Development of the enteric nervous system in the moth. I. Diversity of cell types and the embryonic expression of FMRFamide-related neuropeptides

The enteric nervous system (ENS) of the larval moth Manduca sexta consists of two small ganglia and several nerve networks that lie superficially along the alimentary tract. Within this system are approximately 600 neurons that exhibit a spectrum of biochemical and morphological characteristics and that express these features in a definable sequence during development. The accessibility of both the neural and nonneural components of the moth ENS throughout embryogenesis makes it a potentially useful model in which to examine the developmental regulation of transmitter phenotype. In this paper, we have focused on the differentiation of the enteric plexus (EP) cells, a heterogeneous population of enteric neurons that are distributed across the foregut-midgut boundary. Unlike many neurons of the CNS in insects, the cells of the enteric plexus are not uniquely identifiable. While the total number of EP cells is constant, their locations vary significantly from animal to animal. However, several distinct classes of neurons can be identified within this population on the basis of morphology and transmitter phenotype, including one class that contains substances related to the molluscan peptide Phe-Met-Arg-Phe-amide (FMRFamide). Expression of this FMRFamide-like material within the enteric plexus is position-specific, occurring only in neurons on the midgut and not in those on the foregut. FMRFamide-like immunoreactivity first appears in approximately one-third of these cells at 65% of development; this pattern is retained without apparent modification throughout subsequent embryonic and postembryonic development. In the following paper, we describe the sequence of stereotyped cell migration that precedes the expression of this peptidergic phenotype and that underlies the formation of the enteric plexus during embryogenesis.     

Development of the enteric nervous system in the moth. II. Stereotyped cell migration precedes the differentiation of embryonic neurons

The enteric plexus is a discrete portion of the enteric nervous system (ENS) in the larval moth Manduca sexta. It consists of a stereotyped array of nerves extending across specific regions of both the foregut and midgut. Within these nerves are approximately 400 neurons (the EP cells), which do not appear to be uniquely identifiable but exhibit a spectrum of morphological and biochemical phenotypes. In this report we have described the morphogenetic events by which the enteric plexus is created during embryogenesis and have characterized the morphological differentiation of the EP neurons. In particular, we have demonstrated a prominent role for stereotyped cellular migration in the formation of this region of the ENS. The neurons of the enteric plexus arise from the dorsal epithelium of the foregut in the form of a dense, triangular packet. Between 40 and 65% of embryogenesis, the cells of this packet become progressively dispersed by a sequence of migratory events: an initial, slow phase of migration that is circumferentially directed around the foregut, and a rapid, dispersing phase by which the EP cells achieve their mature distributions across the foregut and midgut surface. These migratory phases occur along defined pathways on the gut and result in cellular translocations of up to 250 microns. In the early phase, some migrating neurons extend long axons in stereotyped directions, while others retain a simple bipolar morphology. Neurons of both morphological types are interspersed throughout the initial packet of cells and participate equally in the migratory process. Toward the end of migration, cells with the simpler morphology also extend axons along predictable pathways. Several additional subtypes subsequently differentiate in various regions within the plexus. The expression of specific peptidergic substances (related to the molluscan peptide Phe-Met-Arg-Phe-NH2, as described in the accompanying paper (P. F. Copenhaver and P. H. Taghert, 1988, Dev. Biol. 130, 70-84) commences within the EP cell population only after these migratory phases are complete and can be correlated with the outcome of cellular migration: only neurons that navigate onto the midgut regions of the plexus subsequently exhibit the peptidergic phenotype. This system should provide an excellent model with which to examine the mechanisms underlying the migratory process and the potential roles of cellular migration in regulating neuronal differentiation.     

Evidence for neural progenitor cells in the human adult enteric nervous system

Putative neural stem cells have been identified within the enteric nervous system (ENS) of adult rodents and cultured from human myenteric plexus. We conducted studies to identify neural stem cells or progenitor cells within the submucosa of adult human ENS. Jejunum tissue was removed from adult human subjects undergoing gastric bypass surgery. The tissue was immunostained, and confocal images of ganglia in the submucosal plexus were collected to identify protein gene product 9.5 (PGP 9.5) - immunoractive neurons and neuronal progenitor cells that coexpress PGP 9.5 and nestin. In addition to PGP-9.5-positive/nestin-negative neuronal cells within ganglia, we observed two other types of cells: (1) cells in which PGP 9.5 and nestin were co-localized, (2) cells negative for both PGP 9.5 and nestin. These observations suggest that the latter two types of cells are related to a progenitor cell population and are consistent with the concept that the submucosa of human adult ENS contains stem cells capable of maintenance and repair within the peripheral nervous system.     

Transient expression of the calcitonin receptor by enteric neurons of the embryonic and early post-natal mouse

Calcitonin receptor-immunoreactivity (CTR-ir) was found in enteric neurons of the mouse gastrointestinal tract from embryonic day 13.5 (E13.5) to post-natal day 28 (P28). CTR-ir occurred in cell bodies in ganglia of the myenteric plexus extending from the esophagus to the colon and in nerve cells of the submucosal ganglia of the small and large intestines. CTR-ir was also found in vagal nerve trunks and mesenteric nerves. Counts in the ileal myenteric plexus revealed CTR-ir in 80% of neurons. CTR-ir was clearly evident in the cell bodies of enteric neurons by E15.5. The immunoreactivity reached maximum intensity between P1.5 and P12 but was weaker at P18 and barely detectable at P28. The receptor was detected in nerve processes in the intestine for only a brief period around E17.5, when it was present in one to two axonal processes per villus in the small intestine. In late gestation and soon after birth, CTR-ir was also evident in the mucosal epithelium. The perinatal expression of CTR within the ENS suggests that the calcitonin/CTR system may have a role in the maturation of enteric neurons. Signals may reach enteric neurons in milk, which contains high levels of calcitonin.     

RGMa inhibits neurite outgrowth of neuronal progenitors from murine enteric nervous system via the neogenin receptor in vitro

The enteric nervous system (ENS) in vertebrate embryos is formed by neural crest-derived cells. During development, these cells undergo extensive migration from the vagal and sacral regions to colonize the entire gut, where they differentiate into neurons and glial cells. Guidance molecules like netrins, semaphorins, slits, and ephrins are known to be involved in neuronal migration and axon guidance. In the CNS, the repulsive guidance molecule (RGMa) has been implicated in neuronal differentiation, migration, and apoptosis. Recently, we described the expression of the subtypes RGMa and RGMb and their receptor neogenin during murine gut development. In the present study, we investigated the influence of RGMa on neurosphere cultures derived from fetal ENS. In functional in vitro assays, RGMa strongly inhibited neurite outgrowth of differentiating progenitors via the receptor neogenin. The repulsive effect of RGMa on processes of differentiated enteric neural progenitors could be demonstrated by collapse assay. The influence of the RGM receptor on ENS was also analyzed in neogenin knockout mice. In the adult large intestine of mutants we observed disturbed ganglia formation in the myenteric plexus. Our data indicate that RGMa may be involved in differentiation processes of enteric neurons in the murine gut.     

<Emphasis Type="SmallCaps">l</Emphasis>-Aspartate-immunoreactive neurons in the rat enteric nervous system

l-Aspartate (l-Asp) is an excitatory neurotransmitter in the central nervous system. In the present study, we demonstrate, for the first time, the presence of l-Asp in a particular neuronal cell class in the enteric nervous system (ENS). Scattered l-Asp-immunoreactive neuronal cell bodies and nerve fibers were found extensively in both the myenteric and submucosal plexus throughout the small and large intestines. Many l-Asp-immunoreactive nerve fibers, which originated from intrinsic nerve cell bodies, were found in the ganglia and interconnecting nerve bundles. Electron microscopy revealed that l-Asp-immunoreactive terminals frequently formed synaptic contacts with intrinsic nerve cells, suggesting that some l-Asp-immunoreactive neurons might function as interneurons. These results suggest that l-Asp-immunoreactive neurons play a significant role within the ENS to control intestinal functions. The presence of enteric l-Asp-immunoreactive neurons provides strong support for the proposal that l-Asp is a neuromodulator in the rat ENS.     

Non-cell-autonomous effects of Ret deletion in early enteric neurogenesis

Neural crest cells (NCCs) form at the dorsal margin of the neural tube and migrate along distinct pathways throughout the vertebrate embryo to generate multiple cell types. A subpopulation of vagal NCCs invades the foregut and colonises the entire gastrointestinal tract to form the enteric nervous system (ENS). The colonisation of embryonic gut by NCCs has been studied extensively in chick embryos, and genetic studies in mice have identified genes crucial for ENS development, including Ret. Here, we have combined mouse embryo and organotypic gut culture to monitor and experimentally manipulate the progenitors of the ENS. Using this system, we demonstrate that lineally marked intestinal ENS progenitors from E11.5 mouse embryos grafted into the early vagal NCC pathway of E8.5 embryos colonise the entire length of the gastrointestinal tract. By contrast, similar progenitors transplanted into Ret-deficient host embryos are restricted to the proximal foregut. Our findings establish an experimental system that can be used to explore the interactions of NCCs with their cellular environment and reveal a previously unrecognised non-cell-autonomous effect of Ret deletion on ENS development.     

The emergence of neural activity and its role in the development of the enteric nervous system

The enteric nervous system (ENS) is a vital part of the autonomic nervous system that regulates many gastrointestinal functions, including motility and secretion. All neurons and glia of the ENS arise from neural crest-derived cells that migrate into the gastrointestinal tract during embryonic development. It has been known for many years that a subpopulation of the enteric neural crest-derived cells expresses pan-neuronal markers at early stages of ENS development. Recent studies have demonstrated that some enteric neurons exhibit electrical activity from as early as E11.5 in the mouse, with further maturation of activity during embryonic and postnatal development. This article discusses the maturation of electrophysiological and morphological properties of enteric neurons, the formation of synapses and synaptic activity, and the influence of neural activity on ENS development.     

Requirement of signalling by receptor tyrosine kinase RET for the directed migration of enteric nervous system progenitor cells during mammalian embryogenesis

The majority of neurones and glia of the enteric nervous system (ENS) are derived from the vagal neural crest. Shortly after emigration from the neural tube, ENS progenitors invade the anterior foregut and, migrating in a rostrocaudal direction, colonise in an orderly fashion the rest of the foregut, the midgut and the hindgut. We provide evidence that activation of the receptor tyrosine kinase RET by glial cell line-derived neurotrophic factor (GDNF) is required for the directional migration of ENS progenitors towards and within the gut wall. We find that neural crest-derived cells present within foetal small intestine explants migrate towards an exogenous source of GDNF in a RET-dependent fashion. Consistent with an in vivo role of GDNF in the migration of ENS progenitors, we demonstrate that Gdnf is expressed at high levels in the gut of mouse embryos in a spatially and temporally regulated manner. Thus, during invasion of the foregut by vagal-derived neural crest cells, expression of Gdnf was restricted to the mesenchyme of the stomach, ahead of the invading NC cells. Twenty-four hours later and as the ENS progenitors were colonising the midgut, Gdnf expression was upregulated in a more posterior region - the caecum anlage. In further support of a role of endogenous GDNF in enteric neural crest cell migration, we find that in explant cultures GDNF produced by caecum is sufficient to attract NC cells residing in more anterior gut segments. In addition, two independently generated loss-of-function alleles of murine Ret, Ret.k- and miRet51, result in characteristic defects of neural crest cell migration within the developing gut. Finally, we identify phosphatidylinositol-3 kinase and the mitogen-activated protein kinase signalling pathways as playing crucial roles in the migratory response of enteric neural crest cells to GDNF.     

Enteric nervous system progenitors are coordinately controlled by the G protein-coupled receptor EDNRB and the receptor tyrosine kinase RET

The enteric nervous system (ENS) in vertebrates is derived mainly from vagal neural crest cells that enter the foregut and colonize the entire wall of the gastrointestinal tract. Failure to completely colonize the gut results in the absence of enteric ganglia (Hirschsprung's disease). Two signaling systems mediated by RET and EDNRB have been identified as critical players in enteric neurogenesis. We demonstrate that interaction between these signaling pathways controls ENS development throughout the intestine. Activation of EDNRB specifically enhances the effect of RET signaling on the proliferation of uncommitted ENS progenitors. In addition, we reveal novel antagonistic roles of these pathways on the migration of ENS progenitors. Protein kinase A is a key component of the molecular mechanisms that integrate signaling by the two receptors. Our data provide strong evidence that the coordinate and balanced interaction between receptor tyrosine kinases and G protein-coupled receptors controls the development of the nervous system in mammals.     

Development of the mammalian enteric nervous system.

The mammalian enteric nervous system is derived from neural crest cells which invade the foregut and hindgut mesenchyme. It has been established that signalling molecules produced by the mesenchyme of the gut wall play a critical role in the development of the mammalian enteric nervous system. Recent studies have characterised further the role of such molecules and have identified novel extracellular and intracellular signals that are critical for enteric ganglia formation.     

Pleiotropic effects of the bone morphogenetic proteins on development of the enteric nervous system

Formation of the enteric nervous system (ENS) from migratory neural crest-derived cells that colonize the primordial gut involves a complex interplay among different signaling molecules. The bone morphogenetic proteins (BMPs), specifically BMP2 and BMP4, play a particularly important role in virtually every stage of gut and ENS development. BMP signaling helps to pattern both the anterior-posterior axis and the radial axis of the gut prior to colonization by migratory crest progenitor cells. BMP signaling then helps regulate the migration of enteric neural crest-derived precursors as they colonize the fetal gut and form ganglia. BMP2 and -4 promote differentiation of enteric neurons in early fetal ENS development and glia at later stages. A major role for BMP signaling in the ENS is regulation of responses to other growth factors. Thus BMP signaling first regulates neurogenesis by modulating responses to GDNF and later gliogenesis through its effects on GGF-2 responses. Furthermore, BMPs promote growth factor dependency for survival of ENS neurons (on NT-3) and glia (on GGF-2) by inducing TrkC (neurons) and ErbB3 (glia). BMP signaling limits total neuron numbers, favoring the differentiation of later born neuronal phenotypes at the expense of earlier born ones thus influencing the neuronal composition of the ENS and the glia/neuron ratio. BMP2 and -4 also promote gangliogenesis via modification of neural cell adhesion molecules and promote differentiation of the circular and then longitudinal smooth muscles. Disruption of BMP signaling leads to defects in the gut and in ENS function commensurate with these complex developmental roles.     

Cellular changes in the enteric nervous system during ageing

The intrinsic neurons of the gut, enteric neurons, have an essential role in gastrointestinal functions. The enteric nervous system is plastic and continues to undergo changes throughout life, as the gut grows and responds to dietary and other environmental changes. Detailed analysis of changes in the ENS during ageing suggests that enteric neurons are more vulnerable to age-related degeneration and cell death than neurons in other parts of the nervous system, although there is considerable variation in the extent and time course of age-related enteric neuronal loss reported in different studies. Specific neuronal subpopulations, particularly cholinergic myenteric neurons, may be more vulnerable than others to age-associated loss or damage. Enteric degeneration and other age-related neuronal changes may contribute to gastrointestinal dysfunction that is common in the elderly population. Evidence suggests that caloric restriction protects against age-associated loss of enteric neurons, but recent advances in the understanding of the effects of the microbiota and the complex interactions between enteric ganglion cells, mucosal immune system and intestinal epithelium indicate that other factors may well influence ageing of enteric neurons. Much remains to be understood about the mechanisms of neuronal loss and damage in the gut, although there is evidence that reactive oxygen species, neurotrophic factor dysregulation and/or activation of a senescence associated phenotype may be involved. To date, there is no evidence for ongoing neurogenesis that might replace dying neurons in the ageing gut, although small local sites of neurogenesis would be difficult to detect. Finally, despite the considerable evidence for enteric neurodegeneration during ageing, and evidence for some physiological changes in animal models, the ageing gut appears to maintain its function remarkably well in animals that exhibit major neuronal loss, indicating that the ENS has considerable functional reserve.     

In Situ Ca2+ Imaging of the Enteric Nervous System

Reflex behaviors of the intestine are controlled by the enteric nervous system (ENS). The ENS is an integrative network of neurons and glia in two ganglionated plexuses housed in the gut wall. Enteric neurons and enteric glia are the only cell types within the enteric ganglia. The activity of enteric neurons and glia is responsible for coordinating intestinal functions. This protocol describes methods for observing the activity of neurons and glia within the intact ENS by imaging intracellular calcium (Ca²⁺) transients with fluorescent indicator dyes. Our technical discussion focuses on methods for Ca²⁺ imaging in whole-mount preparations of the myenteric plexus from the rodent bowel. Bulk loading of ENS whole-mounts with a high-affinity Ca²⁺ indicator such as Fluo-4 permits measurements of Ca²⁺ responses in individual neurons or glial cells. These responses can be evoked repeatedly and reliably, which permits quantitative studies using pharmacological tools. Ca²⁺ responses in cells of the ENS are recorded using a fluorescence microscope equipped with a cooled charge-coupled device (CCD) camera. Fluorescence measurements obtained using Ca²⁺ imaging in whole-mount preparations offer a straightforward means of characterizing the mechanisms and potential functional consequences of Ca²⁺ responses in enteric neurons and glial cells.     

Enteric neurons show a primary cilium

The primary cilium is a non‐motile cilium whose structure is 9+0. It is involved in co‐ordinating cellular signal transduction pathways, developmental processes and tissue homeostasis. Defects in the structure or function of the primary cilium underlie numerous human diseases, collectively termed ciliopathies. The presence of single cilia in the central nervous system (CNS) is well documented, including some choroid plexus cells, neural stem cells, neurons and astrocytes, but the presence of primary cilia in differentiated neurons of the enteric nervous system (ENS) has not yet been described in mammals to the best of our knowledge. The enteric nervous system closely resembles the central nervous system. In fact, the ultrastructure of the ENS is more similar to the CNS ultrastructure than to the rest of the peripheral nervous system. This research work describes for the first time the ultrastructural characteristics of the single cilium in neurons of rat duodenum myenteric plexus, and reviews the cilium function in the CNS to propose the possible role of cilia in the ENS cells.     

Co-localization of zinc transporter 3 (ZnT3) with sensory neuromediators and/or neuromodulators in the enteric nervous system of the porcine esophagus

Zinc transporter 3 (ZnT3) is one of the zinc transporters family. It is closely connected to the nervous system, where enables the transport of zinc ions from the cytoplasm to synaptic vesicles. This substance has been described within the central and peripheral nervous system, especially in the enteric nervous system (ENS). The aim of the present study was to describe the co-localization of ZnT3 with selected neuromediators and/or neuromodulators participating in sensory stimuli conduction in neurons of the ENS within the porcine esophagus. Co-localization of ZnT3 with substance P (SP), leucine enkephalin (LENK) and calcitonin gene-related peptide (CGRP) was studied using standard double-immunofluorescence technique. The obtained results show that ZnT3, SP and/or LENK may occur in the same enteric neurons, and the degree of co-localization of these substances clearly depends on the fragment of esophagus studied and the type of enteric ganglia. In contrast, the co-localization of ZnT3 with CGRP was not observed during the present investigation. The obtained results suggest that ZnT3 in the ENS may be involved in the conduction of sensory and/or pain stimuli.     

BMP signaling is necessary for neural crest cell migration and ganglion formation in the enteric nervous system

The enteric nervous system (ENS) is derived from neural crest cells that migrate along the gastrointestinal tract to form a network of neurons and glia that are essential for regulating intestinal motility. Despite the number of genes known to play essential roles in ENS development, the molecular etiology of congenital disorders affecting this process remains largely unknown. To determine the role of bone morphogenetic protein (BMP) signaling in ENS development, we first examined the expression of bmp2, bmp4, and bmprII during hindgut development and find these strongly expressed in the ENS. Moreover, functional BMP signaling, demonstrated by the expression of phosphorylated Smad1/5/8, is present in the enteric ganglia. Inhibition of BMP activity by noggin misexpression within the developing gut, both in ovo and in vitro, inhibits normal migration of enteric neural crest cells. BMP inhibition also leads to hypoganglionosis and failure of enteric ganglion formation, with crest cells unable to cluster into aggregates. Abnormalities of migration and ganglion formation are the hallmarks of two human intestinal disorders, Hirschsprung's disease and intestinal neuronal dysplasia. Our results support an essential role for BMP signaling in these aspects of ENS development and provide a basis for further investigation of these proteins in the etiology of neuro-intestinal disorders.