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.     

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.     

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.     

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.     

Enteric nervous system development: analysis of the selective developmental potentialities of vagal and sacral neural crest cells using quail-chick chimeras

The majority of the enteric nervous system (ENS) is derived from vagal neural crest cells (NCC). For many years, the contribution from a second region of the neuraxis (the sacral neural crest) to the ENS has been less clear, with conflicting reports appearing in the literature. To resolve this longstanding issue, we documented the spatiotemporal migration and differentiation of vagal and sacral-derived NCC within the developing chick embryo using quail-chick grafting and antibody labelling. Results showed that vagal NCC colonised the entire length of the gut in a rostrocaudal direction. The hindgut, the region of the gastrointestinal tract most frequently affected in developmental disorders, was found to be colonised in a complex manner. Vagal NCC initially migrated within the submucosa, internal to the circular muscle layer, before colonising the myenteric plexus region. In contrast, sacral NCC, which colonised the hindgut in a caudorostral direction, were primarily located in the myenteric plexus region from where they subsequently migrated to the submucosa. We also observed that sacral NCC migrated into the hindgut in significant numbers only after vagal-derived cells had colonised the entire length of the gut. This suggested that to participate in ENS formation, sacral cells may require an interaction with vagal-derived cells, or with factors or signalling molecules released by them or their progeny. To investigate this possible inter-relationship, we ablated sections of vagal neural crest (NC) to prevent the rostrocaudal migration of ENS precursors and, thus, create an aganglionic hindgut model. In the same NC ablated animals, quail-chick sacral NC grafts were performed. In the absence of vagal-derived ganglia, sacral NCC migrated and differentiated in an apparently normal manner. Although the numbers of sacral cells within the hindgut was slightly higher in the absence of vagal-derived cells, the increase was not sufficient to compensate for the lack of enteric ganglia. As vagal NCC appear to be more invasive than sacral NCC, since they colonise the entire length of the gut, we investigated the ability of transplanted vagal cells to colonise the hindgut by grafting the vagal NC into the sacral region. We found that when transplanted, vagal cells retained their invasive capacity and migrated into the hindgut in large numbers. Although sacral-derived cells normally contribute a relatively small number of precursors to the post-umbilical gut, many heterotopic vagal cells were found within the hindgut enteric plexuses at much earlier stages of development than normal. Heterotopic grafting of invasive vagal NCC into the sacral neuraxis may, therefore, be a means of rescuing an aganglionic hindgut phenotype.     

Expression analysis of BMP2, BMP5, BMP10 in human colon tissues from Hirschsprung disease patients

Objective: Bone morphogenetic proteins (BMPs) are members of the transforming growth factor β (TGF β) superfamily. BMP2, BMP5 and BMP10 exert their biological functions by interacting with membrane bound receptors belonging to the serine/threonine kinase family. Hirschsprung disease (HSCR) is characterized by the absence of intramural ganglion cells in the nerve plexuses of the distal gut. However, putative Notch function in enteric nervous system (ENS) development and the etiology of HSCR is unknown. Methods: Aganglionic and ganglionic colon segment tissues of 50 HSCR patients were investigated for the expression pattern of BMP2, BMP5 and BMP10 using real-time RT-PCR, Western blot analysis and immunohistochemical staining. Results: The mRNA levels of BMP2, BMP5 and BMP10 in the stenotic colon segment from HSCR patients were significantly higher than those in the normal ones. Similar increased expressions of them in the stenotic colon segments were detected by Western blotting coupled with densitometry analysis. Lastly, immunohistologicl stain showed significant BMP2, 5 and 10 increases in mucous and muscular layers from stenotic colon segments compared to normal segments. Conclusions: BMP2, BMP5 and BMP10 are elevated in the stenotic colon segment of HSCR, and BMPs signaling plays a pivotal role in the development of HSCR.     

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.     

Correlation Between Genetic Variations in Hox Clusters and Hirschsprung's Disease

Interactions between migrating neural crest cells and the environment of the gut are crucial for the development of the enteric nervous system (ENS). A key signalling mediator is the RET-receptor-tyrosine-kinase which, when defective, causes Hirschprung's disease (HSCR, colon aganglionosis). RET mutations alone cannot account for the variable HSCR phenotype, invoking interactions with as yet unknown, and probably inter-related, loci involved in ENS development. Homeobox ( HOX ) genes have a major role in gut development as depicted by the enteric Hox code. We investigated whether DNA alterations in HOX genes, either alone or in combination with RET , are implicated in HSCR. Genotyping effort was minimized by applying the HapMap data on Han Chinese from Beijing (CHB). 194 HSCR patients and 168 controls were genotyped using Sequenom technology for 72 tag, single nucleotide polymorphisms (SNPs) distributed along the HOX clusters. The HapMap frequencies were compared to those in our population and standard statistics were used for frequency comparisons. The multifactor-dimensionality-reduction method was used for multilocus analysis, in which RET promoter SNP genotypes were included. Genetic interactions were found between two HOX loci (5'- HOXA13 and 3'UTR- HOXB7 ) and the RET loci tested. Minor allele frequencies (MAF) of the SNPs tested in our sample were not significantly different from those reported by HapMap when the sample sizes of the populations compared were considered. This is the first evaluation of the HOX genes in HSCR and the first application of HapMap data in a Chinese population. The interacting HOX loci may affect the penetrance of the RET risk allele. HapMap data for the CHB population correlated well with the general Chinese population.     

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.     

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.     

To learn,to remember,to forget—How smart is the gut?

The enteric nervous system (ENS) resides within the gut wall and autonomously controls gut functions through coordinated activation of sensory, inter and motor neurons. Its activity is modulated by the enteric immune and endocrine system as well as by afferent and efferent nerves of the parasympathetic and sympathetic nervous system. The ENS is often referred to as the second brain and hence is able to perform sophisticated tasks. We review the evidence that the “smartness” of the ENS may even extend to its ability to learn and to memorize. Examples for habituation, sensitization, conditioned behaviour and long‐term facilitation are evidence for various forms of implicit learning. Moreover, we discuss how this may change not only basic Neurogastroenterology but also our understanding of development of gut diseases and chronic disorders in gut functions. At the same time, we identify open questions and future challenges to confirm learning, memory and memory deficits in the gut. Despite some remaining experimental challenges, we are convinced that the gut is able to learn and are tempted to answer the question with: Yes, the gut is smart.     

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.     

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.     

Novel mutations at RET ligand genes preventing receptor activation are associated to Hirschsprung's disease

Hirschsprung disease (HSCR) is a developmental disorder characterized by the absence of ganglion cells along variable lengths of the distal gastrointestinal tract. The major susceptibility gene for the disease is the RET proto-oncogene, which encodes a receptor tyrosine kinase activated by the glial cell-derived neurotrophic factor (GDNF) family ligands. We analyzed the coding sequence of GDNF, NTRN, and, for the first time, ARTN and PSPN in HSCR patients and detected several novel variants potentially involved in the pathogenesis of HSCR. In vitro functional analysis revealed that the variant R91C in PSPN would avoid the correct expression and secretion of the mature protein. Moreover, this study also highlighted the role of both this variant and F127L in NRTN in altering RET activation by a significant reduction in phosphorylation. To support the role of PSPN R91C in HSCR phenotype, enteric nervous system (ENS) progenitors were isolated from human postnatal gut tissues and expression of GFRα4, the main co-receptor for PSPN, was demonstrated. This suggests that not only GDNF and NRTN but also PSPN might promote survival of precursor cells during ENS development. In summary, we report for the first time the association of PSPN gene with HSCR and confirm the involvement of NRTN in the disease, with the identification of novel variants in those genes. Our results suggest that the biological consequence of the mutations NTRN F127L and PSPN R91C would be a reduction in the activation of RET-dependent signaling pathways, leading to a defect in the proliferation, migration, and/or differentiation process of neural crest cells within the developing gut and thus to the typical aganglionosis of the HSCR phenotype.     

The Ret(C620R) mutation affects renal and enteric development in a mouse model of Hirschsprung's disease

In rare families RET tyrosine kinase receptor substitutions located in exon 10 (especially at positions 609, 618, and 620) can concomitantly cause the MEN 2A (multiple endocrine neoplasia type 2A) or FMTC (familial medullary thyroid carcinoma) cancer syndromes, and Hirschsprung's disease (HSCR). No animal model mimicking the co-existence of the MEN 2 pathology and HSCR is available, and the association of these activating mutations with a developmental defect still represents an unresolved problem. The aim of this work was to investigate the significance of the RET(C620R) substitution in the pathogenesis of both gain- and loss-of-function RET-associated diseases. We report the generation of a line of mice carrying the C620R mutation in the Ret gene. Although Ret(C620R) homozygotes display severe defects in kidney organogenesis and enteric nervous system development leading to perinatal lethality. Ret(C620R) heterozygotes recapitulate features characteristic of HSCR including hypoganglionosis of the gastrointestinal tract. Surprisingly, heterozygotes do not show any defects in the thyroid that might be attributable to a gain-of-function mutation. The Ret(C620R) allele is responsible for HSCR and affects the development of kidneys and the enteric nervous system (ENS). These mice represent an interesting model for studying new therapeutic approaches for the treatment of HSCR disease.     

Advances in Molecular Genetics of Hirschsprung's Disease

Hirschsprung's disease (HSCR) is a developmental disorder of the enteric nervous system, which occurs due to the failure of neural crest cells to fully colonize the gut during embryonic development. It is characterized by the absence of the enteric ganglia in a variable length of the intestine. Substantial progress has been made in understanding the genetic basis of HSCR with the help of advanced genetic analysis techniques and animal models. More than 11 genes have been found to be involved in the pathogenesis of HSCR. The RET gene is the most important susceptibility gene involved in HSCR with both coding and non‐ coding sequence mutations. Due to phenotypic diversity and genetic complexity observed in HSCR, mutational analysis has limited practical value in genetic counseling and clinical practice. In this review, we discuss the progress that has been made in understanding the molecular genetics of HSCR and summarize the currently identified genes as well as interactions between pathways and gene‐modifying loci in HSCR. Anat Rec, 2012. © 2012 Wiley Periodicals, Inc.     

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.     

In vitro formation of enteric neural network structure in a gut-like organ differentiated from mouse embryonic stem cells

Using an embryoid body (EB) culture system, we developed a functional organ-like cluster--a "gut"--from mouse embryonic stem (ES) cells (ES gut). Each ES gut exhibited spontaneous contractions but did not exhibit distinct peristalsis-like movements. In these spontaneously contracting ES guts, dense distributions of interstitial cells of Cajal (c-kit [a transmembrane receptor that has tyrosine kinase activity]-positive cells; gut pacemaker cells) and smooth muscle cells were discernibly identified; however, enteric neural ganglia were absent in the spontaneously differentiated ES gut. By adding brain-derived neurotrophic factor (BDNF) only during EB formation, we for the first time succeeded in in vitro formation of enteric neural ganglia with connecting nerve fiber tracts (enteric nervous system [ENS]) in the ES gut. The ES gut with ENS exhibited strong peristalsis-like movements. During EB culture in BDNF(+) medium, we detected each immunoreactivity associated with the trk proto-oncogenes (trkB; BDNF receptors) and neural crest marker, proto-oncogene tyrosine-protein kinase receptor ret precursor (c-ret), p75, or sox9. These results indicated that the present ENS is differentiated from enteric neural crest-derived cells. Moreover, focal stimulation of ES guts with ENS elicited propagated increases in intracellular Ca(2+) concentration ([Ca(2+)](i)) at single or multiple sites that were attenuated by atropine or abolished by tetrodotoxin. These results suggest in vitro formation of physiologically functioning enteric cholinergic excitatory neurons. We for the first time succeeded in the differentiation of functional neurons in ENS by exogenously adding BDNF in the ES gut, resulting in generation of distinct peristalsis-like movements.