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.     

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.     

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.     

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.     

Effect of Gdnf haploinsufficiency on rate of migration and number of enteric neural crest-derived cells.

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into the foregut and then colonize the entire length of the gastrointestinal tract. Previous studies have demonstrated that glial cell line-derived neurotrophic factor (GDNF) promotes the migration of enteric neural crest-derived cells (ENCs) in vitro, but a role for GDNF in the migration of ENCs in vivo has yet to be demonstrated. In this study, the effects of Gdnf haploinsufficiency on ENC rate of migration and number during mid embryonic development were examined. Although the entire gut of embryonic Gdnf(+/-) mice was colonized, a significant delay in the migration of ENCs along the embryonic hindgut was found. However, significant effects of Gdnf haploinsufficiency on ENC number were detected before the stage at which migration defects were first evident. As previous studies have shown a relationship between ENC number and migration, the effects of Gdnf haploinsufficiency on migration may be due to an indirect effect on cell number and/or a direct effect of GDNF on ENC migration. Gdnf haploinsufficiency did not cause any detectable change in the rate of neuronal differentiation of ENCs.     

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.     

Balancing neural crest cell intrinsic processes with those of the microenvironment in Tcof1 haploinsufficient mice enables complete enteric nervous system formation

The enteric nervous system (ENS) comprises a complex neuronal network that regulates peristalsis of the gut wall and secretions into the lumen. The ENS is formed from a multipotent progenitor cell population called the neural crest, which is derived from the neuroepithelium. Neural crest cells (NCCs) migrate over incredible distances to colonize the entire length of the gut and during their migration they must survive, proliferate and ultimately differentiate. The absence of an ENS from variable lengths of the colon results in Hirschsprung's disease (HSCR) or colonic aganglionosis. Mutations in about 12 different genes have been identified in HSCR patients but the complex pattern of inheritance and variable penetrance suggests that additional genes or modifiers must be involved in the etiology and pathogenesis of this disease. We discovered that Tcof1 haploinsufficiency in mice models many of the early features of HSCR. Neuroepithelial apoptosis diminished the size of the neural stem cell pool resulting in reduced NCC numbers and their delayed migration along the gut from E10.5 to E14.5. Surprisingly however, we observe continued and complete colonization of the entire colon throughout E14.5-E18.5, a period in which the gut is considered to be non- or less-permissive to NCC. Thus, we reveal for the first time that reduced NCC progenitor numbers and delayed migration do not unequivocally equate with a predisposition for the pathogenesis of HSCR. In fact, these deficiencies can be overcome by balancing NCC intrinsic processes of proliferation and differentiation with extrinsic influences of the gut microenvironment.     

肠易激综合征早期生活事件模型下的大鼠结肠肠神经可塑性研究

目的从神经可塑性角度探讨肠易激综合征的可能发病机制、临床特征及治疗方法。方法雄性SD大鼠出生后连续13 d每天分离3 h。腹壁撤退反射实验用来检测内脏痛觉过敏。肠神经系统结构可塑性可以通过铺片技术和免疫荧光技术,比较近端结肠神经节(HuD阳性细胞)的大小和数目以及胶质细胞(GFAP)的变化来检测。检测近端结肠多组肌间神经丛和黏膜下神经丛肠神经递质类型(ChAT-、VIP-、nNOS-、calbindin-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,新生期母婴分离可以诱导肠神经结构改变,导致神经元肥大和增生、胶质细胞与神经元的比例增高。神经递质方面,新生期母婴分离组P75和TrkA表达(黏膜下神经丛、肌间神经丛)较正常组均明显增加。ChAT在肌间神经丛表达明显增加,VIP在黏膜下神经丛表达降低,nNOS在肌间神经丛表达增高,Cabindin表达未见明显异常。结论新生期母婴分离可以引起大鼠结肠肠神经可塑性的长期改变。新生期母婴分离诱导的内脏高敏感性中存在肠神经系统可塑性。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。     

Cell adhesion molecule L1 affects the rate of differentiation of enteric neurons in the developing gut

The enteric nervous system arises predominantly from vagal level neural crest cells that migrate into and along the developing gut. As the neural crest‐derived cells migrate within the gut, a subpopulation begins to differentiate into enteric neurons. Here, we show that the differentiation of neural crest‐derived cells into enteric neurons is delayed in L1‐deficient mice, compared with littermate controls. However, glial cell differentiation is not affected in L1‐deficient mice. These mice also show a delay in the differentiation of a neurotransmitter‐specific subtype of enteric neuron within the gastrointestinal tract. Together, these results suggest a role for the cell adhesion molecule, L1, in the differentiation of neural crest‐derived cells into enteric neurons within the developing enteric nervous system. Developmental Dynamics 238:708–715, 2009. © 2009 Wiley‐Liss, Inc.     

Focal,but not global,cerebral ischaemia causes loss of myenteric neurons and upregulation of vasoactive intestinal peptide in mouse ileum

Reduced blood flow to the brain induces cerebral ischaemia, potentially causing central injury and peripheral complications including gastrointestinal (GI) dysfunction. The pathophysiology behind GI symptoms is suspected to be neuropathy in the enteric nervous system (ENS), which is essential in regulating GI function. This study investigates if enteric neuropathy occurs after cerebral ischaemia, by analysing neuronal survival and relative numbers of vasoactive intestinal peptide (VIP) and neuronal nitric oxide synthase (nNOS) expressing neurons in mouse ileum after three types of cerebral ischaemia. Focal cerebral ischaemia, modelled by permanent middle cerebral artery occlusion (pMCAO) and global cerebral ischaemia, modelled with either transient occlusion of both common carotid arteries followed by reperfusion (GCIR) or chronic cerebral hypoperfusion (CCH) was performed on C56BL/6 mice. Sham‐operated mice for each ischaemia model served as control. Ileum was collected after 1–17 weeks, depending on model, and analysed using morphometry and immunocytochemistry. For each group, intestinal mucosa and muscle layer thicknesses, neuronal numbers and relative proportions of neurons immunoreactive (IR) for nNOS or VIP were estimated. No alterations in mucosa or muscle layer thicknesses were noted in any of the groups. Loss of myenteric neurons and an increased number of VIP‐IR submucous neurons were found in mouse ileum 7 days after pMCAO. None of the global ischaemia models showed any alterations in neuronal survival or relative numbers of VIP‐ and nNOS‐IR neurons. We conclude that focal cerebral ischaemia and global cerebral ischaemia influence enteric neuronal survival differently. This is suggested to reflect differences in peripheral neuro‐immune responses.     

Consequences of intestinal inflammation on the enteric nervous system: neuronal activation induced by inflammatory mediators

The ENS is responsible for the regulation and control of all gastrointestinal functions. Because of this critical role, and probably as a consequence of its remarkable plasticity, the ENS is often relatively well preserved in conditions where the architecture of the intestine is seriously disrupted, such as in IBD. There are structural and functional changes in the enteric innervation in animal models of experimental intestinal inflammation and in IBD. These include both up and down regulation of transmitter expression and the induction of new genes in enteric neurons. Using Fos expression as a surrogate marker of neuronal activation it is now well established that enteric neurons (and also enteric glia) respond to inflammation. Whether this "activation" is limited to a short-term functional response, such as increased neuronal excitability, or reflects a long-term change in some aspect of the neuronal phenotype (or both) has yet to be firmly established, but it appears that enteric neurons are highly plastic in their response to inflammation.     

Nitric oxide regenerates the normal colonic peristaltic activity in mdx dystrophic mouse

We demonstrated in vitro that the colonic peristaltic activity is modified in dystrophin-deficient mdx mouse indicating a defect in the enteric nervous system (ENS). Since nitric oxide (NO) has been proposed as a putative inhibitory mediator of ENS, here we have examined the effects of both L-Arginine (L-Arg) and Nomega-nitro-L-arginine methyl ester (L-NAME) on the peristaltic activity of mdx mouse distal colon. The motor pattern of colonic segment showed irregular peristaltic waves. L-Arg (10(-7) - 10(-5) M) induced the peristaltic activity to slow down. At a concentration of 10(-5) M, L-Arg produced hypomotility, characterised by a decrease in amplitude, frequency and ejected fluid volume. Conversely, L-NAME elicited hypermotility, this effect being reversed once again by the subsequent addition of L-Arg. Interestingly the addition of 10(-5) M L-Arg to the organ bath led to the normal progression, in an oral to aboral direction, of 90% of the peristaltic waves. This last result strongly suggests that exogenous application of L-Arg restores the integrative circuits of the ENS responsible for programming and co-ordinating peristaltic activity in the distal colon of mdx mouse.     

Short-chain fatty acids decrease the frequency of spontaneous contractions of longitudinal muscle via enteric nerves in rat distal colon

Short-chain fatty acids (SCFAs) produced by the bacterial fermentation of carbohydrates in the cecum and proximal colon are reported to modify colonic motility as a luminal factor. Besides the physical stimuli in the distal colon, SCFAs in the intestinal lumen also seem to affect colonic motility under physiological concentrations. This study therefore used fasted rats to investigate the effect of SCFAs on the spontaneous contractions of longitudinal muscle (LM) in rat distal colon, including mucosa in vitro. The frequency of spontaneous contractions of LM strips from the distal colon was 9.4 +/- 0.5 contractions/20 min. The exogenous addition of >5 mM SCFAs decreased the frequency of spontaneous contractions of the LM to 6.1 +/- 0.8 contractions/20 min. Among SCFAs, only acetate elicited this inhibitory response. TTX and the combination of hexamethonium and granisetron abolished SCFA-induced inhibitory response, suggesting that this inhibitory response is mediated via the ENS, including nicotinic and 5-HT(3) receptors. In conclusion, it is suggested that SCFAs in rat distal colon decrease the frequency of spontaneous contractions of the LM and that SCFAs may contribute to colonic motility, including the peristaltic reflex, by regulating the frequency of spontaneous contractions of the LM through the enteric nervous system (ENS).     

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.     

Differing effects of NT-3 and GDNF on dissociated enteric ganglion cells exposed to hydrogen peroxide in vitro

Oxidative stress is widely recognized to contribute to neuronal death during various pathological conditions and ageing. In the enteric nervous system (ENS), reactive oxygen species have been implicated in the mechanism of age-associated neuronal loss. The neurotrophic factors, neurotrophin 3 (NT-3) and glial cell line-derived neurotrophic factor (GDNF), are important in the development of enteric neurons and continue to be expressed in the gut throughout life. It has therefore been suggested that they may have a neuroprotective role in the ENS. We investigated the potential of NT-3 and GDNF to prevent the death of enteric ganglion cells in dissociated cell culture after exposure to hydrogen peroxide (H(2)O(2)). H(2)O(2) treatment resulted in a dose-dependent death of enteric neurons and glial cells, as demonstrated by MTS assay, bis-benzimide and propidium iodide staining and immunolabelling. Cultures treated with NT-3 prior to exposure showed reduced cell death compared to untreated control or GDNF-treated cultures. GDNF treatment did not affect neuronal survival in H(2)O(2)-treated cultures. These results suggest that NT-3 is able to enhance the survival of enteric ganglion cells exposed to oxidative stress.     

The developmental genetics of Hirschsprung's disease

Hirschsprung's disease (HSCR), also known as aganglionic megacolon, derives from a congenital malformation of the enteric nervous system (ENS). It displays an incidence of 1 in 5000 live births with a 4:1 male to female sex ratio. Clinical signs include severe constipation and distended bowel due to a non‐motile colon. If left untreated, aganglionic megacolon is lethal. This severe congenital condition is caused by the absence of colonic neural ganglia and thus lack of intrinsic innervation of the colon due in turn to improper colonization of the developing intestines by ENS progenitor cells. These progenitor cells are derived from a transient stem cell population called neural crest cells (NCC). The genetics of HSCR is complex and can involve mutations in multiple genes. However, it is estimated that mutations in known genes account for less than half of the cases of HSCR observed clinically. The male sex bias is currently unexplained. The objective of this review is to provide an overview of the pathophysiology and genetics of HSCR, within the context of our current knowledge of NCC development, sex chromosome genetics and laboratory models.     

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.     

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.     

Reduced endothelin converting enzyme-1 and endothelin-3 mRNA in the developing bowel of male mice may increase expressivity and penetrance of Hirschsprung disease-like distal intestinal aganglionosis.

Hirschsprung disease (distal intestinal aganglionosis, HSCR) is a multigenic disorder with incomplete penetrance, variable expressivity, and a strong male gender bias. Recent studies demonstrated that these genetic patterns arise because gene interactions determine whether enteric nervous system (ENS) precursors successfully proliferate and migrate into the distal bowel. We now demonstrate that male gender bias in the extent of distal intestinal aganglionosis occurs in mice with Ret dominant-negative mutations (RetDN) that mimic human HSCR. We hypothesized that male gender bias could result from reduced expression of a gene already known to be essential for ENS development. Using quantitative real-time polymerase chain reaction (PCR) we demonstrated reduced levels of endothelin converting enzyme-1 and endothelin-3 mRNA in the male mouse bowel at the time that ENS precursors migrate into the colon. Other HSCR-associated genes are expressed at comparable levels in male and female mice. Testosterone and Mullerian inhibiting substance had no deleterious effect on ENS precursor development, but adding EDN3 peptide to E11.5 male RetDN heterozygous mouse gut explants in organ culture significantly increased the rate of ENS precursor migration through the bowel.