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.     

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.     

Intestinal smooth muscle is required for patterning the enteric nervous system

The development of the enteric nervous system (ENS) and intestinal smooth muscle occurs in a spatially and temporally correlated manner, but how they influence each other is unknown. In the developing mid‐gut of the chick embryo, we find that α‐smooth muscle actin expression, indicating early muscle differentiation, occurs after the arrival of migrating enteric neural crest‐derived cells (ENCCs). In contrast, hindgut smooth muscle develops prior to ENCC arrival. Smooth muscle development is normal in experimentally aganglionic hindguts, suggesting that proper development and patterning of the muscle layers does not rely on the ENS. However, inhibiting early smooth muscle development severely disrupts ENS patterning without affecting ENCC proliferation or apoptosis. Our results demonstrate that early intestinal smooth muscle differentiation is required for patterning the developing ENS.     

Trans-mesenteric neural crest cells are the principal source of the colonic enteric nervous system

Cell migration is fundamental to organogenesis. During development, the enteric neural crest cells (ENCCs) that give rise to the enteric nervous system (ENS) migrate and colonize the entire length of the gut, which undergoes substantial growth and morphological rearrangement. How ENCCs adapt to such changes during migration, however, is not fully understood. Using time-lapse imaging analyses of mouse ENCCs, we show that a population of ENCCs crosses from the midgut to the hindgut via the mesentery during a developmental time period in which these gut regions are transiently juxtaposed, and that such 'trans-mesenteric' ENCCs constitute a large part of the hindgut ENS. This migratory process requires GDNF signaling, and evidence suggests that impaired trans-mesenteric migration of ENCCs may underlie the pathogenesis of Hirschsprung disease (intestinal aganglionosis). The discovery of this trans-mesenteric ENCC population provides a basis for improving our understanding of ENS development and pathogenesis.     

Lumbo-sacral neural crest contributes to the avian enteric nervous system independently of vagal neural crest.

Most of the avian enteric nervous system is derived from the vagal neural crest, but a minority of the neural cells in the hindgut, and to an even lesser extent in the midgut, are of lumbo-sacral crest origin. Since the lumbo-sacral contribution was not detected or deemed negligible in the absence of vagal cells, it had been hypothesised that lumbo-sacral neural crest cells require vagal crest cells to contribute to the enteric nervous system. In contrast, zonal aganglionosis, a rare congenital human bowel disease led to the opposite suggestion, that lumbo-sacral cells could compensate for the absence of vagal cells to construct a complete enteric nervous system. To test these notions, we combined E4 chick midgut and hindgut, isolated prior to arrival of neural precursors, with E1. 7 chick vagal and/or E2.7 quail lumbo-sacral neural tube as crest donors, and grafted these to the chorio-allantoic membrane of E9 chick hosts. Double and triple immuno-labelling for quail cells (QCPNA), neural crest cells (HNK-1), neurons and neurites (neurofilament) and glial cells (GFAP) indicated that vagal crest cells produced neurons and glia in large ganglia throughout the entire intestinal tissues. Lumbo-sacral crest contributed small numbers of neurons and glial cells in the presence or absence of vagal cells, chiefly in colorectum, but not in nearby small intestinal tissue. Thus for production of enteric neural cells the avian lumbo-sacral neural crest neither requires the vagal neural crest, nor significantly compensates for its lack. However, enteric neurogenesis of lumbo-sacral cells requires the hindgut microenvironment, whereas that of vagal cells is not restricted to a particular intestinal region.     

Enteric glial cells express full-length TrkB and depend on TrkB expression for normal development

The embryonic development of the enteric nervous system (ENS) from neural crest precursor cells requires neurotrophic signaling. Neurotrophins (NTs) are a family of growth factors that bind Trk receptors to signal diverse functions, including development and maintenance of different cell populations in the peripheral nervous system. In this study we investigated the expression and cell localization of TrkB, the high affinity receptor for brain-derived neurotrophic factor and NT-4, in the murine ENS using Western blot and immunohistochemistry. The results demonstrate that enteric glial cells within the ENS express full-length TrkB at all stages tested. The ENS of TrkB deficient mice have reduced expression of glial cell markers, and a disarrangement of glial cells and the plexular neuropil. These results strongly suggest TrkB has essential roles in the normal development and maintenance of glial cells in the ENS.     

Immunophenotypic characterization of enteric neural crest cells in the developing avian colorectum

Background: The enteric nervous system (ENS) develops from neural crest‐derived cells that migrate along the intestine to form two plexuses of neurons and glia. While the major features of ENS development are conserved across species, minor differences exist, especially in the colorectum. Given the embryologic and disease‐related importance of the distal ENS, the aim of this study was to characterize the migration and differentiation of enteric neural crest‐derived cells (ENCCs) in the colorectum of avian embryos. Results: Using normal chick embryos and vagal neural tube transplants from green fluorescent protein (GFP) ‐transgenic chick embryos, we find ENCCs entering the colon at embryonic day (E) 6.5, with colonization complete by E8. Undifferentiated ENCCs at the wavefront express HNK‐1, N‐cadherin, Sox10, p75, and L1CAM. By E7, differentiation begins in the proximal colon, with L1CAM and Sox10 becoming restricted to neuronal and glial lineages, respectively. By E8, multiple markers of differentiation are expressed along the entire colorectum. Conclusions: Our results establish the pattern of ENCC migration and differentiation in the chick colorectum, demonstrate the conservation of marker expression across species, highlight a range of markers, including neuronal cell adhesion molecules, which label cells at the wavefront, and provide a framework for future studies in avian ENS development. Developmental Dynamics 241:842–851, 2012. © 2012 Wiley Periodicals, Inc.     

Enteric Neurogenesis During Life Span Under Physiological and Pathophysiological Conditions

The enteric nervous system (ENS) controls gastrointestinal key functions and is mainly characterized by two ganglionated plexus located in the gut wall: the myenteric plexus and the submucous plexus. The ENS harbors a high number and diversity of enteric neurons and glial cells, which generate neuronal circuitry to regulate intestinal physiology. In the past few years, the pivotal role of enteric neurons in the underlying mechanism of several intestinal diseases was revealed. Intestinal diseases are associated with neuronal death that could in turn compromise intestinal functionality. Enteric neurogenesis and regeneration is therefore a crucial aspect within the ENS and could be revealed not only during embryogenesis and early postnatal periods, but also in the adulthood. Enteric glia and/or enteric neural precursor/progenitor cells differentiate into enteric neurons, both under homeostatic and pathologic conditions beyond the perinatal period. The unique role of the intestinal microbiota and serotonin signaling in postnatal and adult neurogenesis has been shown by several studies in health and disease. In this review article, we will mainly focus on different recent studies, which advanced the concept of postnatal and adult ENS neurogenesis. Moreover, we will discuss the key factors and underlying mechanisms, which promote enteric neurogenesis. Finally, we will shortly describe neurogenesis of transplanted enteric neural progenitor cells. Anat Rec, 302:1345–1353, 2019. © 2019 Wiley Periodicals, Inc.     

Hoxb3 vagal neural crest-specific enhancer element for controlling enteric nervous system development.

The neural and glial cells of the intrinsic ganglia of the enteric nervous system (ENS) are derived from the hindbrain neural crest at the vagal level. The Hoxb3 gene is expressed in the vagal neural crest and in the enteric ganglia of the developing gut during embryogenesis. We have identified a cis-acting enhancer element b3IIIa in the Hoxb3 gene locus. In this study, by transgenic mice analysis, we examined the tissue specificity of the b3IIIa enhancer element using the lacZ reporter gene, with emphasis on the vagal neural crest cells and their derivatives in the developing gut. We found that the b3IIIa-lacZ transgene marks only the vagal region and not the trunk or sacral region. Using cellular markers, we showed that the b3IIIa-lacZ transgene was expressed in a subset of enteric neuroblasts during early development of the gut, and the expression was maintained in differentiated neurons of the myenteric plexus at later stages. The specificity of the b3IIIa enhancer in directing gene expression in the developing ENS was further supported by genetic analysis using the Dom mutant, a spontaneous mouse model of Hirschsprung's disease characterized by the absence of enteric ganglia in the distal gut. The colonization of lacZ-expressing cells in the large intestine was incomplete in all the Dom/b3IIIa-lacZ hybrid mutants we examined. To our knowledge, this is the only vagal neural crest-specific genetic regulatory element identified to date. This element could be used for a variety of genetic manipulations and in establishing transgenic mouse models for studying the development of the ENS.     

Gut feelings: Studying enteric nervous system development,function, and disease in the zebrafish model system

The enteric nervous system (ENS) is the largest part of the peripheral nervous system and is entirely neural crest–derived. It provides the intrinsic innervation of the gut, controlling different aspects of gut function, such as motility. In this review, we will discuss key points of Zebrafish ENS development, genes, and signaling pathways regulating ENS development, as well as contributions of the Zebrafish model system to better understand ENS disorders. During their migration, enteric progenitor cells (EPCs) display a gradient of developmental states based on their proliferative and migratory characteristics, and show spatiotemporal heterogeneity based on gene expression patterns. Many genes and signaling pathways that regulate the migration and proliferation of EPCs have been identified, but later stages of ENS development, especially steps of neuronal and glial differentiation, remain poorly understood. In recent years, Zebrafish have become increasingly important to test candidate genes for ENS disorders (e.g., from genome‐wide association studies), to identify environmental influences on ENS development (e.g., through large‐scale drug screens), and to investigate the role the gut microbiota play in ENS development and disease. With its unique advantages as a model organism, Zebrafish will continue to contribute to a better understanding of ENS development, function, and disease. Developmental Dynamics 247:268–278, 2018. © 2017 Wiley Periodicals, Inc.     

Gdnf is mitogenic,neurotrophic, and chemoattractive to enteric neural crest cells in the embryonic colon

Glial‐derived neurotrophic factor (Gdnf) is required for morphogenesis of the enteric nervous system (ENS) and it has been shown to regulate proliferation, differentiation, and survival of cultured enteric neural crest–derived cells (ENCCs). The goal of this study was to investigate its in vivo role in the colon, the site most commonly affected by intestinal neuropathies such as Hirschsprung's disease. Gdnf activity was modulated in ovo in the distal gut of avian embryos using targeted retrovirus‐mediated gene overexpression and retroviral vector‐based gene silencing. We find that Gdnf has a pleiotropic effect on colonic ENCCs, promoting proliferation, inducing neuronal differentiation, and acting as a chemoattractant. Down‐regulating Gdnf similarly induces premature neuronal differentiation, but also inhibits ENCC proliferation, leading to distal colorectal aganglionosis with severe proximal hypoganglionosis. These results indicate an important role for Gdnf signaling in colonic ENS formation and emphasize the critical balance between proliferation and differentiation in the developing ENS. Developmental Dynamics 240:1402–1411, 2011. © 2011 Wiley‐Liss, Inc.     

The single nucleotide polymorphisms in Smad-interacting protein 1 gene contribute to its ectopic expression and susceptibility in Hirschsprung's disease

Hirschsprung's disease (HSCR) is the third most common congenital disorder of the gastrointestinal tract. It is an anomalous enteric nervous system (ENS) characterized by the absence of ganglion cells in the myenteric and submucosal plexuses. It has been reported that the Smad-interacting protein 1(SIP1) is critical in embryonic development of ENS for its regulation on neural crest cells. In the present study, we analyzed 3 polymorphisms of the SIP1 gene rs41292293 (exon5), rs34961586 (exon6) and rs13017697 (exon8) to determine their potential contributions to the susceptibility of HSCR. Allele frequencies and genotype distributions were analyzed by sequence analysis in 107 HSCR patients and 107 normal controls. The SIP1 expression was carried out by using real-time PCR, western blot and immunohistochemistry. Polymorphic analysis indicated that the genotype distributions and allele frequencies in SIP1 gene rs41292293, rs34961586 and rs13017697 were statistically different between HSCR and normal controls. The expression analysis revealed that SIP1 was ectopically expressed in the aganglionic segments; neither the mRNA nor the protein levels demonstrated that the difference compared with those was in the normal segments. In conclusion, the single nucleotide polymorphisms in SIP1 gene rs41292293, rs34961586 and rs13017697 are associated with the ectopic expression of this gene in human HSCR and contribute to the susceptibility of this disease in population.     

BMPRIA is a promising marker for evaluating ganglion cells in the enteric nervous system--a pilot study

Congenital disorders of the enteric nervous system (ENS) comprise a large group of conditions characterized by abnormalities in the number, size, or location of enteric ganglia. Their diagnosis requires careful histological evaluation of intestinal biopsies to determine the presence and morphology of these cells. Based on the recently discovered role of bone morphogenetic proteins (BMPs) in ENS development, we examined the expression of the ligands, BMP2 and BMP4, and their receptors, BMPRIA, BMPRIB, and BMPRII, during formation of the human ENS. The spatiotemporal expression pattern of these proteins suggests a role for BMP signaling in human ENS formation. We find BMPRIA, in particular, strongly and specifically expressed in all ganglion cells of the ENS at every age examined, from fetus to adult. Moreover, BMPRIA immunohistochemistry consistently allowed the identification of ganglion cells in rectal biopsies from patients with Hirschsprung disease, intestinal neuronal dysplasia, and immature ganglion cells. We propose that BMPRIA immunohistochemistry may be a promising new tool for the identification of enteric ganglion cells in the evaluation of patients with neurointestinal disorders.     

Local modulation of intestinal ion transport by enteric neurons

Principles of autonomic nervous system control of intestinal ion transport need to include the newer concepts of the enteric nervous system (ENS). Based on studies of nervous control of the myenteric plexus, it is likely that ENS control of intestinal transport occurs through local mechanisms. In vitro transport studies and a limited number of radioreceptor-binding studies in mucosal cells support the notion that putative neurotransmitters alter transport by acting directly with mucosal receptors. In vivo and in vitro studies cannot alone uncover the indirect transport effects that neurotransmitters may have when they interact with enteric neurons. Studies focused on uptake and release of neurotransmitters suggest that norepinephrine (NE)-induced absorption may be modulated by local NE presynaptic neuronal mechanisms. Endogenous NE release may be enhanced by nicotinic and angiotensin II agents but decreased by muscarinic and alpha-adrenergic agents or prostaglandins. Presynaptic neuronal mechanisms that modulate endogenous acetylcholine (ACh) release and ACh-induced secretion are less well defined. Intestinal transport may be controlled by negative feedback, interneuronal, or transsynaptic presynaptic mechanisms. We propose that transport is controlled by a balance between the principal neurotransmitters NE and ACh. These neurotransmitters may be modulated by neuroactive peptides located either in neurons or in enteroendocrine cells. Efferent neurons may modulate release of neuropeptides from enteroendocrine cells into the luminal or antiluminal sides of mucosal cells. Intestinal transport also may be controlled by luminal factors that cause neuropeptide release from enteroendocrine cells or by specialized luminal receptors acting on sensory afferent neurons and intrinsic neuronal reflexes. Therefore, local modulation of intestinal transport by the ENS represents a finely tuned neuronal system with complex interrelations similar to many found in the central nervous system.     

Spatiotemporal mapping of vascularization and innervation in the fetal murine intestine

Background : In mice, the intestinal tube develops from the splanchopleure before embryonic day 9.5. Subsequent patterning of nerves and blood vessels is critical for normal digestive function. A hierarchical branching vascular network allows for efficient nutrient absorption, while the complex enteric nervous system regulates intestinal motility as well as secretion, absorption, and blood flow. Despite the well‐recognized significance of these systems, the precise mechanisms by which they develop have not been clearly established in mammals. Results : Using a novel whole‐mount immunohistochemical protocol, we visualize the pattern of intestinal neurovascular development in mice between embryonic day 10.5 and birth. In particular, we focus on the development and remodeling of the enteric vascular plexus, the migration and organization of enteric neural crest‐derived cells, and the integration of peripheral sympathetic nerves with the enteric nervous system. These correlative data lead us to hypothesize a functional interaction between migrating neural crest‐derived cells and endothelial cells of the primary capillary plexus, as well as a subsequent interaction between developing peripheral autonomic nerves and differentiated neural crest‐derived cells. Conclusions: These studies provide useful anatomical data for continuing investigations on the functional mechanisms underlying intestinal organogenesis. Developmental Dynamics 244:56–68, 2015. Published 2014. This article is a U.S. Government work and is in the public domain in the USA     

神经生长因子通路调节大鼠结肠的肠神经系统可塑性

目的探讨神经生长因子（NGF）是否可以诱导新生期母婴分离（NMS）模型下的肠神经系统（ENS）可塑性。方法雄性SD大鼠出生后行NMS。每天NMS前10min腹腔注射K252a（非特异性神经生长因子受体TrK拮抗剂）阻断NGF信号,对正常新生鼠每天注射NGF模拟NMS诱导的肠神经可塑性。8周后腹壁撤退反射检测内脏痛觉过敏。通过铺片技术和免疫荧光技术。比较各组近端结肠神经节（HuD阳性细胞）大小和数目以及胶质细胞的变化。检测肌间神经丛和粘膜下神经丛肠神经递质类型（ChAT,VIP,nNOS,Cab,TrKA,P75阳性细胞）,分析神经递质的可塑性变化。结果新生期应激可致成年鼠内脏敏感性增高,NGF可诱导内脏敏感性增高,K252a能使之部分缓解。NGF可以诱导部分神经结构重排、近端肌间神经丛ChAT的增高,所有结果经统计学分析,差异有统计学意义。结论早期生活事件是引起成年后肠神经系统可塑性改变的重要原因。这种可塑性变化可能是依赖NGF通路调节。     

Nervous system development in normal and atresic chick embryo intestine: an immunohistochemical study

Intestinal motility disorders are a common complication after surgery for neonatal intestinal atresia. Although intestinal atresia causes alterations in the enteric nervous system, especially in its inner structures (nervous fibers in the mucosa, submucous and deep muscular plexuses), how these alterations develop is unclear. The chick model is a useful research tool for investigating the ontogenesis of the enteric nervous system and the pathogenesis of congenital bowel diseases. More information is needed on the overlap between the developing enteric nervous system and intestinal atresia. Because vasoactive intestinal polypeptide and substance P are typical intestinal neuropeptides, and vasoactive intestinal polypeptide acts as a modulator in neurodevelopment and an inhibitor of smooth muscle cell proliferation, our aim in this study was to investigate the distribution of their immunoreactivity in the developing enteric nervous system of normal and experimental chick models. We studied gut specimens excised from normal chick embryos (aged 12–20 days) and experimental chick embryos (aged 15–20 days) that underwent surgical intervention on day 12 to induce intestinal atresia (atresic embryos) or simply to grasp the bowel loop (sham-operated embryos). In normal chick embryos we showed vasoactive intestinal polypeptide and substance P immunoreactivity from day 12 in the submucous and myenteric plexuses. The distribution of peptide immunoreactivity differed markedly in atresic and normal or sham-operated gut embryos. These differences especially affected the inner structures of the enteric nervous system of specimens proximal to atresia and were related to the severity of dilation. Because nerve structures in the gut wall mucosa and submucous and deep muscular plexuses play a role in motility control and stretch sensation in the intestinal wall, our findings in the chick embryo may help to explain how gut motility disorders develop after surgery for neonatal intestinal atresia.     

How to innervate a simple gut: familiar themes and unique aspects in the formation of the insect enteric nervous system.

Like the vertebrate enteric nervous system (ENS), the insect ENS consists of interconnected ganglia and nerve plexuses that control gut motility. However, the insect ENS lies superficially on the gut musculature, and its component cells can be individually imaged and manipulated within cultured embryos. Enteric neurons and glial precursors arise via epithelial-to-mesenchymal transitions that resemble the generation of neural crest cells and sensory placodes in vertebrates; most cells then migrate extensive distances before differentiating. A balance of proneural and neurogenic genes regulates the morphogenetic programs that produce distinct structures within the insect ENS. In vivo studies have also begun to decipher the mechanisms by which enteric neurons integrate multiple guidance cues to select their pathways. Despite important differences between the ENS of vertebrates and invertebrates, common features in their programs of neurogenesis, migration, and differentiation suggest that these relatively simple preparations may provide insights into similar developmental processes in more complex systems.