Appearance of neurons and glia with respect to the wavefront during colonization of the avian gut by neural crest cells.

The enteric nervous system is formed by neural crest cells that migrate, proliferate, and differentiate into neurons and glia distributed in ganglia along the gastrointestinal tract. In the developing embryo some enteric crest cells cease their caudal movements, whereas others continue to migrate. Subsequently, the enteric neurons form a reticular network of ganglia interconnected by axonal projections. We studied the developing avian gut to characterize the pattern of migration of the crest cells, and the relationship between migration and differentiation. Crest cells at the leading edge of the migratory front appear as strands of cells; isolated individual crest cells are rarely seen. In the foregut and midgut, these strands are located immediately beneath the serosa. In contrast, crest cells entering the colon appear first in the deeper submucosal mesenchyme and later beneath the serosa. As the neural crest wavefront passes caudally, the crest cell cords become highly branched, forming a reticular lattice that presages the mature organization of the enteric nervous system. Neurons and glia first appear within the strands at the advancing wavefront. Later neurons are consistently located at the nodes where branches of the lattice intersect. In the most rostral foregut and in the colon, some neurons initially appear in close association with extrinsic nerve fibers from the vagus and Remak's nerve, respectively. We conclude that crest cells colonize the gut as chains of cells and that, within these chains, both neurons and glia appear close to the wavefront.     

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.     

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     

Intraganglionic macrophages: a new population of cells in the enteric ganglia

The enteric nervous system shares embryological, morphological, neurochemical, and functional features with the central nervous system. In addition to neurons and glia, the CNS includes a third component, microglia, which are functionally and immunophenotypically similar to macrophages, but a similar cell type has not previously been identified in enteric ganglia. In this study we identify a population of macrophages in the enteric ganglia, intermingling with the neurons and glia. These intraganglionic macrophages (IMs) are highly ramified and express the hematopoietic marker CD45, major histocompatibility complex (MHC) class II antigen, and chB6, a marker specific for B cells and microglia in avians. These IMs do not express antigens typically associated with T cells or dendritic cells. The CD45⁺/ChB6⁺/MHCII⁺ signature supports a hematopoietic origin and this was confirmed using intestinal chimeras in GFP‐transgenic chick embryos. The presence of green fluorescent protein positive (GFP⁺⁾/CD45⁺ cells in the intestinal graft ENS confirms that IMs residing within enteric ganglia have a hematopoietic origin. IMs are also found in the ganglia of CSF1R^GFP chicken and CX3CR1^GFP mice. Based on the expression pattern and location of IMs in avians and rodents, we conclude that they represent a novel non‐neural crest‐derived microglia‐like cell population within the enteric ganglia.     

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.     

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.     

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.     

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.     

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.     

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.     

The origin and differentiation of enteric neurons of the intestine of the fowl embryo

Neuroblasts were identified histologically in the intestine of the fowl embryo of stages 25 to 35 (Hamburger and Hamilton, ′51). These cells occurred rostral to the umbilicus at stage 26, and appeared caudally in a wave that reached the coprodeum at stage 34. The presence of histologically unidentifiable neuronal precursors was studied by grafting onto the chorio-allantoic membrane (CAM) short lengths of gut from embryos of stage 24 to 34. After growth in isolation on the CAM, the resultant gut grafts were examined histologically for enteric neurons. In these grafts, neuronal differentiation occurred in the gut as a wave moving caudally at about 40 ?m/hr. This wave preceded by less than 12 hours the normal appearance of neuroblasts at any particular level of the intestine. The findings are discussed with regard to previous reports on the origin of enteric neurons. The most likely conclusion is that all enteric neurons in the fowl are derived from vagal neural crest. The results obtained with grafts containing neurons of Remak's nerve, in addition to intestine, suggest that lumbo-sacral neural crest may give rise to some enteric supporting cells which, in the absence of normal enteric plexuses, differentiate as pigment cells. If lumbo-sacral neural crest cells contribute to post-umbilical enteric neurons, their differentiation requires the presence of vagal cells. A comparison of enteric neuronal density and distribution in short and long colo-rectal grafts suggests that only the advancing front of the neuronal precursor wave is necessary to populate the remainder of the gut. This high proliferative potential in the wave-front appears to decline on reaching the most caudal colo-rectum and coprodeum.     

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.     

Expression of Ret-, p75(NTR)-, Phox2a-, Phox2b-, and tyrosine hydroxylase-immunoreactivity by undifferentiated neural crest-derived cells and different classes of enteric neurons in the embryonic mouse gut.

Cells of the enteric nervous system are derived from the neural crest. Probes to a number of molecules identify neural crest-derived cells within the gastrointestinal tract of embryonic mice prior to their differentiation into neurons and glial cells. However, it is unclear whether the different markers are identifying all neural crest-derived cells. In this study the distribution of p75(NTR)-immunoreactivity was compared with that of Ret-, Phox2a-, Phox2b-, and tyrosine hydroxylase (TH) in undifferentiated neural crest-derived cells in the E10.5-E13.5 mouse intestine. Neural crest-derived cells colonise the embryonic mouse gut in a rostral-to-caudal wave between E9.5-E14, and differentiation into enteric neurons also occurs in a rostral-to-caudal wave. Thus, the most caudal neural crest-derived cells within the gut are undifferentiated. These most caudal neural crest-derived cells co-expressed p75(NTR)-, Phox2b- and Ret-immunoreactivity; at E10.5 a sub-population was also TH-positive. The most caudal cells did not show Phox2a-immunoreactivity at any stage. However, a sub-population of cells, which was rostral to the undifferentiated neural crest-derived cells, was Phox2a-positive, and these are likely to be cells beginning to differentiate along a neuronal lineage. The expression of Ret-, Phox2a-, Phox2b- and p75(NTR)-immunoreactivity by two classes of enteric neurons that differentiate prior to birth was also examined. Nitric oxide synthase (NOS) neurons showed Phox2b and Ret immunoreactivity at all ages, and Phox2a and p75(NTR) immunoreactivity only transiently. Calcitonin gene-related peptide (CGRP) neurons showed Phox2b and Ret-immunoreactivity, but not Phox2a immunoreactivity. It is concluded that all undifferentiated neural crest-derived cells initially express Phox2b, Ret, and p75(NTR); a sub-population of these cells also expresses TH transiently. Those cells that are beginning to differentiate along a neuronal lineage maintain their expression of Phox2b and Ret, and they start to express Phox2a, but down-regulate p75(NTR); those cells that differentiate along a glial lineage down-regulate Ret and maintain their expression of p75(NTR). Dev Dyn 1999;216:137-152.     

Suppression of the melanogenic potential of migrating neural crest‐derived cells by the branchial arches

The development of melanocytes from neural crest‐derived precursors that migrate along the dorsolateral pathway has been attributed to the selection of this route by cells that are fate‐restricted to the melanocyte lineage. Alternatively, melanocytes could arise from nonspecified cells that develop in response to signals encountered while these cells migrate, or at their final destinations. In most animals, the bowel, which is colonized by crest‐derived cells that migrate through the caudal branchial arches, contains no melanocytes; however, the enteric microenvironment does not prevent melanocytes from developing from crest‐derived precursors placed experimentally into the bowel wall. To test the hypothesis that the branchial arches remove the melanogenic potential from the crest‐derived population that colonizes the gut, the Silky fowl (in which the viscera are pigmented) was studied. Sources of crest included Silky fowl and quail vagal and truncal neural folds/tubes, which were cultured or explanted to chorioallantoic membranes alone or together with branchial arches or limb buds from Silky fowl, White Leghorn, or quail embryos. Crest and mesenchyme‐derived cells were distinguished by using the quail nuclear marker. Melanocytes developed from Silky fowl and quail crest‐derived cells. Melanocyte development from both sources was inhibited by quail and White Leghorn branchial arches (and limb buds), but melanocyte development was unaffected by branchial arch (and limb buds) from Silky fowl. These observations suggest that a factor(s) that is normally expressed in the branchial arches, and is lacking in animals with the Silky mutation, prevents cells with a melanogenic potential from colonizing the bowel. Anat Rec 268:16–26, 2002. © 2002 Wiley‐Liss, Inc.     

Regionally defective colonization of the terminal bowel by the precursors of enteric neurons in lethal spotted mutant mice

In order to gain insight into the process of colonization of the bowel by the neural crest-derived precursors of enteric neurons, the development of the enteric nervous system was examined in lethal spotted mutant mice, a strain in which a segment of bowel is congenitally aganglionic. In addition, nerve fibers within the ganglionic and aganglionic zones of the gut of adult mutant mice were investigated with respect to their content of acetylcholinesterase, immunoreactive substance P, vasoactive intestinal polypeptide and serotonin, and their ability to take up [³Hserotonin. In both the fetal gut of developing mutant mice and in the mature bowel of adult animals abnormalities were limited to the terminal 2 mm of colon. The enteric nervous system in the proximal alimentary tract was indistinguishable from that of control animals for all of the parameters examined. In the terminal bowel, the normal plexiform pattern of the innervation and ganglion cell bodies were replaced by a coarse reticulum of nerve fibers that stained for acetylcholineserase and were continuous with extrinsic nerves running between the colon and the pelvic plexus. These coarse nerve bundles contained greatly reduced numbers of fibers that displayed substance P- and vasoactive intestinal polypeptide-like immunoreactivity, but a serotonergic innervation was totally missing from the aganglionic bowel. During development, acetylcholineserase and uptake of [³Hserotonin appeared in neural elements in the foregut of mutant mice on the 12th day of embryonic life (E12), about the same time these markers appeared in the forgut in normal mice. By day E14, neurons expressing one or the other marker were recognizable as far distally as about 2 mm from the anus. The appearance of neurons in segments of gut grown for 2 weeks as expiants in culture was used as an assay for the presence of neuronal progenitor cells in the segments of fetal bowel at the time of explantation. Both acetyl- cholinesterase activity and uptake of [³Hserotonin developed in neuronsin vitro in expiants of proximal bowel between days E10 and E17. At all times, however, the terminal 2mm of mutant but not normal fetal gut gave rise to aneuronal cultures. In some mutant mice rare, small, ectopically-situated pelvic ganglia were found just outside aganglionic segments of fetal colon. Uptake of [³Hserotonin, normally a marker for intrinsic enteric neurites, was found in these ganglia.The experiments suppport the hypothesis that the terminal 2 mm of the gut in lethal spotted mutant mice is intrinsically abnormal and thus cannot be colonized by the precursors of enteric neurons. The defect seems to be specific in that both cells and processes of intrinsic enteric neurons, including all serotonergic and most peptidergic neurites, seem to be excluded from the abnormal region while extrinsic nerve fibers, including sympathetic and sensory axons, are able to enter the aganglionic zones. Since examination of neural progenitor cells has failed to reveal a significant proximo-distal displacement of these cells through the enteric tube during development of the murine bowel, a defect in the migration of precursor cells down the alimentary tract to the terminal gut seems unlikely to be substantially involved in the pathogenesis of aganglionosis. This conclusion is supported by the normal enteric nervous system in proximal regions of the mutant gut and the presence of enteric type neurons outside of, but at the same level as the aganglionic region.     

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.     

Three-dimensional slice cultures from murine fetal gut for investigations of the enteric nervous system.

Three-dimensional intestinal cultures offer new possibilities for the examination of growth potential, analysis of time specific gene expression, and spatial cellular arrangement of enteric nervous system in an organotypical environment. We present an easy to produce in vitro model of the enteric nervous system for analysis and manipulation of cellular differentiation processes. Slice cultures of murine fetal colon were cultured on membrane inserts for up to 2 weeks without loss of autonomous contractility. After slice preparation, cultured tissue reorganized within the first days in vitro. Afterward, the culture possessed more than 35 cell layers, including high prismatic epithelial cells, smooth muscle cells, glial cells, and neurons analyzed by immunohistochemistry. The contraction frequency of intestinal slice culture could be modulated by the neurotransmitter serotonin and the sodium channel blocker tetrodotoxin. Coculture experiments with cultured neurospheres isolated from enhanced green fluorescent protein (eGFP) transgenic mice demonstrated that differentiating eGFP-positive neurons were integrated into the intestinal tissue culture. This slice culture model of enteric nervous system proved to be useful for studying cell-cell interactions, cellular signaling, and cell differentiation processes in a three-dimensional cell arrangement.