Dedicated macrophages organize and maintain the enteric nervous system.

Correct development and maturation of the enteric nervous system (ENS) is critical for survival1. At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood2. Here we demonstrate that resident macrophages of the muscularis externa (MMϕ) refine the ENS early in life by pruning synapses and phagocytosing enteric neurons. Depletion of MMϕ before weaning disrupts this process and results in abnormal intestinal transit. After weaning, MMϕ continue to interact closely with the ENS and acquire a neurosupportive phenotype. The latter is instructed by transforming growth factor-ß produced by the ENS; depletion of the ENS and disruption of transforming growth factor-ß signalling result in a decrease in neuron-associated MMϕ associated with loss of enteric neurons and altered intestinal transit. These findings introduce a new reciprocal cell-cell communication responsible for maintenance of the ENS and indicate that the ENS, similarly to the brain, is shaped and maintained by a dedicated population of resident macrophages that adapts its phenotype and transcriptome to the timely needs of the ENS niche.

Cells of the human intestinal tract mapped across space and time.

The cellular landscape of the human intestinal tract is dynamic throughout life, developing in utero and changing in response to functional requirements and environmental exposures. Here, to comprehensively map cell lineages, we use single-cell RNA sequencing and antigen receptor analysis of almost half a million cells from up to 5 anatomical regions in the developing and up to 11 distinct anatomical regions in the healthy paediatric and adult human gut. This reveals the existence of transcriptionally distinct BEST4 epithelial cells throughout the human intestinal tract. Furthermore, we implicate IgG sensing as a function of intestinal tuft cells. We describe neural cell populations in the developing enteric nervous system, and predict cell-type-specific expression of genes associated with Hirschsprung's disease. Finally, using a systems approach, we identify key cell players that drive the formation of secondary lymphoid tissue in early human development. We show that these programs are adopted in inflammatory bowel disease to recruit and retain immune cells at the site of inflammation. This catalogue of intestinal cells will provide new insights into cellular programs in development, homeostasis and disease.

Down syndrome mouse models have an abnormal enteric nervous system.

Children with trisomy 21 (Down syndrome [DS]) have a 130-fold increased incidence of Hirschsprung Disease (HSCR), a developmental defect where the enteric nervous system (ENS) is missing from distal bowel (i.e., distal bowel is aganglionic). Treatment for HSCR is surgical resection of aganglionic bowel, but many children have bowel problems after surgery. Post-surgical problems like enterocolitis and soiling are especially common in children with DS. To determine how trisomy 21 affects ENS development, we evaluated the ENS in two DS mouse models, Ts65Dn and Tc1. These mice are trisomic for many chromosome 21 homologous genes, including Dscam and Dyrk1a, which are hypothesized to contribute to HSCR risk. Ts65Dn and Tc1 mice have normal ENS precursor migration at E12.5 and almost normal myenteric plexus structure as adults. However, Ts65Dn and Tc1 mice have markedly reduced submucosal plexus neuron density throughout the bowel. Surprisingly, the submucosal neuron defect in Ts65Dn mice is not due to excess Dscam or Dyrk1a, since normalizing copy number for these genes does not rescue the defect. These findings suggest the possibility that the high frequency of bowel problems in children with DS and HSCR may occur because of additional unrecognized problems with ENS structure.

Identification of Genes Associated With Hirschsprung Disease, Based on Whole-Genome Sequence Analysis, and Potential Effects on Enteric Nervous System Development.

BACKGROUND & AIMS: Hirschsprung disease, or congenital aganglionosis, is believed to be oligogenic-that is, caused by multiple genetic factors. We performed whole-genome sequence analyses of patients with Hirschsprung disease to identify genetic factors that contribute to disease development and analyzed the functional effects of these variants. METHODS: We performed whole-genome sequence analyses of 443 patients with short-segment disease, recruited from hospitals in China and Vietnam, and 493 ethnically matched individuals without Hirschsprung disease (controls). We performed genome-wide association analyses and gene-based rare-variant burden tests to identify rare and common disease-associated variants and study their interactions. We obtained induced pluripotent stem cell (iPSC) lines from 4 patients with Hirschsprung disease and 2 control individuals, and we used these to generate enteric neural crest cells for transcriptomic analyses. We assessed the neuronal lineage differentiation capability of iPSC-derived enteric neural crest cells using an in vitro differentiation assay. RESULTS: We identified 4 susceptibility loci, including 1 in the phospholipase D1 gene (PLD1) (P = 7.4 × 10-7). The patients had a significant excess of rare protein-altering variants in genes previously associated with Hirschsprung disease and in the ß-secretase 2 gene (BACE2) (P = 2.9 × 10-6). The epistatic effects of common and rare variants across these loci provided a sensitized background that increased risk for the disease. In studies of the iPSCs, we observed common and distinct pathways associated with variants in RET that affect risk. In functional assays, we found variants in BACE2 to protect enteric neurons from apoptosis. We propose that alterations in BACE1 signaling via amyloid ß precursor protein and BACE2 contribute to pathogenesis of Hirschsprung disease. CONCLUSIONS: In whole-genome sequence analyses of patients with Hirschsprung disease, we identified rare and common variants associated with disease risk. Using iPSC cells, we discovered some functional effects of these variants.

The Drosophila Ret gene functions in the stomatogastric nervous system with the Maverick TGFß ligand and the Gfrl co-receptor.

The RET receptor tyrosine kinase is crucial for the development of the enteric nervous system (ENS), acting as a receptor for Glial cell line-derived neurotrophic factor (GDNF) via GFR co-receptors. Drosophila has a well-conserved RET homolog (Ret) that has been proposed to function independently of the Gfr-like co-receptor (Gfrl). We find that Ret is required for development of the stomatogastric (enteric) nervous system in both embryos and larvae, and its loss results in feeding defects. Live imaging analysis suggests that peristaltic waves are initiated but not propagated in mutant midguts. Examination of axons innervating the midgut reveals increased branching but the area covered by the branches is decreased. This phenotype can be rescued by Ret expression. Additionally, Gfrl shares the same ENS and feeding defects, suggesting that Ret and Gfrl might function together via a common ligand. We identified the TGFß family member Maverick (Mav) as a ligand for Gfrl and a Mav chromosomal deficiency displayed similar embryonic ENS defects. Our results suggest that the Ret and Gfrl families co-evolved before the separation of invertebrate and vertebrate lineages.

Ascl1 Is Required for the Development of Specific Neuronal Subtypes in the Enteric Nervous System.

The enteric nervous system (ENS) is organized into neural circuits within the gastrointestinal wall where it controls the peristaltic movements, secretion, and blood flow. Although proper gut function relies on the complex neuronal composition of the ENS, little is known about the transcriptional networks that regulate the diversification into different classes of enteric neurons and glia during development. Here we redefine the role of Ascl1 (Mash1), one of the few regulatory transcription factors described during ENS development. We show that enteric glia and all enteric neuronal subtypes appear to be derived from Ascl1-expressing progenitor cells. In the gut of Ascl1(-/-) mutant mice, neurogenesis is delayed and reduced, and posterior gliogenesis impaired. The ratio of neurons expressing Calbindin, TH, and VIP is selectively decreased while, for instance, 5-HT(+) neurons, which previously were believed to be Ascl1-dependent, are formed in normal numbers. Essentially the same differentiation defects are observed in Ascl1(KINgn2) transgenic mutants, where the proneural activity of Ngn2 replaces Ascl1, demonstrating that Ascl1 is required for the acquisition of specific enteric neuronal subtype features independent of its role in neurogenesis. In this study, we provide novel insights into the expression and function of Ascl1 in the differentiation process of specific neuronal subtypes during ENS development. SIGNIFICANCE STATEMENT: The molecular mechanisms underlying the generation of different neuronal subtypes during development of the enteric nervous system are poorly understood despite its pivotal function in gut motility and involvement in gastrointestinal pathology. This report identifies novel roles for the transcription factor Ascl1 in enteric gliogenesis and neurogenesis. Moreover, independent of its proneurogenic activity, Ascl1 is required for the normal expression of specific enteric neuronal subtype characteristics. Distinct enteric neuronal subtypes are formed in a temporally defined order, and we observe that the early-born 5-HT(+) neurons are generated in Ascl1(-/-) mutants, despite the delayed neurogenesis. Enteric nervous system progenitor cells may therefore possess strong intrinsic control over their specification at the initial waves of neurogenesis.

NEDL2 regulates enteric nervous system and kidney development in its Nedd8 ligase activity-dependent manner.

The GDNF (Glial cell line-derived neurotrophic factor)/Ret/Akt signaling pathway is essential to the development of ENS (enteric nervous system) as well as kidney. We previously showed that the HECT-type E3 ligase NEDL2 (Nedd4-like ligase 2) is required for the ENS development by activating GDNF/Ret/Akt. However, the underlying mechanism remains unknown. Here we show that in addition to ENS, NEDL2 is also pivotal for kidney development since about 1/3 of Nedl2-deficient mice displayed postnatal unilateral or bilateral kidney hydronephrosis. Double knockout of Nedl1 and Nedl2 in mice leads to postnatal lethal within 2 weeks and the phenotypes resemble those of Nedl2 single knockout mice. Surprisingly, its close member NEDL1 is dispensable for ENS and kidney function and the reason is lack of NEDL1 expression in these systems during early development. Furthermore, biochemical analysis indicated that NEDL2 appears to act like a scaffold protein to recruit SHC, Grb2, PI3K (p110 and p85), PDK1 and Akt together to promote the signaling transduction. Intriguingly, we found that NEDL2 harbours intrinsic Nedd8 ligase activity with cysteine 1341 as the core site. NEDL2 upregulates GDNF-stimulated Akt activity dependent of its Nedd8 ligase activity but not its ubiquitin ligase activity. These findings demonstrate that NEDL2 but not NEDL1 is required for ENS and kidney development in a unique Nedd8 ligase-dependent manner.

Neuronal Differentiation in Schwann Cell Lineage Underlies Postnatal Neurogenesis in the Enteric Nervous System.

Elucidation of the cellular identity of neuronal precursors provides mechanistic insights into the development and pathophysiology of the nervous system. In the enteric nervous system (ENS), neurogenesis persists from midgestation to the postnatal period. Cellular mechanism underlying the long-term neurogenesis in the ENS has remained unclear. Using genetic fate mapping in mice, we show here that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate in the postnatal period and contributes to the ENS. We found SCP-derived neurogenesis in the submucosal region of the small intestine in the absence of vagal neural crest-derived ENS precursors. Under physiological conditions, SCPs comprised up to 20% of enteric neurons in the large intestine and gave rise mainly to restricted neuronal subtypes, calretinin-expressing neurons. Genetic ablation of Ret, the signaling receptor for glial cell line-derived neurotrophic factor, in SCPs caused colonic oligoganglionosis, indicating that SCP-derived neurogenesis is essential to ENS integrity. Identification of Schwann cells as a physiological neurogenic source provides novel insight into the development and disorders of neural crest-derived tissues. SIGNIFICANCE STATEMENT: Elucidating the cellular identity of neuronal precursors provides novel insights into development and function of the nervous system. The enteric nervous system (ENS) is innervated richly by extrinsic nerve fibers, but little is known about the significance of extrinsic innervation to the structural integrity of the ENS. This report reveals that a subset of Schwann cell precursors (SCPs), which invades the gut alongside the extrinsic nerves, adopts a neuronal fate and differentiates into specific neuronal subtypes. SCP-specific ablation of the Ret gene leads to colonic oligoganglionosis, demonstrating a crucial role of SCP-derived neurogenesis in ENS development. Cross-lineage differentiation capacity in SCPs suggests their potential involvement in the development and pathology of a wide variety of neural crest-derived cell types.

A protein tyrosine kinase receptor, c-RET signaling pathway contributes to the enteric neurogenesis induced by a 5-HT4 receptor agonist at an anastomosis after transection of the gut in rodents.

We previously reported that a serotonin 4 (5-HT4) receptor agonist, mosapride citrate (MOS), increased the number of c-RET-positive cells and levels of c-RET mRNA in gel sponge implanted in the necks of rats. The 5-HT4 receptor is a G protein coupled receptor (GPCR) coupled to G protein Gs-cAMP cascades. We investigated the possibility that 5-HT4 receptor activation induced c-RET activation and/or PKA activation by elevating cAMP levels. Rodents were orally administered MOS by adding it to drinking water for 2 weeks after enteric nerve circuit insult via gut transection and anastomosis, together with the RET inhibitors withaferin A (WA) and RPI-1 or the PKA inhibitor H89. We then examined PGP9.5-positive cells in the newly formed granulation tissue at the anastomotic site. MOS significantly increased the number of new neurons, but not when co-administered with WA or RPI-1. Co-administration of H89 failed to alter MOS-induced increases in neurogenesis. In conclusion, the c-RET signaling pathway contributes to enteric neurogenesis facilitated by MOS, though the contribution of PKA activation seems unlikely.

Development of myenteric cholinergic neurons in ChAT-Cre;R26R-YFP mice.

Cholinergic neurons are the major excitatory neurons of the enteric nervous system (ENS), and include intrinsic sensory neurons, interneurons, and excitatory motor neurons. Cholinergic neurons have been detected in the embryonic ENS; however, the development of these neurons has been difficult to study as they are difficult to detect prior to birth using conventional immunohistochemistry. In this study we used ChAT-Cre;R26R-YFP mice to examine the development of cholinergic neurons in the gut of embryonic and postnatal mice. Cholinergic (YFP+) neurons were first detected at embryonic day (E)11.5, and the proportion of cholinergic neurons gradually increased during pre- and postnatal development. At birth, myenteric cholinergic neurons comprised less than half of their adult proportions in the small intestine (25% of myenteric neurons were YFP+ at P0 compared to 62% in adults). The earliest cholinergic neurons appear to mainly project anally. Projections into the presumptive circular muscle were first observed at E14.5. A subpopulation of cholinergic neurons coexpress calbindin through embryonic and postnatal development, but only a small proportion coexpressed neuronal nitric oxide synthase. Our study shows that cholinergic neurons in the ENS develop over a protracted period of time.

Ex vivo neurogenesis within enteric ganglia occurs in a PTEN dependent manner.

A population of multipotent stem cells capable of differentiating into neurons and glia has been isolated from adult intestine in humans and rodents. While these cells may provide a pool of stem cells for neurogenesis in the enteric nervous system (ENS), such a function has been difficult to demonstrate in vivo. An extensive study by Joseph et al. involving 108 rats and 51 mice submitted to various insults demonstrated neuronal uptake of thymidine analog BrdU in only 1 rat. Here we introduce a novel approach to study neurogenesis in the ENS using an ex vivo organotypic tissue culturing system. Culturing longitudinal muscle and myenteric plexus tissue, we show that the enteric nervous system has tremendous replicative capacity with the majority of neural crest cells demonstrating EdU uptake by 48 hours. EdU(+) cells express both neuronal and glial markers. Proliferation appears dependent on the PTEN/PI3K/Akt pathway with decreased PTEN mRNA expression and increased PTEN phosphorylation (inactivation) corresponding to increased Akt activity and proliferation. Inhibition of PTEN with bpV(phen) augments proliferation while LY294002, a PI3K inhibitor, blocks it. These data suggest that the ENS is capable of neurogenesis in a PTEN dependent manner.

Zic2 is required for enteric nervous system development and neurite outgrowth: a mouse model of enteric hyperplasia and dysplasia.

From a forward genetic screen in mice we isolated a novel Zic2 allele with abnormal aggregation of meconium in the gastrointestinal tract. Zic2(m1Nisw) mutant embryos show an increase in the number of enteric neurons in vivo and disorganization of the neurite network. Explant culture of Zic2(m1Nisw) gastrointestinal tract show extensive neurite outgrowth, suggesting that Zic2 is a negative regulator of nerve fiber growth. These studies demonstrate a previously unknown function of Zic2 and provide a novel animal model of enteric nervous system dysplasia and hyperplasia.

Netrin-1 in the developing enteric nervous system and colorectal cancer.

Netrin-1 is a well-characterised chemoattractant involved in neuronal guidance in the developing enteric nervous system (ENS), but it is also a regulator of tumorigenesis. Two of its well-characterised receptors, deleted in colorectal cancer (DCC) and uncoordinated-5 homolog (UNC-5H), belong to a family of dependence receptors that transmit either pro- or anti-apoptosis signals depending on the availability of ligand, in this case netrin-1. This review summarises these two effects of netrin-1 and highlights the additional research needed information about to allow better utilisation of netrin-1 as a therapeutic target for axonal regeneration in the context of colorectal cancer.

Expression level of Hand2 affects specification of enteric neurons and gastrointestinal function in mice.

BACKGROUND & AIMS: Hand2 is a basic helix-loop-helix transcription factor required for terminal differentiation of enteric neurons. We studied Hand2 haploinsufficient mice, to determine whether reduced expression of Hand2 allows sufficient enteric neurogenesis for survival, but not for development of a normal enteric nervous system (ENS). METHODS: Enteric transcripts that encode Hand2 and the neuron-specific embryonic lethal abnormal vision proteins HuB, HuC, and HuD were quantified. Immunocytochemistry was used to identify and quantify neurons. Apoptosis was analyzed with the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling procedure. Intracellular microelectrodes were used to record inhibitory junction potentials. Gastrointestinal transit and colonic motility were measured in vivo. RESULTS: Levels of of enteric Hand2 transcripts were associated with genotypes of mice, in the following order: Hand2(+/+) > Hand2(LoxP/+) > Hand2(+/-) > Hand2(LoxP/-). Parallel reductions were found in expression of HuD and in regional and phenotypic manners. Numbers of neurons, numbers of neuronal nitric oxide synthase(+) and calretinin(+), but not substance P(+) or vasoactive intestinal peptide(+) neurons, decreased. No effects were observed in stomach or cecum. Apoptosis was not detected, consistent with the concept that Hand2 inhibits neuronal differentiation, rather than regulates survival. The amplitude of inhibitory junction potentials in colonic circular muscle was similar in Hand2 wild-type and haploinsufficient mice, although in haploinsufficient mice, the purinergic component was reduced and a nitrergic component appeared. The abnormal ENS of haploinsufficient mice slowed gastrointestinal motility but protected mice against colitis. CONCLUSIONS: Reduced expression of factors required for development of the ENS can cause defects in the ENS that are subtle enough to escape detection yet cause significant abnormalities in bowel function.

Transcriptional networks and signaling pathways that govern vertebrate intestinal development.

The vertebrate intestine is a complex and highly specialized organ comprising tissues derived from all three germ layers. While a description of the morphological events underlying the consolidation and organization of the endoderm, mesoderm, and ectoderm-derived cells into a multi-layered, continuously renewing organ has been available for several decades, only recently has a strong genetic framework for this process started to emerge, and as yet it remains incomplete. This review highlights the roles played by a number of transcription factors and signaling pathways in the development of the vertebrate intestine from the moment the definitive endoderm is formed. These molecular pathways often interact with each other and play multiple roles at different stages of intestinal formation. What is currently attracting considerable attention in the field is the realization that the deregulated activities of these same pathways often play a key role in the initiation and progression of a number of complex, serious intestinal diseases, including inflammatory bowel disease and cancer.

Development of the enteric nervous system and its role in intestinal motility during fetal and early postnatal stages.

Motility patterns in the mature intestine require the coordinated interaction of enteric neurons, gastrointestinal smooth muscle, and interstitial cells of Cajal. In Hirschsprung's disease, the aganglionic segment causes functional obstruction, and thus the enteric nervous system (ENS) is essential for gastrointestinal motility after birth. Here we review the development of the ENS. We then focus on motility patterns in the small intestine and colon of fetal mice and larval zebrafish, where recent studies have shown that the first intestinal motility patterns are not neurally mediated. Finally, we review the development of gastrointestinal motility in humans.

Enteric nervous system development: Recent progress and future challenges.

The enteric nervous system is the largest subdivision of the peripheral nervous system that plays a critical role in digestive functions. Despite considerable progress over the last 15 years in understanding the molecular and cellular mechanisms that control the development of the enteric nervous system, several questions remain unanswered. The present review will focus on recent progress on understanding the development of the mammalian enteric nervous system and highlight interesting directions of future research.

Genetic model system studies of the development of the enteric nervous system, gut motility and Hirschsprung's disease.

The enteric nervous system (ENS) is the largest and most complicated subdivision of the peripheral nervous system. Its action is necessary to regulate many of the functions of the gastrointestinal tract including its motility. Whilst the ENS has been studied extensively by developmental biologists, neuroscientists and physiologists for several decades it has only been since the early 1990s that the molecular and genetic basis of ENS development has begun to emerge. Central to this understanding has been the use of genetic model organisms. In this article, we will discuss recent advances that have been achieved using both mouse and zebrafish model genetic systems that have led to new insights into ENS development and the genetic basis of Hirschsprung's disease.

Development of enteric and vagal innervation of the zebrafish (Danio rerio) gut.

The autonomic nervous system develops following migration and differentiation of precursor cells originating in the neural crest. Using immunohistochemistry on intact zebrafish embryos and larvae we followed the development of the intrinsic enteric and extrinsic vagal innervation of the gut. At 3 days postfertilization (dpf), enteric nerve cell bodies and fibers were seen mainly in the middle and distal intestine, while the innervation of the proximal intestine was scarcer. The number of fibers and cell bodies gradually increased, although a large intraindividual variation was seen in the timing (but not the order) of development. At 11-13 dpf most of the proximal intestine received a similar degree of innervation as the rest of the gut. The main intestinal branches of the vagus were similarly often already well developed at 3 dpf, entering the gut at the transition between the proximal and middle intestine and projecting posteriorly along the length of the gut. Subsequently, fibers branching off the vagus innervated all regions of the gut. The presence of several putative enteric neurotransmitters was suggested by using markers for neurokinin A (NKA), pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal polypeptide (VIP), nitric oxide, serotonin (5-hydroxytryptamine, 5-HT), and calcitonin gene-related peptide (CGRP). The present results corroborate the belief that the enteric innervation is well developed before the onset of feeding (normally occurring around 5-6 dpf). Further, the more detailed picture of how development proceeds at stages previously not examined suggests a correlation between increasing innervation and more regular and elaborated motility patterns.

Occurrence of two NOS isoforms in the developing gut of sea bass Dicentrarchus labrax (L.).

In this work we have examined the appearance and distribution of nitric oxide synthase (NOS), with histochemical, immunohistochemical and biochemical methods, during development of the sea bass (Dicentrarchus labrax) gut. The data showed that both the calcium-calmodulin dependent neuronal isoform (nNOS) and calcium-independent inducible isoform (iNOS) are present in the larval gut of sea bass. The nNOS-immunoreactivity was present in the epithelial cells and enteric nerve cells of gut both in the 8-day-old specimens and in the 24-day-old-larvae. In the adult nNOS-immunoreactivity disappeared from epithelial cells, remaining in the wall intramural neurons and fibers. The iNOS-immunoreactivity was present in the epithelial cells of 24-day-old-larvae and was not detectable in the adult gut. Western blot analysis and determination of NOS activity also demonstrated the presence of the two NOS isoforms, nNOS and iNOS, in the gut of 24-day-old specimens. The presumably different roles played by the two isoforms of enzyme are discussed. The presence of nNOS isoform in the gut enteric neurons of the same larval stages of D. labrax in which we previously demonstrated the presence of substance P and Vasoactive Intestinal Polypeptide (VIP), may suggest that all these three components of the motility control system are already present in the larval phase. Nitric oxide (NO) may be also involved in the early immune response. The present results on the occurrence of iNOS isoform in epithelial gut cells of the same regions in which the gut-associated lymphoid tissue (GALT) will differentiate, may suggest for NO a role in early defence mechanisms, before the establishment of immune responses in GALT. Finally, the developmental and regional differences in nNOS and iNOS expression also suggest a regulatory role in development and differentiation of the sea bass gut.