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.     

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.     

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.     

Genetic screen for mutations affecting development and function of the enteric nervous system.

An intact enteric nervous system is required for normal gastrointestinal tract function. Several human conditions result from decreased innervation by enteric neurons; however, the genetic basis of enteric nervous system development and function is incompletely understood. In an effort to increase our understanding of the mechanisms underlying enteric nervous system development, we screened mutagenized zebrafish for changes in the number or distribution of enteric neurons. We also established a motility assay and rescreened mutants to learn whether enteric neuron number is correlated with gastrointestinal motility in zebrafish. We describe mutations isolated in our screen that affect enteric neurons specifically, as well as mutations that affect other neural crest derivatives or have pleiotropic effects. We show a correlation between the severity of enteric neuron loss and gastrointestinal motility defects. This screen provides biological tools that serve as the basis for future mechanistic studies.     

Neural stem cells for the treatment of disorders of the enteric nervous system: strategies and challenges.

The main goal of this review is to summarize the status of the research in the field of stem cells transplantation, as it is applicable to the treatment of gastrointestinal motility. This field of research has advanced tremendously in the past 10 years, and recent data produced in our laboratories as well as others is contributing to the excitement on the use of neural stem cells (NSC) as a valuable therapeutic approach for disorders of the enteric nervous system characterized by a loss of critical neuronal subpopulations. There are several sources of NSC, and here we describe therapeutic strategies for NSC transplantation in the gut. These include using NSC as a relatively nonspecific cellular replacement strategy in conditions where large populations of neurons or their subsets are missing or destroyed. As with many other recent "breakthroughs" stem cell therapy may eventually prove to be overrated. However, at the present time, it does appear to provide the hope for a true cure for many currently intractable diseases of both the central and the peripheral nervous system. Certainly more extensive research is needed in this field. We hope that our review will encourage new investigators in entering this field of research ad contribute to our knowledge of the potentials of NSC and other cells for the treatment of gastrointestinal dysmotility.     

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.     

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.     

Biology of the adult enteric neural stem cell.

An increasing body of evidence has accumulated in recent years supporting the existence of neural stem cells in the adult gut. There are at least three groups that have obtained them using different methodologies and have described them in vitro. There is a growing amount of knowledge on their biology, but many questions are yet unanswered. Among these questions is whether these cells are part of a permanent undifferentiated pool or are recruited in a regular basis; in addition, the factors and genes involved in their survival, proliferation, migration, and differentiation are largely unknown. Finally, with between 10 and 20% of adults suffering from diseases involving the enteric nervous system, most notably irritable bowel syndrome and gastroesophageal reflux, what is the possible role of enteric nervous stem cells in health and disease? Developmental Dynamics 236:20–32, 2007. © 2006 Wiley‐Liss, Inc.     

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.     

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.     

Intestinal macrophages and their interaction with the enteric nervous system in health and inflammatory bowel disease

Over the past decades, there has been an increasing understanding of cellular and molecular mechanisms that mediate modulation of the immune system by the autonomic nervous system. The discovery that vagal nerve stimulation (VNS) attenuates endotoxin‐induced experimental sepsis paved the way for further studies investigating neuro‐immune interaction. In particular, great attention is now given to intestinal macrophages: several studies report the existence of both intrinsic and extrinsic neural mechanisms by which intestinal immune homoeostasis can be regulated in different layers of the intestine, mainly by affecting macrophage activation through neurotransmitter release. Given the important role of inflammation in numerous disease processes, such as inflammatory bowel disease (IBD), cholinergic anti‐inflammatory mechanisms are under intense investigation both from a basic and clinical science perspective in immune‐mediated diseases such as IBD. This review discusses recent insights on the cross‐talk between enteric neurons and the immune system, especially focusing on macrophages, and provides an overview of basic and translational aspects of the cholinergic anti‐inflammatory response as therapeutic alternative to reinstall immune homoeostasis in intestinal chronic inflammation.     

Expression of p75<Superscript>NTR</Superscript> and Trk neurotrophin receptors in the enteric nervous system of human adults

Recent evidence suggests that the expression of p75NTR and Trk neurotrophin receptors is essential for neuronal survival, not only during development, but also in adulthood. The aim of the present study was to investigate the cell localization and distribution of p75NTR and Trk receptors in the normal adult human enteric nervous system (ENS) using double-label immunohistochemistry. Immunoreactivity for p75NTR was observed in a few neurons, whereas Trk immunoreactivity was present in a higher percentage of neurons. Strong expression of both types of neurotrophin receptors was found in the enteric glia. In addition, Trk immunoreactivity was localized to nerve endings in the lamina propria, muscularis mucosa and along or between circular and longitudinal smooth muscle layers, as well as to the enteric epithelium. Furthermore, polynuclear cells, mast cells and folliculoreticular cells in the germinal layer of lymph nodes displayed p75NTR and/or Trk immunopositivity. Expression of neurotrophin receptors in an inflammatory tissue sample was much more intense compared with that of normal tissue samples. These results suggest that neurotrophin receptors, through interactions with neurotrophins, may play a critical role in functional integrity of the ENS during adulthood.     

Behavior of enteric neural crest-derived cells varies with respect to the migratory wavefront.

Neural crest-derived cells colonize the entire gastrointestinal tract. The migration of these enteric neural crest-derived cells (ENCCs) occurs by their formation of cellular strands that extend into the intestinal mesenchyme. We have studied the behavior of crest cells that underlies the formation and extension of these strands by time-lapse microscopy. ENCCs expressing fluorescent marker molecules were visualized in situ in the embryonic mouse and chick gut. The major contributor to strand extension is from cells located within a region approximately 300 microm behind (rostral to) the most caudal cells in the migratory wavefront. Cells in the region immediately behind the leading cell of the strand either move intermittently in parallel with the leading cell, or advance caudally toward the wavefront over other ENCCs. Another addition to the strands arises from isolated cells located caudal to the wavefront. These cells showed a range of behavior including attachment and separation from the strands. The extending strands converged to form nodes, and then diverged along independent paths to form new strands, a behavior suggestive of attraction and repulsion. This behavior is probably responsible for the unique reticulated arrangement of ganglia in the enteric nervous system. As cells become positioned farther behind the wavefront, they exhibit more restricted movement and varied trajectories. We conclude that ENCCs exhibit different behaviors, depending on their position with respect to the wavefront. These different behaviors suggest a critical role for cell-cell interaction in the migratory process.     

A neuronal subpopulation in the mammalian enteric nervous system expresses TrkA and TrkC neurotrophin receptor-like proteins

Increasing evidence suggests that, in addition to peripheral sensory and sympathetic neurons, the enteric neurons are also under the control of neurotrophins. Recently, neurotrophin receptors have been detected in the developing and adult mammalian enteric nervous system (ENS). Nevertheless, it remains to be established whether neurotrophin receptors are expressed in all enteric neurons and/or in glial cells and whether expression is a common feature in the enteric nervous system of all mammals or if interspecific differences exist. Rabbit polyclonal antibodies against Trk proteins (regarded as essential constituents of the high-affinity signal-transducing neurotrophin receptors) and p75 protein (considered as a low-affinity pan-neurotrophin receptor) were used to investigate the cell localization of these proteins in the ENS of adult man, horse, cow, sheep, pig, rabbit, and rat. Moreover, the percentage of neurons displaying immunoreactivity (IR) for each neurotrophin receptor protein was determined. TrkA-like IR and TrkC-like IR were observed in a neuronal subpopulation in both the myenteric and submucous plexuses, from esophagus to rectum in humans, and in the jejunum-ileum of the other species. Many neurons, and apparently all glial cells, in the human and rat enteric nervous system also displayed p75 IR. TrkB-like IR was found restricted to the glial cells of all species studied, with the exception of humans, in whom IR was mainly in glial cells and a small percentage of enteric neurons (about 5%). These findings indicate that the ENS of adult mammals express neuronal TrkA and TrkC, glial TrkB, and neuronal-glial p75, this pattern of distribution being similar in all examined species. Thus, influence of specific neurotrophins on their cognate receptors may be considered in the physiology and/or pathology of the adult ENS. Anat. Rec. 251:360–370, 1998. © 1998 Wiley-Liss, Inc.     

The development of avian enteric nervous system: distribution of artemin immunoreactivity

Among the factors that control neural crest cell precursors within the enteric nervous system, the ligands of the glial cell line-derived neurotrophic factor family (GFL) seem to be the most influential. Artemin, a member of the GFLs, was previously described only in the oesophagus and stomach of mouse embryos. In this study, the presence and distribution of artemin is reported in duck embryos and adults. Artemin immunoreactivity was apparent in the intestinal tract at embryonic day 7 (d7), firstly in the myenteric plexus and then in the submucous plexus. Later, artemin immunoreactive nerve fibres were also seen in the longitudinal muscle plexus, the circular muscle plexus, the plexus of the muscularis mucosa and in the mucosal plexus. Furthermore, at d7, weak labeling of artemin was detected in neurons and glial cells in the oesophagus, gastric region and duodenum. Subsequently, artemin was also detected in all other intestinal segments. Moreover, during development of the gut in the domestic duck, artemin immunoreactivity decreased in neuronal cell bodies, whilst it increased in neuronal fibres and glial cells. These findings suggest an involvement of artemin in the development and biology of the gut of the domestic duck.