormal migration of sacral neural crest cells in the Dominant megacolon mutant, a mouse model of Hirschsprung's disease

Although the majority of neurons and glia of the enteric nervous system (ENS) are derived from neural crest cells (NCCs) at the vagal (cervico‐thoracic) level, NCCs at the sacral level are also able to migrate to the hindgut of the developing embryo and contribute significantly to the ENS of the colon. Abnormal migration of NCCs may lead to a loss of enteric neurons and glial cells in humans, which is called Hirschsprung's disease (HSCR), and also in the Dominant megacolon mouse (Dom), which is an animal model of HSCR carrying a spontaneous mutation of Sox10. In this study, we aimed to follow the migration of sacral NCCs in the mutant embryos and determine the molecular events that lead to the abnormal migration of sacral NCCs. It was found with in situ cell labeling and whole embryo culture that migrating sacral NCCs tended to aggregate, resulting in their delayed migration toward the hindgut. Genome‐wide profiling of RNA expression with microarrays showed that the expression of the adhesion molecule cadherin 19 (Cdh19) was drastically down‐regulated in mutant sacral NCCs as compared to that in the wild‐type. Results of in situ hybridization with wild‐type embryos at E9.5 to E11.5 indicated that Cdh19 was localized to migrating sacral NCCs and their derivatives which also expressed Sox10. With luciferase assay and chromatin immunoprecipitation, the expression of Cdh19 was found to be directly regulated by Sox10, and the binding site of Sox10 on the promoter of Cdh19 was also identified. When the expression of Cdh19 was knockdown in the primary culture of wild‐type sacral NCCs, their migration ability was reduced, and conversely, upon re‐expression of Cdh19 in the mutant sacral NCCs, their migration ability was restored to a level close to that of wild‐type sacral NCCs. Further experiments with immunoprecipitation showed that the Cdh19 expression modulated NCCs migration via interacting with catenins which are important regulators of the dynamic cytoskeleton of migrating NCC. In summary, the present study showed that the Sox10 mutation in the Dom mutant resulted in the delayed migration of sacral NCCs and significant down‐regulation of the expression of Cdh19 which was a direct downstream target of Sox10. Alterations of Cdh19 expression changed the migration ability of sacral NCCs through interactions with catenins which in turn regulated the dynamic cytoskeleton of migrating NCCs.Support or Funding InformationThis work was supported by a RGC General ResearchFund (CUHK14102214) and a Direct Research Grant from the Faculty of Medicine, TheChinese University of Hong Kong (2014.1.074).

Inactivation of Geminin in neural crest cells affects the generation and maintenance of enteric progenitor cells, leading to enteric aganglionosis

Neural crest cells comprise a multipotent, migratory cell population that generates a diverse array of cell and tissue types, during vertebrate development. Enteric Nervous System controls the function of the gastrointestinal tract and is mainly derived from the vagal and sacral neural crest cells. Deregulation on self-renewal and differentiation of the enteric neural crest cells is evident in enteric nervous system disorders, such as Hirschsprung disease, characterized by the absence of ganglia in a variable length of the distal bowel. Here we show that Geminin is essential for Enteric Nervous System generation as mice that lacked Geminin expression specifically in neural crest cells revealed decreased generation of vagal neural crest cells, and enteric neural crest cells (ENCCs). Geminin-deficient ENCCs showed increased apoptosis and decreased cell proliferation during the early stages of gut colonization. Furthermore, decreased number of committed ENCCs in vivo and the decreased self-renewal capacity of enteric progenitor cells in vitro, resulted in almost total aganglionosis resembling a severe case of Hirschsprung disease. Our results suggest that Geminin is an important regulator of self-renewal and survival of enteric nervous system progenitor cells.

Slit molecules prevent entrance of trunk neural crest cells in developing gut

Neural crest cells emerge from the dorsal neural tube early in development and give rise to sensory and sympathetic ganglia, adrenal cells, teeth, melanocytes and especially enteric nervous system. Several inhibitory molecules have been shown to play important roles in neural crest migration, among them are the chemorepulsive Slit1–3. It was known that Slits chemorepellants are expressed at the entry to the gut, and thus could play a role in the differential ability of vagal but not trunk neural crest cells to invade the gut and form enteric ganglia. Especially since trunk neural crest cells express Robo receptor while vagal do not. Thus, although we know that Robo mediates migration along the dorsal pathway in neural crest cells, we do not know if it is responsible in preventing their entry into the gut. The goal of this study was to further corroborate a role for Slit molecules in keeping trunk neural crest cells away from the gut. We observed that when we silenced Robo receptor in trunk neural crest, the sympathoadrenal (somites 18–24) were capable of invading gut mesenchyme in larger proportion than more rostral counterparts. The more rostral trunk neural crest tended not to migrate beyond the ventral aorta, suggesting that there are other repulsive molecules keeping them away from the gut. Interestingly, we also found that when we silenced Robo in sacral neural crest they did not wait for the arrival of vagal crest but entered the gut and migrated rostrally, suggesting that Slit molecules are the ones responsible for keeping them waiting at the hindgut mesenchyme. These combined results confirm that Slit molecules are responsible for keeping the timeliness of colonization of the gut by neural crest cells.

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.

Proximal Megacolon in an Adult

A44-year-old woman, born and residing in the United States, presented with medically nonresponsive chronic constipation, requiring 1 gallon of polyethylene glycol electrolyte solution weekly to induce bowel movements. She consistently experienced incomplete evacuation. She was hospitalized once for suspected sigmoid volvulus that spontaneously resolved. Physical examination showed a distended tympanitic abdomen with palpable stool in the left lower quadrant. Rectal examination revealed a high resting anal sphincter tone and no coordinated perineal descent during simulation of defecation; these features were confirmed on anorectal manometry (high squeeze anal sphincter pressure and requirement of 200 g to facilitate 50-mL balloon evacuation from the rectum). Gastric emptying was normal, and colonic transit was delayed. She was diagnosed with pelvic floor dyssynergia and symptoms improved somewhat after biofeedback therapy. A plain abdominal radiograph (Figure A) showed a dilated colon, confirmed on computerized tomography scan. Sagittal views of the abdominal computerized tomography (Figure B) showed an elongated, dilated proximal colon with a decompressed rectum. Direct endoscopic visualization of the intersection of the dilated and nondilated colon showed no mass or constrictions. Because of persistent symptoms and megacolon, she underwent colectomy at another hospital. Figure C shows the resected specimen with a dilated ascending and transverse colon, normal-caliber descending colon and rectum, and no constricted transition zone between dilated and normal-caliber segments. No pathologic examination of the colon was available. The enteric nervous system is derived from vagal and sacral neural crest cells. Vagal neural crest cells migrate caudally through the entire length of the gastrointestinal tract and provide the majority of neurons and glia. The sacral neural crest cells migrate rostrally and contribute to the innervation of the distal colon and rectum. Colonic dysganglionosis presents as a continuous spectrum, ranging anatomically from hypoganglionosis in colonic sections of different lengths and distributions to a localized constriction with proximal dilatation in Hirschsprung disease (HD). Hypoganglionosis is a decrease vs a complete lack of ganglia, and it can present as localized or generalized megacolon, or it may occur concomitantly with HD. These disorders present with long-term constipation refractory to medical therapy. Although approximately 95% of HD cases are diagnosed before the age of 5, less severe cases may present after 10 years of age and are considered adult HD.

Enteric nervous system specific deletion of Foxd3 disrupts glial cell differentiation and activates compensatory enteric progenitors

The enteric nervous system (ENS) arises from the coordinated migration, expansion and differentiation of vagal and sacral neural crest progenitor cells. During development, vagal neural crest cells enter the foregut and migrate in a rostro-to-caudal direction, colonizing the entire gastrointestinal tract and generating the majority of the ENS. Sacral neural crest contributes to a subset of enteric ganglia in the hindgut, colonizing the colon in a caudal-to-rostral wave. During this process, enteric neural crest-derived progenitors (ENPs) self-renew and begin expressing markers of neural and glial lineages as they populate the intestine. Our earlier work demonstrated that the transcription factor Foxd3 is required early in neural crest-derived progenitors for self-renewal, multipotency and establishment of multiple neural crest-derived cells and structures including the ENS. Here, we describe Foxd3 expression within the fetal and postnatal intestine: Foxd3 was strongly expressed in ENPs as they colonize the gastrointestinal tract and was progressively restricted to enteric glial cells. Using a novel Ednrb-iCre transgene to delete Foxd3 after vagal neural crest cells migrate into the midgut, we demonstrated a late temporal requirement for Foxd3 during ENS development. Lineage labeling of Ednrb-iCre expressing cells in Foxd3 mutant embryos revealed a reduction of ENPs throughout the gut and loss of Ednrb-iCre lineage cells in the distal colon. Although mutant mice were viable, defects in patterning and distribution of ENPs were associated with reduced proliferation and severe reduction of glial cells derived from the Ednrb-iCre lineage. Analyses of ENS-lineage and differentiation in mutant embryos suggested activation of a compensatory population of Foxd3-positive ENPs that did not express the Ednrb-iCre transgene. Our findings highlight the crucial roles played by Foxd3 during ENS development including progenitor proliferation, neural patterning, and glial differentiation and may help delineate distinct molecular programs controlling vagal versus sacral neural crest development.

Sacral neural crest‐derived cells enter the aganglionic colon of Ednrb−/− mice along extrinsic nerve fibers

Both vagal and sacral neural crest cells contribute to the enteric nervous system in the hindgut. Because it is difficult to visualize sacral crest cells independently of vagal crest, the nature and extent of the sacral crest contribution to the enteric nervous system are not well established in rodents. To overcome this problem we generated mice in which only the fluorescent protein-labeled sacral crest are present in the terminal colon. We found that sacral crest cells were associated with extrinsic nerve fibers. We investigated the source, time of appearance, and characteristics of the extrinsic nerve fibers found in the aganglionic colon. We observed that the pelvic ganglion neurons contributed a number of extrinsic fibers that travel within the hindgut between circular and longitudinal muscles and within the submucosa and serosa. Sacral crest-derived cells along these fibers diminished in number from fetal to postnatal stages. A small number of sacral crest-derived cells were found between the muscle layers and expressed the neuronal marker Hu. We conclude that sacral crest cells enter the hindgut by advancing on extrinsic fibers and, in aganglionic preparations, they form a small number of neurons at sites normally occupied by myenteric ganglia. We also examined the colons of ganglionated preparations and found sacral crest-derived cells associated with both extrinsic nerve fibers and nascent ganglia. Extrinsic nerve fibers serve as a route of entry for both rodent and avian sacral crest into the hindgut.

Migration and transcriptional profiling of sacral neural crest derivatives in the lower urinary tract

Migration and transcriptional profiling of sacral neural crest derivatives in the lower urinary tract

Analysis of the Sacral Neural Crest Cell Contribution to the Hindgut Enteric Nervous System in the Mouse Embryo

The majority of the enteric nervous system is derived from the vagal neural crest, with a second contribution, which is restricted to the post-umbilical gut, originating from the sacral neural crest. In mammals, although sacral neural crest cells (NCCs) have been shown to enter the hindgut, information on their development and role remains scant. Our aim was to determine the migratory routes of sacral NCCs to the hindgut, their timing and site of entry into the gut, and their migratory behaviors and differentiation within the hindgut. We used in situ cell labeling, whole embryo culture, immunofluorescence, organotypic culture, and time-lapse live-cell imaging in mouse embryos. Sacral NCCs emigrated from the neural tube at embryonic day 9.5, accumulated bilateral to the hindgut to form prospective pelvic ganglia at embryonic day 11.5, and from there entered the distal hindgut through its ventrolateral side at embryonic day 13.5. They then migrated along nerve fibers extending from the pelvic ganglia toward the proximal hindgut, intermingling with rostrocaudally migrating vagal NCCs to differentiate into neurons and glia. In organotypic culture, genetically labeled sacral and vagal NCCs displayed different capabilities of entering the hindgut, implying differences in their intrinsic migratory properties. Time-lapse live-cell imaging on explants ex vivo showed that sacral NCCs migrated along nerve fibers and exhibited different migratory behaviors from vagal NCCs. Murine sacral NCCs are a distinct group of cells that migrate along defined pathways from neural tube to hindgut. They exhibit discrete migratory behaviors within the gut mesenchyme and contribute neurons and glial cells to the hindgut enteric nervous system.

The development of excitatory neurons in the chick cloaca

The enteric nervous system of the chick embryo hindgut is derived from the vagal and sacral neural tube. Nerve cells from these regions produce various neuronal phenotypes, including inhibitory and excitatory nerve cells, providing intrinsic innervation to the chick embryo cloaca. We hypothesised that the vagal and sacral neural tubes provide the cloaca, with phenotypically similar nerve cells. The aim of our study was to investigate the origin of excitatory neurotransmission in the developing cloaca. Chicken embryos were incubated until the 10-12 somite stage (ss). To study the vagal neural tube contribution to the cloaca, this region was microsurgically ablated in ovo and replaced with the corresponding region from age-matched quail embryos. To study the sacral neural tube contribution to the cloaca, the vagal neural tube was ablated at the 10-12 ss, but not replaced with quail neural tube, thus, only the sacral neural crest cells remained. All embryos were harvested at E12 and E14, embedded in paraffin wax and serially sectioned. Immunohistochemistry was carried out on all embryos using human natural killer-1, quail non-chick perinuclear, choline acetyltransferase (ChAT) and substance P (SubP) antibodies. Choline acetyltransferase- and SubP-positive neurons were seen to originate in both the vagal and the sacral neural tube. The vagal neural tube provided the majority of the nerve cells to the chick embryo cloaca and expressed both ChAT and SubP in both the myenteric and submucosal plexus. The sacral neural tube contributed a lesser amount of nerve cells to the chick embryo cloaca, but was also seen to produce both ChAT- and SubP-positive nerve cells in both ganglionated plexus. This study shows, for the first time, that the excitatory ChAT- and SubP-expressing neurons in the developing cloaca originate in both the vagal and the sacral neural tube. These results highlight the origin of phenotypically similar nerve cells in both regions of the neural tube, providing new insights into the developmental origin of the intrinsic innervation of the persistent cloaca.