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Inflammatory postoperative conditions of equine colic (acute abdomen) contribute not only to increased client cost, patient discomfort, and hospitalization time, but in many cases, prove to be life-threatening. A unique population of intestinal cells, enteric glia, are increasingly acknowledged for their roles in sensing the gastrointestinal environment and communicating with surrounding cell types. Interactions between enteric glia and intestinal epithelia may prove critical in establishing how equine enteric glia can alter the mucosal barrier to modulate inflammation in health and colic. To study this interaction, we present a method of establishing primary equine enteric glial cultures from equine jejunum and exposing the cultures to inflammatory conditions known to be present in colic. Primary enteric glial cultures were obtained from adult horses euthanized for reasons unrelated to colic. Intestinal villi and lamina propria were micro-dissected to expose the submucosa. The isolated submucosa underwent enzymatic digestion with collagenase, protease, and bovine serum albumin for 2-3 h. Next, mechanical digestion involving centrifugation, pipetting, and cell strainers (40-100 µm), yielded a pellet used for plating on 0.05 mg/mL poly-L-lysine-coated wells at a concentration of ~400,000 cells/300 µL of media. Following confluence and first passage, the enteric glial cells were then exposed to equine recombinant IL-1β (0, 10, 25 ng) for 24 h. To model epithelial-glial interactions at the time of colic, medium conditioned by either control or treated enteric glia was added directly to confluent equine jejunal monolayers while measuring transepithelial electrical resistance (TEER) using a dual-electrode EndOhm chamber. These data demonstrate just one of many potential impactful applications of equine enteric glial culture.

While the enteric nervous system (ENS) is highly dynamic during development, the extent to which it is capable of repair remains unclear. In this issue of Neuron, Stavely et al. 1 show that enteric neurons can reinnervate damaged regions to regain functionality using a glial positioning system (GPS) as their guide., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Here, we establish that plasticity exists within the postnatal enteric nervous system by demonstrating the reinnervation potential of post-mitotic enteric neurons (ENs). Employing BAF53b-Cre mice for selective neuronal tracing, the reinnervation capabilities of mature postnatal ENs are shown across multiple model systems. Isolated ENs regenerate neurites in vitro, with neurite complexity and direction influenced by contact with enteric glial cells (EGCs). Nerve fibers from transplanted ENs exclusively interface and travel along EGCs within the muscularis propria. Resident EGCs persist after Cre-dependent ablation of ENs and govern the architecture of the myenteric plexus for reinnervating ENs, as shown by nerve fiber projection tracing. Transplantation and optogenetic experiments in vivo highlight the rapid reinnervation potential of post-mitotic neurons, leading to restored gut muscle contractile activity within 2 weeks. These studies illustrate the structural and functional reinnervation capacity of post-mitotic ENs and the critical role of EGCs in guiding and patterning their trajectories., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

During enteric nervous system (ENS) development, pioneering wavefront enteric neural crest cells (ENCCs) initiate gut colonization. However, the molecular mechanisms guiding their specification and niche interaction are not fully understood. We used single-cell RNA sequencing and spatial transcriptomics to map the spatiotemporal dynamics and molecular landscape of wavefront ENCCs in mouse embryos. Our analysis shows a progressive decline in wavefront ENCC potency during migration and identifies transcription factors governing their specification and differentiation. We further delineate key signaling pathways (ephrin-Eph, Wnt-Frizzled, and Sema3a-Nrp1) utilized by wavefront ENCCs to interact with their surrounding cells. Disruptions in these pathways are observed in human Hirschsprung's disease gut tissue, linking them to ENS malformations. Additionally, we observed region-specific and cell-type-specific transcriptional changes in surrounding gut tissues upon wavefront ENCC arrival, suggesting their role in shaping the gut microenvironment. This work offers a roadmap of ENS development, with implications for understanding ENS disorders., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

The human enteric nervous system, ENS, is a large network of glial and neuronal cell types with remarkable neurotransmitter diversity. The ENS controls bowel motility, enzyme secretion, and nutrient absorption and interacts with the immune system and the gut microbiome. Consequently, developmental and acquired defects of the ENS are responsible for many human diseases and may contribute to symptoms of Parkinson's disease. Limitations in animal model systems and access to primary tissue pose significant experimental challenges in studies of the human ENS. Here, a detailed protocol is presented for effective in vitro derivation of the ENS lineages from human pluripotent stem cells, hPSC, using defined culture conditions. Our protocol begins with directed differentiation of hPSCs to enteric neural crest cells within 15 days and yields diverse subtypes of functional enteric neurons within 30 days. This platform provides a scalable resource for developmental studies, disease modeling, drug discovery, and regenerative applications.

Regenerative cell therapy to replenish the missing neurons and glia in the aganglionic segment of Hirschsprung disease represents a promising treatment option. However, the success of cell therapies for this condition are hindered by poor migration of the transplanted cells. This limitation is in part due to a markedly less permissive extracellular environment in the postnatal gut than that of the embryo. Coordinated interactions between enteric neural crest-derived cells (ENCDCs) and their local environment drive migration along the embryonic gut during development of the enteric nervous system. Modifying transplanted cells, or the postnatal extracellular environment, to better recapitulate embryonic ENCDC migration could be leveraged to improve the engraftment and coverage of stem cell transplants. We compared the transcriptomes of ENCDCs from the embryonic intestine to that of postnatal-derived neurospheres and identified 89 extracellular matrix (ECM)-associated genes that are differentially expressed. Agrin, a heparin sulfate proteoglycan with a known inhibitory effect on ENCDC migration, was highly over-expressed by postnatal-derived neurospheres. Using a function-blocking antibody and a shRNA-expressing lentivirus, we show that inhibiting agrin promotes ENCDC migration in vitro and following cell transplantation ex vivo and in vivo. This enhanced migration is associated with an increased proportion of GFAP + cells, whose migration is especially enhanced., (© The Author(s) 2024. Published by Oxford University Press.)

The enteric nervous system (ENS) comprises a complex network of neurons whereby a subset appears to be dopaminergic although the characteristics, roles, and implications in disease are less understood. Most investigations relating to enteric dopamine (DA) neurons rely on immunoreactivity to tyrosine hydroxylase (TH)-the rate-limiting enzyme in the production of DA. However, TH immunoreactivity is likely to provide an incomplete picture. This study herein provides a comprehensive characterization of DA neurons in the gut using a reporter mouse line, expressing a fluorescent protein (tdTomato) under control of the DA transporter (DAT) promoter. Our findings confirm a unique localization of DA neurons in the gut and unveil the discrete subtypes of DA neurons in this organ, which we characterized using both immunofluorescence and single-cell transcriptomics, as well as validated using in situ hybridization. We observed distinct subtypes of DAT-tdTomato neurons expressing co-transmitters and modulators across both plexuses; some of them likely co-releasing acetylcholine, while others were positive for a slew of canonical DAergic markers (TH, VMAT2 and GIRK2). Interestingly, we uncovered a seemingly novel population of DA neurons unique to the ENS which was ChAT/DAT-tdTomato-immunoreactive and expressed Grp, Calcb, and Sst. Given the clear heterogeneity of DAergic gut neurons, further investigation is warranted to define their functional signatures and decipher their implication in disease., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Correct development and maturation of the enteric nervous system (ENS) is critical for survival 1 . At birth, the ENS is immature and requires considerable refinement to exert its functions in adulthood 2 . 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-β produced by the ENS; depletion of the ENS and disruption of transforming growth factor-β 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., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Enteric glia are a unique population of peripheral neuroglia associated with the enteric nervous system (ENS) throughout the digestive tract. The emerging data from the latest glial biology studies unveiled enteric glia as a heterogenic population with plastic and adaptative abilities that display phenotypic and functional changes upon distinct extrinsic cues. This aspect is essential in the dynamic signaling that enteric glia engage with neurons and other neighboring cells within the intestinal wall, such as epithelial, endocrine, and immune cells to maintain local homeostasis. Likewise, enteric glia sense signals from luminal microbes, although the extent of this active communication is still unclear. In this minireview, we discuss the recent findings that support glia-microbes crosstalk in the intestine in health and disease, pointing out the critical aspects that require further investigation., (Copyright © 2023 Elsevier B.V. All rights reserved.)

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition., Competing Interests: Declaration of interests A.M.G. receives research funds from Takeda Pharmaceutical Company., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

The developmental programs that build and sustain animal forms also encode the capacity to sense and adapt to the microbial world within which they evolved. This is abundantly apparent in the development of the digestive tract, which typically harbors the densest microbial communities of the body. Here, we review studies in human, mouse, zebrafish and Drosophila that are revealing how the microbiota impacts the development of the gut and its communication with the nervous system, highlighting important implications for human and animal health., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Glia in the central nervous system exert precise spatial and temporal regulation over neural circuitry on a synapse-specific basis, but it is unclear if peripheral glia share this exquisite capacity to sense and modulate circuit activity. In the enteric nervous system (ENS), glia control gastrointestinal motility through bidirectional communication with surrounding neurons. We combined glial chemogenetics with genetically encoded calcium indicators expressed in enteric neurons and glia to study network-level activity in the intact myenteric plexus of the proximal colon. Stimulation of neural fiber tracts projecting in aboral, oral, and circumferential directions activated distinct populations of enteric glia. The majority of glia responded to both oral and aboral stimulation and circumferential pathways, while smaller subpopulations were activated only by ascending and descending pathways. Cholinergic signaling functionally specifies glia to the descending circuitry, and this network plays an important role in repressing the activity of descending neural pathways, with some degree of cross-inhibition imposed upon the ascending pathway. Glial recruitment by purinergic signaling functions to enhance activity within ascending circuit pathways and constrain activity within descending networks. Pharmacological manipulation of glial purinergic and cholinergic signaling differentially altered neuronal responses in these circuits in a sex-dependent manner. Collectively, our findings establish that the balance between purinergic and cholinergic signaling may differentially control specific circuit activity through selective signaling between networks of enteric neurons and glia. Thus, enteric glia regulate the ENS circuitry in a network-specific manner, providing profound insights into the functional breadth and versatility of peripheral glia., Competing Interests: The authors declare no competing interest., (Copyright © 2021 the Author(s). Published by PNAS.)

Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

The development of the enteric nervous system (ENS) is highly modulated by the synchronized interaction between the enteric neural crest cells (ENCCs) and the neural stem cell niche comprising the gut microenvironment. Genetic defects dysregulating the cellular behaviour(s) of the ENCCs result in incomplete innervation and hence ENS dysfunction. Hirschsprung disease (HSCR) is a rare complex neurocristopathy in which the enteric neural crest-derived cells fail to colonize the distal colon. In addition to ENS defects, increasing evidence suggests that HSCR patients may have intrinsic defects in the niche impairing the extracellular matrix (ECM)-cell interaction and/or dysregulating the cellular niche factors necessary for controlling stem cell behaviour. The niche defects in patients may compromise the regenerative capacity of the stem cell-based therapy and advocate for drug- and niche-based therapies as complementary therapeutic strategies to alleviate/enhance niche-cell interaction. Here, we provide a summary of the current understandings of the role of the enteric neural stem cell niche in modulating the development of the ENS and in the pathogenesis of HSCR. Deciphering the contribution of the niche to HSCR may provide important implications to the development of regenerative medicine for HSCR.

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., (© 2021. The Author(s).)

Neuronal nitric oxide synthase (nNOS) neurons play a fundamental role in inhibitory neurotransmission, within the enteric nervous system (ENS), and in the establishment of gut motility patterns. Clinically, loss or disruption of nNOS neurons has been shown in a range of enteric neuropathies. However, the effects of nNOS loss on the composition and structure of the ENS remain poorly understood. The aim of this study was to assess the structural and transcriptional consequences of loss of nNOS neurons within the murine ENS. Expression analysis demonstrated compensatory transcriptional upregulation of pan neuronal and inhibitory neuronal subtype targets within the Nos1 -/- colon, compared to control C57BL/6J mice. Conventional confocal imaging; combined with novel machine learning approaches, and automated computational analysis, revealed increased interconnectivity within the Nos1 -/- ENS, compared to age-matched control mice, with increases in network density, neural projections and neuronal branching. These findings provide the first direct evidence of structural and molecular remodelling of the ENS, upon loss of nNOS signalling. Further, we demonstrate the utility of machine learning approaches, and automated computational image analysis, in revealing previously undetected; yet potentially clinically relevant, changes in ENS structure which could provide improved understanding of pathological mechanisms across a host of enteric neuropathies., (© 2021. The Author(s).)

Enteric neural stem cells (ENSC) have been identified as a possible treatment for enteric neuropathies. After in vivo transplantation, ENSC and their derivatives have been shown to engraft within colonic tissue, migrate and populate endogenous ganglia, and functionally integrate with the enteric nervous system. However, the mechanisms underlying the integration of donor ENSC, in recipient tissues, remain unclear. Therefore, we aimed to examine ENSC integration using an adapted ex vivo organotypic culture system. Donor ENSC were obtained from Wnt1 cre/+ ;R26R YFP/YFP mice allowing specific labelling, selection and fate-mapping of cells. YFP + neurospheres were transplanted to C57BL6/J (6-8-week-old) colonic tissue and maintained in organotypic culture for up to 21 days. We analysed and quantified donor cell integration within recipient tissues at 7, 14 and 21 days, along with assessing the structural and molecular consequences of ENSC integration. We found that organotypically cultured tissues were well preserved up to 21-days in ex vivo culture, which allowed for assessment of donor cell integration after transplantation. Donor ENSC-derived cells integrated across the colonic wall in a dynamic fashion, across a three-week period. Following transplantation, donor cells displayed two integrative patterns; longitudinal migration and medial invasion which allowed donor cells to populate colonic tissue. Moreover, significant remodelling of the intestinal ECM and musculature occurred upon transplantation, to facilitate donor cell integration within endogenous enteric ganglia. These results provide critical evidence on the timescale and mechanisms, which regulate donor ENSC integration, within recipient gut tissue, which are important considerations in the future clinical translation of stem cell therapies for enteric disease., (© 2021. The Author(s).)

One of the most transformative developments in neurogastroenterology is the realization that many functions normally attributed to enteric neurons involve interactions with enteric glial cells: a large population of peripheral neuroglia associated with enteric neurons throughout the gastrointestinal tract. The notion that glial cells function solely as passive support cells has been refuted by compelling evidence that demonstrates that enteric glia are important homeostatic cells of the intestine. Active signalling mechanisms between enteric glia and neurons modulate gastrointestinal reflexes and, in certain circumstances, function to drive neuroinflammatory processes that lead to long-term dysfunction. Bidirectional communication between enteric glia and immune cells contributes to gastrointestinal immune homeostasis, and crosstalk between enteric glia and cancer stem cells regulates tumorigenesis. These neuromodulatory and immunomodulatory roles place enteric glia in a unique position to regulate diverse gastrointestinal disease processes. In this Review, we discuss current concepts regarding enteric glial development, heterogeneity and functional roles in gastrointestinal pathophysiology and pathophysiology, with a focus on interactions with neurons and immune cells. We also present a working model to differentiate glial states based on normal function and disease-induced dysfunctions., (© 2021. Springer Nature Limited.)

Lipopolysaccharides (LPS), also known as lipoglycans or endotoxins, form part of the outer membrane of Gram-negative bacteria. Previous studies have described the various harmful impacts of LPS on humans and animals. Nevertheless, many aspects of these effects are still not fully explained. One of them is the influence of endotoxins on the neurochemical characterization of neurons within the enteric nervous system (ENS), which is found in the intestinal wall and plays important adaptive roles during pathological processes and exposures. In this study, the impact of a low single dose of Salmonella Enteritidis LPS on the duodenal enteric neurons immunoreactive to substance P (SP), vasoactive intestinal polypeptide (VIP), pituitary adenylate cyclase activating peptide (PACAP-27), and cocaine- and amphetamine-regulated transcript (CART) was studied using a double immunofluorescence technique. During the study, it was shown that even a low dose of LPS affects the number of enteric neurons containing the neuropeptides studied, and these changes were dependent on the type of the enteric plexus. The most visible changes concerned the SP-like immunoreactive (LI) neurons in the outer submucous plexus (LPS caused an increase in the percentage of these neurons from15.74 ± 0.61 to 21.72 ± 0.79%). Furthermore, the VIP-LI neurons in the inner submucous plexus were seen to decrease from 12.64 ± 0.83 to 5.96 ± 0.58%. The mechanisms behind these noted fluctuations are not clear, but it may be connected with the pro-inflammatory and neurotoxic activity of LPS., (© 2020. Springer Science+Business Media, LLC, part of Springer Nature.)

Objective: Enteric glial cells (EGCs) can activate multiple pathways to inhibit the deleterious effects of acute and chronic insults. Our aim was to test the effect of EGCs on hyperglycemia-induced neuron damage and its underlying intracellular mechanisms., Methods: A coculture model composed of EGCs and neuroblastoma cells (SH-SY5Y) was established to examine glial-mediated neuroprotection under high glucose conditions. The cell counting assay kit CCK-8 was used to measure cell viability. Flow cytometry was used to measure the induction of reactive oxygen species (ROS), change of mitochondrial membrane potential (MMP), cell cycle distribution, and apoptosis. The expressions of cyclin D1, cyclin E2, Bax, cleaved caspase-3, AKT, p-AKT, GSK-3β, and p-GSK-3β were tested using western blot., Results: Exposure to high glucose (≥35 mM) reduced the viability of SH-SY5Y cells in a concentration- and time-dependent manner. Meanwhile, enhanced ROS generation and decrease of MMP were observed in SH-SY5Y cells when treated with high glucose. Furthermore, high glucose also caused SH-SY5Y cells arrest in G2 phase and apoptosis, accompanied by decreasing cyclin D1 and E2, and upregulating Bax and cleaved caspase-3. Coculture EGC lines or EGC-conditioned medium with SH-SY5Y prevented the neurotoxic effects. The p-AKT/AKT and p-GSK-3β/GSK-3β ratios were dramatically decreased in SH-SY5Y cells after high glucose incubation, which was restored after coculture with EGCs., Conclusions: EGCs can protect neurons from hyperglycemia-induced injury by activating the Akt/GSK-3β pathway., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

The enteric nervous system is responsible for controlling the gastrointestinal tract (GIT) functions. Enteric neuropathies are highly correlated to the development of several intestinal disturbances. Fluoride (F) is extensively applied for dental health improvement and its ingestion can promote systemic toxicity with mild to severe GIT symptomatology and neurotoxicity. Although F harmful effects have been published, there is no information regarding noxiousness of a high acute F exposure (25 mg F/kg) on enteric neurons and levels of expression of intestinal proteins in the duodenum. Quantitative proteomics of the duodenum wall associated to morphometric and quantitative analysis of enteric neurons displayed F effects of a high acute exposure. F-induced myenteric neuroplasticity was characterized by a decrease in the density of nitrergic neurons and morphometric alterations in the general populations of neurons, nitrergic neurons, and substance P varicosities. Proteomics demonstrated F-induced alterations in levels of expression of 356 proteins correlated to striated muscle cell differentiation; generation of precursor metabolites and energy; NADH and glutathione metabolic process and purine ribonucleoside triphosphate biosynthesis. The neurochemical role of several intestinal proteins was discussed specially related to the modulation of enteric neuroplasticity. The results provide a new perspective on cell signaling pathways of gastrointestinal symptomatology promoted by acute F toxicity.

The esophagus is derived from the anterior portion of the foregut endoderm, which also gives rise to the respiratory system. As it develops, the esophageal lining is transformed from a simple columnar epithelium into a stratified squamous cell layer, accompanied by the replacement of unspecified mesenchyme with layers of muscle cells. Studies in animal models have provided significant insights into the roles of various signaling pathways in esophageal development. More recent studies using human pluripotent stem cells (hPSCs) further demonstrate that some of these signaling pathways are conserved in human esophageal development. In addition, a combination of mouse genetics and hPSC differentiation approaches have uncovered new players that control esophageal morphogenesis. In this Review, we summarize these new findings and discuss how the esophagus is established and matures throughout different stages, including its initial specification, respiratory-esophageal separation, epithelial morphogenesis and maintenance. We also discuss esophageal muscular development and enteric nervous system innervation, which are essential for esophageal structure and function., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background: Hirschsprung disease (HSCR, OMIM 142623) is a rare congenital disorder that results from a failure to fully colonize the gut by enteric precursor cells (EPCs) derived from the neural crest. Such incomplete gut colonization is due to alterations in EPCs proliferation, survival, migration and/or differentiation during enteric nervous system (ENS) development. This complex process is regulated by a network of signaling pathways that is orchestrated by genetic and epigenetic factors, and therefore alterations at these levels can lead to the onset of neurocristopathies such as HSCR. The goal of this study is to broaden our knowledge of the role of epigenetic mechanisms in the disease context, specifically in DNA methylation. Therefore, with this aim, a Whole-Genome Bisulfite Sequencing assay has been performed using EPCs from HSCR patients and human controls., Results: This is the first study to present a whole genome DNA methylation profile in HSCR and reveal a decrease of global DNA methylation in CpG context in HSCR patients compared with controls, which correlates with a greater hypomethylation of the differentially methylated regions (DMRs) identified. These results agree with the de novo Methyltransferase 3b downregulation in EPCs from HSCR patients compared to controls, and with the decrease in the global DNA methylation level previously described by our group. Through the comparative analysis of DMRs between HSCR patients and controls, a set of new genes has been identified as potential susceptibility genes for HSCR at an epigenetic level. Moreover, previous differentially methylated genes related to HSCR have been found, which validates our approach., Conclusions: This study highlights the relevance of an adequate methylation pattern for a proper ENS development. This is a research area that provides a novel approach to deepen our understanding of the etiopathogenesis of HSCR.

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1 + EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Development of the human intestine is not well understood. Here, we link single-cell RNA sequencing and spatial transcriptomics to characterize intestinal morphogenesis through time. We identify 101 cell states including epithelial and mesenchymal progenitor populations and programs linked to key morphogenetic milestones. We describe principles of crypt-villus axis formation; neural, vascular, mesenchymal morphogenesis, and immune population of the developing gut. We identify the differentiation hierarchies of developing fibroblast and myofibroblast subtypes and describe diverse functions for these including as vascular niche cells. We pinpoint the origins of Peyer's patches and gut-associated lymphoid tissue (GALT) and describe location-specific immune programs. We use our resource to present an unbiased analysis of morphogen gradients that direct sequential waves of cellular differentiation and define cells and locations linked to rare developmental intestinal disorders. We compile a publicly available online resource, spatio-temporal analysis resource of fetal intestinal development (STAR-FINDer), to facilitate further work., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

The enteric nervous system (ENS) controls gastrointestinal functions. In large mammals' intestine, it comprises an inner (ISP) and outer (OSP) submucous plexus and a myenteric plexus (MP). This study quantifies enteric neurons in the ISP, OSP, and MP of the pig ascending (AC) and descending colon (DC) using the HuC/D, choline acetyltransferase (ChAT), and neuronal nitric oxide synthase (nNOS) neuronal markers in whole mount preparations with multiple labeling immunofluorescence. We established that the ISP contains the highest number of HuC/D neurons/mm 2 , which were more abundant in AC vs. DC, followed by OSP and MP with similar density in AC and DC. In the ISP, the density of ChAT immunoreactive (IR) neurons was very similar in AC and DC (31% and 35%), nNOS-IR neurons were less abundant in AC than DC (15% vs. 42%, P < 0.001), and ChAT/nNOS-IR neurons were 5% and 10%, respectively. In the OSP, 39-44% of neurons were ChAT-IR in AC and DC, while 45% and 38% were nNOS-IR and 10-12% were ChAT/nNOS-IR (AC vs. DC P < 0.05). In the MP, ChAT-IR neurons were 44% in AC and 54% in DC (P < 0.05), nNOS-IR neurons were 50% in both, and ChAT/nNOS-IR neurons were 12 and 18%, respectively. The ENS architecture with multilayered submucosal plexuses and the distribution of functionally distinct groups of neurons in the pig colon are similar to humans, supporting the suitability of the pig as a model and providing the platform for investigating the mechanisms underlying human colonic diseases.

The enteric nervous system (ENS), which is derived from neural crest, is essential for gut function, and its deficiency causes severe congenital diseases. Since the capacity for ENS regeneration in mammals is limited, additional complementary models would be useful. Here, we show that the ENS in zebrafish larvae at 10-15 days postfertilization is highly regenerative. After laser ablation, the number of enteric neurons recovered to ∼50% of the control by 10 days post-ablation (dpa). Using transgenic lines in which enteric neural crest-derived cells (ENCDCs) and enteric neurons are labeled with fluorescent proteins, we live imaged the regeneration process and found covering by neurites that extended from the unablated area and entry of ENCDCs into the ablated areas by 1-3 dpa. BrdU assays suggested that ∼80% of the enteric neurons and ∼90% of the Sox10-positive ENCDCs therein at 7 dpa were generated through proliferation. Thus, ENS regeneration involves proliferation, entrance and neurogenesis of ENCDCs. This is the first report regarding the regeneration process of the zebrafish ENS. Our findings provide a basis for further in vivo research at single-cell resolution in this vertebrate model., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2021. Published by The Company of Biologists Ltd.)

Background & Aims: Enteric glial cells express type II major histocompatibility complex (MHC-II) molecules in Crohn's disease and Chagas disease, but it is unclear whether the expressed molecules are functional. We examined the capabilities of enteric glia to act as an antigen-presenting cell in vivo and whether glial MHC-II has immunomodulatory effects., Methods: We generated Sox10 CreERT2 ;IAB fl/fl mice to ablate MHC-II in enteric glia after exposure to tamoxifen. We measured phagocytic activity and autophagy activation to assess potential peptide sources loaded onto glial MHC-II and measured T- and B-lymphocyte activation and serum and colonic tissue cytokine levels to study enteric glial immunomodulatory capabilities., Results: Enteric glia express MHC-II molecules in response to a subclinical dose of interferon-γ and lipopolysaccharide in vivo. Glial MHC-II expression contributes to effective B-lymphocyte and T-lymphocyte activation with marked effects on T-helper cell (Th)17 and regulatory T cell subtypes. No effect on Th1 or Th2 subtypes was observed. Enteric glial MHC-II does not have a major effect on serum or colonic tissue cytokine levels but may influence local cytokine levels. Glial MHC-II expression requires the activation of autophagy pathways, but activating autophagy alone is not sufficient to drive glial MHC-II expression., Conclusions: Enteric glia express MHC-II as a mechanism to tune intestinal immune responses. Glial autophagy is triggered in response to proinflammatory stimuli and induces glial antigen presentation, which functions to modulate the activation of T-lymphocyte subsets involved in tolerance. These observations suggest that enteric glia may express MHC-II to maintain immune homeostasis during inflammatory conditions such as Crohn's disease., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Parkinson's disease (PD) is the second most common neurodegenerative disease worldwide. The loss of nigrostriatal dopaminergic neurons produces its characteristic motor symptoms, but PD patients also have non-motor symptoms such as constipation and orthostatic hypotension. The pathological hallmark of PD is the presence of α-synuclein-containing Lewy bodies and neurites in the brain. However, the PD pathology is observed in not only the central nervous system (CNS) but also in parts of the peripheral nervous system such as the enteric nervous system (ENS). Since constipation is a typical prodromal non-motor symptom in PD, often preceding motor symptoms by 10-20 years, it has been hypothesized that PD pathology propagates from the ENS to the CNS via the vagal nerve. Discovery of pharmacological and other methods to halt this progression of neurodegeneration in PD has the potential to improve millions of lives. Astrocytes protect neurons in the CNS by secretion of neurotrophic and antioxidative factors. Similarly, astrocyte-like enteric glial cells (EGCs) are known to secrete neuroprotective factors in the ENS. In this article, we summarize the neuroprotective function of astrocytes and EGCs and discuss therapeutic strategies for the prevention of neurodegeneration in PD targeting neurotrophic and antioxidative molecules in glial cells., Competing Interests: No potential conflict of interest relevant to this article was reported.

The enteric nervous system (ENS) constitutes the largest part of the peripheral nervous system. In recent years, ENS development and its neurogenetic capacity in homeostasis and allostasishave gained increasing attention. Developmentally, the neural precursors of the ENS are mainly derived from vagal and sacral neural crest cell portions. Furthermore, Schwann cell precursors, as well as endodermal pancreatic progenitors, participate in ENS formation. Neural precursorsenherite three subpopulations: a bipotent neuron-glia, a neuronal-fated and a glial-fated subpopulation. Typically, enteric neural precursors migrate along the entire bowel to the anal end, chemoattracted by glial cell-derived neurotrophic factor (GDNF) and endothelin 3 (EDN3) molecules. During migration, a fraction undergoes differentiation into neurons and glial cells. Differentiation is regulated by bone morphogenetic proteins (BMP), Hedgehog and Notch signalling. The fully formed adult ENS may react to injury and damage with neurogenesis and gliogenesis. Nevertheless, the origin of differentiating cells is currently under debate. Putative candidates are an embryonic-like enteric neural progenitor population, Schwann cell precursors and transdifferentiating glial cells. These cells can be isolated and propagated in culture as adult ENS progenitors and may be used for cell transplantation therapies for treating enteric aganglionosis in Chagas and Hirschsprung's diseases.

Acrylamide is one of the harmful substances present in food. The present study aimed to establish the effect of acrylamide supplementation in tolerable daily intake (TDI) dose (0.5 µg/kg b.w./day) and a dose ten times higher than TDI (5 µg/kg b.w./day) on the population of vasoactive intestinal peptide-like immunoreactive (VIP-LI) neurons in the porcine small intestine and the degree of the co-localization of VIP with other neuroactive substances (neuronal nitric oxide synthase (nNOS), substance P (SP), and cocaine- and amphetamine-regulated transcript peptide (CART)). In our work, 15 Danish landrace gilts (5 in each experimental group) received capsules (empty or with low or high doses of acrylamide) for a period of 28 days with their morning feeding. Using double immunofluorescence staining, we established that acrylamide supplementation increased the number of neurons showing immunoreactivity towards VIP in all types of enteric nervous system (ENS) plexuses and fragments of the small intestine studied. Moreover, both doses of acrylamide led to changes in the degree of co-localization of VIP with nNOS, SP, and CART in intramural neurons. The observed changes may be the adaptation of neurons to local inflammation, oxidative stress, or the direct toxic effects of acrylamide on intestinal neurons, also referred to as neuronal plasticity.

Background: The gut is the only organ system with intrinsic neural reflexes. Intrinsic primary afferent neurons (IPANs) of the enteric nervous system initiate intrinsic reflexes, form gut-brain connections, and undergo considerable neuroplasticity to cause digestive diseases. They remain inaccessible to study in mice in the absence of a selective marker. Advillin is used as a marker for primary afferent neurons in dorsal root ganglia. The aim of this study was to test the hypothesis that advillin is expressed in IPANs of the mouse jejunum., Methods: Advillin expression was assessed with immunohistochemistry and using transgenic mice expressing an inducible Cre recombinase under the advillin promoter were used to drive tdTomato and the genetically encoded calcium indicator GCaMP5. These mice were used to characterize the morphology and physiology of advillin-expressing enteric neurons using confocal microscopy, calcium imaging, and whole-cell patch-clamp electrophysiology., Key Results: Advillin is expressed in about 25% of myenteric neurons of the mouse jejunum, and these neurons demonstrate the requisite properties of IPANs. Functionally, they demonstrate calcium responses following mechanical stimuli of the mucosa and during antidromic action potentials. They have Dogiel type II morphology with neural processes that mostly remain within the myenteric plexus, but also project to the mucosa and express NeuN and calcitonin gene-related peptide (CGRP), but not nNOS., Conclusions and Inferences: Advillin marks jejunal IPANs providing accessibility to this important neuronal population to study and model digestive disease., (© 2020 John Wiley & Sons Ltd.)

Background: Intestinal bacteria have been increasingly shown to be involved in early postnatal development. Previous work has shown that intestinal bacteria are necessary for the structural development and intrinsic function of the enteric nervous system in early postnatal life. Furthermore, colonization with a limited number of bacteria appears to be sufficient for the formation of a normal enteric nervous system. We tested the hypothesis that common bacterial components could influence the programming of developing enteric neurons., Methods: The developmental programming of enteric neurons was studied by isolating enteric neural crest-derived cells from the fetal gut of C57Bl/6 mice at embryonic day 15.5. After the establishment of the cell line, cultured enteric neuronal precursors were exposed to increasing concentrations of a panel of bacterial components including lipopolysaccharide, flagellin, and components of peptidoglycan., Key Result: Exposure to bacterial components consistently affected proportions of enteric neuronal precursors that developed into nitrergic neurons. Furthermore, flagellin and D-gamma-Glu-mDAP were found to promote the development of serotonergic neurons. Proportions of dopaminergic neurons remained unchanged. Proliferation of neuronal precursor cells was significantly increased upon exposure to lipopolysaccharide and flagellin, while no significant changes were observed in the proportion of apoptotic neuronal precursors compared to baseline with exposure to any bacterial component., Conclusions and Interfaces: These findings suggest that bacterial components may influence the development of enteric neurons., (© 2020 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

The enteric nervous system (ENS) coordinates diverse functions in the intestine but has eluded comprehensive molecular characterization because of the rarity and diversity of cells. Here we develop two methods to profile the ENS of adult mice and humans at single-cell resolution: RAISIN RNA-seq for profiling intact nuclei with ribosome-bound mRNA and MIRACL-seq for label-free enrichment of rare cell types by droplet-based profiling. The 1,187,535 nuclei in our mouse atlas include 5,068 neurons from the ileum and colon, revealing extraordinary neuron diversity. We highlight circadian expression changes in enteric neurons, show that disease-related genes are dysregulated with aging, and identify differences between the ileum and proximal/distal colon. In humans, we profile 436,202 nuclei, recovering 1,445 neurons, and identify conserved and species-specific transcriptional programs and putative neuro-epithelial, neuro-stromal, and neuro-immune interactions. The human ENS expresses risk genes for neuropathic, inflammatory, and extra-intestinal diseases, suggesting neuronal contributions to disease., Competing Interests: Declaration of Interests A.R. is a co-founder and equity holder of Celsius Therapeutics, equity holder of Immunitas, and, until August, 2020, an SAB member of ThermoFisher Scientific, Syros Pharmaceuticals, Neogene Therapeutics, and Asimov. A.R. is an employee of Genentech Inc. R.J.X. is a co-founder and equity holder of Celsius Therapeutics and Jnana Therapeutics. E.D. is an employee of Bristol-Myers Squibb. E.D., C.S., G.E., O.R.R., R.X., and A.R. are co-inventors on PCT/US2019/055894, which includes the methods of this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

The enteric nervous system (ENS) is derived primarily from the vagal neural crest, a migratory multipotent cell population emerging from the dorsal neural tube between somites 1 and 7. Defects in the development and function of the ENS cause a range of enteric neuropathies, including Hirschsprung disease. Little is known about the signals that specify early ENS progenitors, limiting progress in the generation of enteric neurons from human pluripotent stem cells (hPSCs) to provide tools for disease modeling and regenerative medicine for enteric neuropathies. We describe the efficient and accelerated generation of ENS progenitors from hPSCs, revealing that retinoic acid is critical for the acquisition of vagal axial identity and early ENS progenitor specification. These ENS progenitors generate enteric neurons in vitro and, following in vivo transplantation, achieved long-term colonization of the ENS in adult mice. Thus, hPSC-derived ENS progenitors may provide the basis for cell therapy for defects in the ENS., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

The presence and identity of neural progenitors in the enteric nervous system (ENS) of vertebrates is a matter of intense debate. Here, we demonstrate that the non-neuronal ENS cell compartment of teleosts shares molecular and morphological characteristics with mammalian enteric glia but cannot be identified by the expression of canonical glial markers. However, unlike their mammalian counterparts, which are generally quiescent and do not undergo neuronal differentiation during homeostasis, we show that a relatively high proportion of zebrafish enteric glia proliferate under physiological conditions giving rise to progeny that differentiate into enteric neurons. We also provide evidence that, similar to brain neural stem cells, the activation and neuronal differentiation of enteric glia are regulated by Notch signalling. Our experiments reveal remarkable similarities between enteric glia and brain neural stem cells in teleosts and open new possibilities for use of mammalian enteric glia as a potential source of neurons to restore the activity of intestinal neural circuits compromised by injury or disease., Competing Interests: SM, YO, EF, SB, CP, QX, SH, RK, LC, DW, VP, TH No competing interests declared, CP Carmen Pin is affiliated with AstraZeneca. The author has no financial interests to declare., (© 2020, McCallum et al.)

Hirschsprung disease (HSCR) is a neurocristopathy defined by intestinal aganglionosis due to alterations during the development of the Enteric Nervous System (ENS). A wide spectrum of molecules involved in different signaling pathways and mechanisms have been described in HSCR onset. Among them, epigenetic mechanisms are gaining increasing relevance. In an effort to better understand the epigenetic basis of HSCR, we have performed an analysis for the identification of long non-coding RNAs (lncRNAs) by qRT-PCR in enteric precursor cells (EPCs) from controls and HSCR patients. We aimed to test the presence of a set lncRNAs among 84 lncRNAs in human EPCs, which were previously related with crucial cellular processes for ENS development, as well as to identify the possible differences between HSCR patients and controls. As a result, we have determined a set of lncRNAs with positive expression in human EPCs that were screened for mutations using the exome data from our cohort of HSCR patients to identify possible variants related to this pathology. Interestingly, we identified three lncRNAs with different levels of their transcripts ( SOCS2-AS , MEG3 and NEAT1 ) between HSCR patients and controls. We propose such lncRNAs as possible regulatory elements implicated in the onset of HSCR as well as potential biomarkers of this pathology.

Connections from intrinsic primary afferent neurons (IPANs), to ascending motor and interneurons have been described in guinea pig colon. These mono- and polysynaptic circuits may underlie polarized motor reflexes evoked by local gut stimulation. There is a need to translate findings in guinea pig to mouse, a species increasingly used in enteric neuroscience. Here, mouse distal colon was immunolabeled for CGRP, a marker of putative IPANs. This revealed a combination of large, intensely immunofluorescent axons in myenteric plexus and circular muscle, and thinner varicose axons with less immunofluorescence. The latter formed dense, basket-like varicosity clusters (CGRP+ baskets) that enveloped myenteric nerve cell bodies. Immunolabeling after 4-5 days in organ culture caused loss of large CGRP+ axons, but not varicose CGRP+ fibers and CGRP+ baskets. Baskets were characterized further by triple labeling with CGRP, nitric oxide synthase (NOS) and calretinin (CALR) antibodies. Approximately half (48%) of nerve cell bodies inside CGRP+ baskets lacked both NOS and CALR, while two overlapping populations containing NOS and/or CALR comprised the remainder. Quantitative analysis revealed CGRP+ varicosities were most abundant in baskets, followed by CALR+ varicosities, with a high degree of colocalization between the two markers. Few NOS+ varicosities occurred in baskets. Significantly higher proportions of CALR+ and CGRP+ varicosities colocalized in baskets than in circular muscle. In conclusion, CGRP+ baskets in mouse colon are formed by intrinsic enteric neurons with a neurochemical profile consistent with IPANs and have direct connections to both excitatory and inhibitory neurons., (© 2020 Wiley Periodicals, Inc.)

The enteric nervous system (ENS) is essential for normal gastrointestinal function. Although the embryonic origin of enteric neurons from the neural crest is well established, conflicting evidence exists regarding postnatal enteric neurogenesis. Here, we address this by examining the origin of de novo neurogenesis in the post-embryonic zebrafish ENS. Although new neurons are added during growth and after injury, the larval intestine appears to lack resident neurogenic precursors or classical glia marked by sox10 , plp1a , gfap or s100 Rather, lineage tracing with lipophilic dye or inducible Sox10-Cre suggests that post-embryonic enteric neurons arise from trunk neural crest-derived Schwann cell precursors that migrate from the spinal cord into the intestine. Furthermore, the 5-HT 4 receptor agonist prucalopride increases enteric neurogenesis in normal development and after injury. Taken together, the results suggest that despite the lack of resident progenitors in the gut, post-embryonic enteric neurogenesis occurs via gut-extrinsic Schwann cell precursors during development and injury, and is promoted by serotonin receptor agonists. The absence of classical glia in the ENS further suggests that neural crest-derived enteric glia might have evolved after the teleost lineage.This article has an associated 'The people behind the papers' interview., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The barrier functions of the gastrointestinal tract rely in large part on a single layer of columnar intestinal epithelial cells. These epithelial cells are mediators of intestinal homeostasis, regulating and communicating biochemical signals between underlying stromal cells and luminal cues. The development of representative in vitro models to recapitulate the gastrointestinal epithelium is crucial to understanding cell-cell interactions during intestinal homeostasis and dysfunction. Ideally, models would contain microbiota/immune cells, polarized intestinal architecture, multilayered cellular complexity, extracellular matrix, biochemical cues, and mechanical deformation. This review focuses on historical and state of the art biomaterials and substrates used in the field to establish static and fluidic models of the intestinal epithelium. A discussion of conventional adenocarcinoma colon cancer cell lines, primary intestinal epithelial cells derived from organoids, and stromal support cells such as enteric neurons, myofibroblasts, and immune cells, as well as the importance of increasing cellular complexity and future outlook is included.

Background: Neurons are an integral component of the immune system that functions to coordinate responses to bacterial pathogens. Sensory nociceptive neurons that can detect bacterial pathogens are found throughout the body with dense innervation of the intestinal tract., Methods: In this study, we assessed the role of these nerves in the coordination of host defenses to Citrobacter rodentium. Selective ablation of nociceptive neurons significantly increased bacterial burden 10 days postinfection and delayed pathogen clearance., Results: Because the sensory neuropeptide CGRP (calcitonin gene-related peptide) regulates host responses during infection of the skin, lung, and small intestine, we assessed the role of CGRP receptor signaling during C rodentium infection. Although CGRP receptor blockade reduced certain proinflammatory gene expression, bacterial burden and Il-22 expression was unaffected., Conclusions: Our data highlight that sensory nociceptive neurons exert a significant host protective role during C rodentium infection, independent of CGRP receptor signaling., (© The Author(s) 2020. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail: journals.permissions@oup.com.)

The enteric nervous system (ENS) is a complex network constituted of neurons and glial cells that ensures the intrinsic innervation of the gastrointestinal tract. ENS cells originate from vagal and sacral neural crest cells that are initially located at the border of the neural tube. In birds, sacral neural crest cells (sNCCs) first give rise to an extramural ganglionated structure (the so-called Nerve of Remak [NoR]) and to the pelvic plexus. Later, sNCCs enter the colon mesenchyme to colonize and contribute to the intrinsic innervation of the caudal part of the gut. However, no specific sNCC marker has been described. Here, we report the expression pattern of prospero-related homeobox 1 (PROX1) in the developing chick colon. PROX1 is a homeobox domain transcription factor that plays a role in cell type specification in various tissues. Using in situ hybridization and immunofluorescence techniques, we showed that PROX1 is expressed in sNCCs localized in the NoR and in the pelvic plexus. Then, using real-time quantitative PCR we found that PROX1 displays a strong and highly dynamic expression pattern during NoR development. Moreover, we demonstrated using in vivo cell tracing, that sNCCs are the source of the PROX1-positive cells within the NoR. Our results indicate that PROX1 is the first marker that specifically identifies sNCCs. This might help to better identify the role of the different neural crest cell populations in distal gut innervation, and consequently to improve the diagnosis of diseases linked to incomplete ENS formation, such as Hirschsprung's disease., (© 2019 Wiley Periodicals, Inc.)

Hyperglycemia is one of the main causes of diabetes complications. Gastrointestinal (GI) disturbances are one of the most frequent complications during diabetes. The porcine digestive tract possesses physiological and pathological similarities to the human digestive tract. This also applies to the innervation of the gastrointestinal tract. In this study, the influence of experimentally-inducted hyperglycemia was examined on the expression of vesicular acetylcholine transporter (VAChT), cocaine- and amphetamine-regulated transcript (CART), galanin (GAL), vasoactive intestinal polypeptide (VIP), and calcitonin gene-related peptide (CGRP) in the enteric nervous system (ENS) neurons in the small intestine of the pig. During the current study, an increased number of neurons containing CART, VIP, GAL, and CGRP under streptozotocin injection were observed. The augmentation of expression included all enteric plexuses present in the small intestine. The same results were obtained in the case of VAChT; namely, chronic hyperglycemia led to an increase in the number of neurons utilizing VAChT in all investigated plexuses. The obtained results suggested that the function of neuropeptides studied in this experiment depended on their localization in the ENS structures, as well as part of the GI tract. Diabetes led to alterations in the neurochemical phenotype of small intestine enteric neurons.

Telocytes (TCs) are recently described interstitial cells, present in almost all human organs. Among many other functions, TCs regulate gastrointestinal motility together with the interstitial cells of Cajal (ICCs). TCs and ICCs have close localization in the human myenteric plexus; however, the exact spatial relationship cannot be clearly examined by previously applied double immunofluorescence/confocal microscopy. Data on TCs and submucosal ganglia and their relationship to intestinal nerves are scarce. The aim of the study was to analyse the spatial relationship among these components in the normal human ileum and colon with double CD34/CD117 and CD34/S100 immunohistochemistry and high-resolution light microscopy. TCs were found to almost completely encompass both myenteric and submucosal ganglia in ileum and colon. An incomplete monolayer of ICCs was localized between the TCs and the longitudinal muscle cells in ileum, whereas only scattered ICCs were present on both surfaces of the colonic myenteric ganglia. TC-telopodes were observed within colonic myenteric ganglia. TCs, but no ICCs, were present within and around the interganglionic nerve fascicles, submucosal nerves and mesenterial nerves, but were only observed along small nerves intramuscularly. These anatomic differences probably reflect the various roles of TCs and ICCs in the bowel function., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Objective: Gastrointestinal dysmotility in critically ill patients is important as enteral nutrition is crucial. However, normal gut motility is impaired under conditions of critical illness subsequent to severe insult. Interstitial cells of Cajal (ICC) form an extensive network associated with the myenteric plexus in the enteric nervous system. There are few reports about ICC distribution in critically ill patients. The aim of this study was to evaluate ICC in critically ill patients., Methods: Postmortem colon harvest was obtained from critically ill patients. Control specimens were obtained from patients without bowel movement problems who underwent hemicolectomy. The tissues were stained with c-Kit for ICC. The number of ICC was identified by counting from 10 high-power fields (HPFs)., Results: Specimens from six patients were analyzed and compared with those from six control patients. All patients had abnormalities of crypt architecture and inflammatory cell infiltrations. Mucosal thickness tended to be lower in the critically ill patients than in the controls (147 ± 47 versus 231 ± 127 μm; P = 0.15). Muscle layer thickness tended to be higher in the critically ill patients than in the controls (494 ± 163 versus 394 ± 258 μm; P = 0.44). ICC in the critically ill patients were almost depleted in the colon compared with those in the controls. Significantly fewer ICC were present in the critically ill patients than in the controls (0.45 versus 7.25 cells/HPF; P < 0.05)., Conclusions: Critical illness is associated with diminished numbers of ICC in the colon. This finding could have implications for dysmotility in critically ill patients., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Adolescence is a critical period of development. It is very likely that there is significant maturation of the enteric nervous system (ENS) of the gut during this stage of life, especially since there are substantial changes in factors known to influence the ENS including diet and microbiota during this time, but this remains unknown. To examine maturation of the ENS during adolescence, we performed immunohistochemistry using advanced microscopy and analytical methods to compare enteric neurons and glia of the duodenum and colon of mice taken prior to weaning with those of young adult mice. We found significant changes in the architecture of both myenteric and submucosal plexuses and surprisingly found subsets of enteric cells that co-expressed the pan-neuronal marker, Hu, and either glial markers Sox10 or S100β, not both. About 70% and 35% of all Hu ​+ ​neurons in the submucous plexus of the young adult duodenum and colon respectively also expressed S100β. The proportion of Hu+/Sox10 ​+ ​cells in the duodenal myenteric plexus decreased, while the proportion of Hu+/S100β+ cells in the colonic submucosal plexus increased during adolescence. In the submucous plexus, there were significant increases in the proportions of vasoactive intestinal peptide+ and choline acetyltransferase ​+ ​secretomotor neurons, of neurofilament M (NFM)+ neurons in the colon and of calretinin ​+ ​neurons in the duodenum during adolescence. There were no age-dependent changes in the neurochemistry of various myenteric neuronal subtypes, including those immunoreactive for neuronal nitric oxide synthase (nNOS), Calbindin, Calretinin or NFM. There were significant increases in the somata sizes of Calretinin ​+ ​submucosal and myenteric neurons, and nNOS ​+ ​myenteric neurons, and these enteric neurons received significantly more synaptophysin ​+ ​contacts onto their cell bodies during adolescence. This is the first study showing that enteric neurons and glia in the gut undergo significant changes in their anatomy and chemistry during adolescence. Notably changes in synaptic contacts within the enteric circuitry strongly suggest maturation in gastrointestinal function occurs during this time., Competing Interests: Declaration of competing interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Due to numerous therapeutic applications and high availability, non-steroidal anti-inflammatory drugs (NSAIDs) are the most widely used drugs worldwide. However, long-term use of these drugs can lead to damage to the gastrointestinal mucosa. The enteric nervous system (ENS), which is part of the autonomic nervous system, controls most aspects of gastrointestinal activity. Enteric neurons are characterized by considerable chemical plasticity and the appearance of a pathological factor results in a change in the synthesis of neurotransmitters. The purpose of this study was to determine the effects of naproxen on expression of biologically active substances by intramural neurons supplying the porcine duodenum. The study was performed on eight immature pigs of the Pietrain x Duroc race (approximately 20kg of body weight). The animals were divided into two groups - a control (C group) and an experimental group (N group). Group C (n=4) consisted of animals which received empty gelatine capsules. Group N (n=4) was composed of pigs who received naproxen orally for 28 days, approximately one hour before feeding. After this time, animals from both groups were euthanized. Frozen sections (14μm thickness) were then prepared from the collected duodenum and subjected to double immunofluorescence staining. Antibodies against the neuronal marker PGP 9.5 and against vasoactive intestinal polypeptide (VIP), substance P (SP), neuronal nitric oxide synthase (nNOS), galanin (GAL), pituitary adenylate cyclase-activating polypeptide (PACAP) and cocaine- and amphetamine- regulated transcript peptide (CART) were used as primary antibodies. The polyclonal donkey anti-rabbit, anti-mouse and anti-guinea pig IgG antibodies - Alexa Fluor 488 and 546 - were also used for staining. Analysis of the results obtained with a fluorescence microscope showed a significant increase in the number of nNOS-, VIP-, GAL-, PACAP- and CART-immunoreactive ganglionated neurons and a decrease in the number of SP-positive neurons in the myenteric and submucosal plexuses of the porcine duodenum. The obtained results indicate the participation of enteric neurotransmitters in the neuronal duodenal response to naproxen-induced inflammation., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Hirschsprung disease (HSCR) is characterized by absence of the enteric nervous system (ENS) in the distal bowel. Despite removal of the aganglionic segment, gastrointestinal (GI) problems persist. Cell therapy offers potential treatment but use of genetic models is limited by their poor survival. We have developed a novel model of aganglionosis in which enteric neural crest-derived cells (ENCDCs) express diphtheria toxin (DT) receptor. Local DT injection into the colon wall results in focal, specific, and sustained ENS ablation without altering GI transit or colonic contractility, allowing improved survival over other aganglionosis models. Focal ENS ablation leads to increased smooth muscle and mucosal thickness, and localized inflammation. Transplantation of ENCDCs into this region leads to engraftment, migration, and differentiation of enteric neurons and glial cells, with restoration of normal architecture of the colonic epithelium and muscle, reduction in inflammation, and improved survival.

Background & Aims: It has been a challenge to develop fully functioning cells from human pluripotent stem cells (hPSCs). We investigated how activation of hedgehog signaling regulates derivation of enteric neural crest (NC) cells from hPSCs., Methods: We analyzed transcriptomes of mouse and hPSC-derived enteric NCs using single-cell RNA sequencing (scRNA-seq) to identify the changes in expression associated with lineage differentiation. Intestine tissues were collected from Tg(GBS-GFP), Sufu f/f; Wnt1-cre , Ptch1 +/- , and Gli3 Δ699/Δ699 mice and analyzed by flow cytometry and immunofluorescence for levels of messenger RNAs encoding factors in the hedgehog signaling pathway during differentiation of enteric NCs. Human NC cells (HNK-1 + p75 NTR+ ) were derived from IMR90 and UE02302 hPSC lines. hPSCs were incubated with a hedgehog agonist (smoothened agonist [SAG]) and antagonists (cyclopamine) and analyzed for differentiation. hPSC-based innervated colonic organoids were derived from these hPSC lines and analyzed by immunofluorescence and neuromuscular coupling assay for expression of neuronal subtype markers and assessment of the functional maturity of the hPSC-derived neurons, respectively., Results: Single-cell RNA sequencing analysis showed that neural fate acquisition by human and mouse enteric NC cells requires reduced expression of NC- and cell cycle-specific genes and up-regulation of neuronal or glial lineage-specific genes. Activation of the hedgehog pathway was associated with progression of mouse enteric NCs to the more mature state along the neuronal and glial lineage differentiation trajectories. Activation of the hedgehog pathway promoted development of cultured hPSCs into NCs of greater neurogenic potential by activating expression of genes in the neurogenic lineage. The hedgehog agonist increased differentiation of hPSCs into cells of the neuronal lineage by up-regulating expression of GLI2 target genes, including INSM1, NHLH1, and various bHLH family members. The hedgehog agonist increased expression of late neuronal markers and neuronal activities in hPSC-derived neurons., Conclusions: In enteric NCs from humans and mice, activation of hedgehog signaling promotes differentiation into neurons by promoting cell-state transition, expression of genes in the neurogenic lineage, and functional maturity of enteric neurons., (Copyright © 2019 AGA Institute. Published by Elsevier Inc. All rights reserved.)