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Gut peristaltic movements transport ingested materials along the gut axis, which is critical for food digestion and nutrient absorption. While a large amount of studies have been devoted to analyzing the physiological functions of peristalsis in adults, little is known about how the peristaltic system is established during embryogenesis. In recent years, the chicken developing gut has emerged as an excellent model, in which specific sites along the gut axis can be genetically labeled enabling live imaging and optogenetic analyses. This review provides an overview of recent progress in optogenetic studies of gut peristalsis. Analyses with an improved channelrhodopsin-2 variant demonstrated that the peristalsis can artificially be generated in the developing gut. These studies unveiled novel functional coordination between different regions along the gut axis. In addition, imaging with GCaMP6s, a genetically encoded calcium indicator, enabled a fine mapping of developmental changes in the peristaltic patterns as Ca2+ signals. These advanced techniques will broaden our knowledge of how embryonic peristalsis is established at the cellular and molecular level, leading to the understanding of physiological and pathological processes in adult peristalsis., (© 2024 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Despite a distinct developmental origin, extraembryonic cells in mice contribute to gut endoderm and converge to transcriptionally resemble their embryonic counterparts. Notably, all extraembryonic progenitors share a non-canonical epigenome, raising several pertinent questions, including whether this landscape is reset to match the embryonic regulation and if extraembryonic cells persist into later development. Here we developed a two-colour lineage-tracing strategy to track and isolate extraembryonic cells over time. We find that extraembryonic gut cells display substantial memory of their developmental origin including retention of the original DNA methylation landscape and resulting transcriptional signatures. Furthermore, we show that extraembryonic gut cells undergo programmed cell death and neighbouring embryonic cells clear their remnants via non-professional phagocytosis. By midgestation, we no longer detect extraembryonic cells in the wild-type gut, whereas they persist and differentiate further in p53-mutant embryos. Our study provides key insights into the molecular and developmental fate of extraembryonic cells inside the embryo., (© 2024. The Author(s).)

Hirschsprung disease (HSCR) is associated with deficiency of the receptor tyrosine kinase RET, resulting in loss of cells of the enteric nervous system (ENS) during fetal gut development. The major contribution to HSCR risk is from common sequence variants in RET enhancers with additional risk from rare coding variants in many genes. Here, we demonstrate that these RET enhancer variants specifically alter the human fetal gut development program through significant decreases in gene expression of RET, members of the RET-EDNRB gene regulatory network (GRN), other HSCR genes, with an altered transcriptome of 2,382 differentially expressed genes across diverse neuronal and mesenchymal functions. A parsimonious hypothesis for these results is that beyond RET's direct effect on its GRN, it also has a major role in enteric neural crest-derived cell (ENCDC) precursor proliferation, its deficiency reducing ENCDCs with relative expansion of non-ENCDC cells. Thus, genes reducing RET proliferative activity can potentially cause HSCR. One such class is the 23 RET-dependent transcription factors enriched in early gut development. We show that their knockdown in human neuroblastoma SK-N-SH cells reduces RET and/or EDNRB gene expression, expanding the RET-EDNRB GRN. The human embryos we studied had major remodeling of the gut transcriptome but were unlikely to have had HSCR: thus, genetic or epigenetic changes in addition to those in RET are required for aganglionosis., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Chatterjee et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Over the past 5 years, systematic investigation of the mesenteric organ has expanded and shown that the mesentery is the organ in and on which all abdominal digestive organs develop and remain connected to. In turn, this observation has clarified the anatomical foundation of the abdomen and the fundamental order at that level. Findings related to the shape and development of the mesentery have illuminated its function, advancing our understanding of the pathobiology, diagnosis, and treatment of several abdominal and systemic diseases. Inclusion of the mesentery in surgical resections alters the course of benign and malignant diseases. Mesenteric-based scoring systems can enhance the radiological interpretation of abdominal disease. Emerging findings reconcile observations across scientific and clinical fields and have been assimilated into reference curricula and practice guidelines. This Review summarises the developmental, anatomical, and clinical advances made since the mesentery was redesignated as an organ in 2016., Competing Interests: Declaration of interests All authors declare no competing interests., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

The spatial organization of gut microbiota influences both microbial abundances and host-microbe interactions, but the underlying rules relating bacterial dynamics to large-scale structure remain unclear. To this end, we studied experimentally and theoretically the formation of three-dimensional bacterial clusters, a key parameter controlling susceptibility to intestinal transport and access to the epithelium. Inspired by models of structure formation in soft materials, we sought to understand how the distribution of gut bacterial cluster sizes emerges from bacterial-scale kinetics. Analyzing imaging-derived data on cluster sizes for eight different bacterial strains in the larval zebrafish gut, we find a common family of size distributions that decay approximately as power laws with exponents close to -2, becoming shallower for large clusters in a strain-dependent manner. We show that this type of distribution arises naturally from a Yule-Simons-type process in which bacteria grow within clusters and can escape from them, coupled to an aggregation process that tends to condense the system toward a single massive cluster, reminiscent of gel formation. Together, these results point to the existence of general, biophysical principles governing the spatial organization of the gut microbiome that may be useful for inferring fast-timescale dynamics that are experimentally inaccessible., Competing Interests: BS, RP No competing interests declared, (© 2021, Schlomann and Parthasarathy.)

The human fetal immune system begins to develop early during gestation; however, factors responsible for fetal immune-priming remain elusive. We explored potential exposure to microbial agents in utero and their contribution toward activation of memory T cells in fetal tissues. We profiled microbes across fetal organs using 16S rRNA gene sequencing and detected low but consistent microbial signal in fetal gut, skin, placenta, and lungs in the 2 nd trimester of gestation. We identified several live bacterial strains including Staphylococcus and Lactobacillus in fetal tissues, which induced in vitro activation of memory T cells in fetal mesenteric lymph node, supporting the role of microbial exposure in fetal immune-priming. Finally, using SEM and RNA-ISH, we visualized discrete localization of bacteria-like structures and eubacterial-RNA within 14 th weeks fetal gut lumen. These findings indicate selective presence of live microbes in fetal organs during the 2 nd trimester of gestation and have broader implications toward the establishment of immune competency and priming before birth., Competing Interests: Declaration of interests The authors declare no competing interests., (Crown Copyright © 2021. Published by Elsevier Inc. All rights reserved.)

While the colonization of the embryonic gut by neural crest cells has been the subject of intense scrutiny over the past decades, we are only starting to grasp the morphogenetic transformations of the enteric nervous system happening in the fetal stage. Here, we show that enteric neural crest cell transit during fetal development from an isotropic cell network to a square grid comprised of circumferentially-oriented cell bodies and longitudinally-extending interganglionic fibers. We present ex-vivo dynamic time-lapse imaging of this isotropic-to-nematic phase transition and show that it occurs concomitantly with circular smooth muscle differentiation in all regions of the gastrointestinal tract. Using conditional mutant embryos with enteric neural crest cells depleted of β1-integrins, we show that cell-extracellular matrix anchorage is necessary for ganglia to properly reorient. We demonstrate by whole mount second harmonic generation imaging that fibrous, circularly-spun collagen I fibers are in direct contact with neural crest cells during the orientation transition, providing an ideal orientation template. We conclude that smooth-muscle associated extracellular matrix drives a critical reorientation transition of the enteric nervous system in the mammalian fetus.

Euphorbiae pekinensis Radix (EP) is effective in treating various diseases, but it's toxicity is a major obstacle in use in clinical. Although EP was processed with vinegar to reduce it's toxicity, the detailed mechanism of toxicity in EP have not been clearly delineated. This study investigate the toxicity attenuation-mechanism of Euphorbiae pekinensis after being processed with vinegar (VEP) and the toxic mechanism of four compounds from EP on zebrafish embryos. The contents of four compounds decreased obviously in VEP. Correspondingly, slower development on embryos can be seen as some symptoms like reduction of heart rate, liver area and gastrointestinal peristalsis after exposed to the compounds. Some obvious pathological signals such as pericardial edema and yolk sac edema were observed. Furthermore, the compounds could increase the contents of MDA and GSH-PX and induce oxidative damage by inhibiting the activity of SOD. Also, four compounds could provoke apoptosis by up-regulating the expression level of p53, MDM2, Bax, Bcl-2 and activating the activity of caspase-3, caspase-9. In conclusion, the four compounds play an important role in the toxicity attenuation effects of VEP, which may be related to the apoptosis induction and oxidative damage. This would contribute to the clinical application and further toxicity-reduction mechanism research., (Copyright © 2021 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: The Drosophila hindgut is commonly used model for studying various aspects of organogenesis like primordium establishment, further specification, patterning, and morphogenesis. During embryonic development of Drosophila, many transcriptional activators are involved in the formation of the hindgut. The transcription factor Orthopedia (Otp), a member of the 57B homeobox gene cluster, is expressed in the hindgut and nervous system of developing Drosophila embryos, but due to the lack of mutants no functional analysis has been conducted yet., Results: We show that two different otp transcripts, a hindgut-specific and a nervous system-specific form, are present in the Drosophila embryo. Using an Otp antibody, a detailed expression analysis during hindgut development was carried out. Otp was not only expressed in the embryonic hindgut, but also in the larval and adult hindgut. To analyse the function of otp, we generated the mutant otp allele otp GT by ends-out gene targeting. In addition, we isolated two EMS-induced otp alleles in a genetic screen for mutants of the 57B region. All three otp alleles showed embryonic lethality with a severe hindgut phenotype. Anal pads were reduced and the large intestine was completely missing. This phenotype is due to apoptosis in the hindgut primordium and the developing hindgut., Conclusion: Our data suggest that Otp is another important factor for hindgut development of Drosophila. As a downstream factor of byn Otp is most likely present only in differentiated hindgut cells during all stages of development rather than in stem cells.

The first contractile waves in the developing embryonic gut are purely myogenic; they only involve smooth muscle. Here, we provide evidence for a transition from smooth muscle to interstitial cell of Cajal (ICC)-driven contractile waves in the developing chicken gut. In situ hybridization staining for anoctamin-1 (ANO1), a known ICC marker, shows that ICCs are already present throughout the gut, as from embryonic day (E)7. We devised a protocol to reveal ICC oscillatory and propagative calcium activity in embryonic gut whole mount and found that the first steady calcium oscillations in ICCs occur on (E14). We show that the activation of ICCs leads to an increase in contractile wave frequency, regularity, directionality, and velocity between E12 and E14. We finally demonstrate that application of the c-KIT antagonist imatinib mesylate in organ culture specifically depletes the ICC network and inhibits the transition to a regular rhythmic wave pattern. We compare our findings to existing results in the mouse and predict that a similar transition should take place in the human fetus between 12 and 14 wk of development. Together, our results point to an abrupt physiological transition from smooth muscle mesenchyme self-initiating waves to ICC-driven motility in the fetus and clarify the contribution of ICCs to the contractile wave pattern. NEW & NOTEWORTHY We reveal a sharp transition from smooth muscle to interstitial cell of Cajal (ICC)-driven motility in the chicken embryo, leading to higher-frequency, more rhythmic contractile waves. We predict the transition to happen between 12 and 14 embryonic wk in humans. We image for the first time the onset of ICC activity in an embryonic gut by calcium imaging. We show the first KIT and anoctamin-1 (ANO1) in situ hybridization micrographs in the embryonic chicken gut.

Precise extrinsic afferent (visceral sensory) and efferent (sympathetic and parasympathetic) innervation of the gut is fundamental for gut-brain cross talk. Owing to the limitation of intrinsic markers to distinctively visualize the three classes of extrinsic axons, which intimately associate within the gut mesentery, detailed information on the development of extrinsic gut-innervating axons remains relatively sparse. Here, we mapped extrinsic innervation of the gut and explored the relationships among various types of extrinsic axons during embryonic development in mice. Visualization with characterized intrinsic markers revealed that visceral sensory, sympathetic, and parasympathetic axons arise from different anatomic locations, project in close association via the gut mesentery, and form distinctive innervation patterns within the gut from embryonic day (E)10.5 to E16.5. Genetic ablation of visceral sensory trajectories results in the erratic extension of both sympathetic and parasympathetic axons, implicating that afferent axons provide an axonal scaffold to route efferent axons. Coculture assay further confirmed the attractive effect of sensory axons on sympathetic axons. Taken together, our study provides key information regarding the development of extrinsic gut-innervating axons occurring through heterotypic axonal interactions and provides an anatomic basis to uncover neural circuit assembly in the gut-brain axis (GBA). SIGNIFICANCE STATEMENT Understanding the development of extrinsic innervation of the gut is essential to unravel the bidirectional neural communication between the brain and the gut. Here, with characterized intrinsic markers targeting vagal sensory, spinal sensory, sympathetic, and parasympathetic axons, respectively, we comprehensively traced the spatiotemporal development of extrinsic axons to the gut during embryonic development in mice. Moreover, in line with the somatic nervous system, pretarget sorting via heterotypic axonal interactions is revealed to play critical roles in patterning extrinsic efferent trajectories to the gut. These findings provide basic anatomic information to explore the mechanisms underlying the process of assembling neural circuitry in the gut-brain axis (GBA)., (Copyright © 2020 the authors.)

Background: The homeobox genes Pdx and Cdx are widespread across the animal kingdom and part of the small ParaHox gene cluster. Gene expression patterns suggest ancient roles for Pdx and Cdx in patterning the through-gut of bilaterian animals although functional data are available for few lineages. To examine evolutionary conservation of Pdx and Cdx gene functions, we focus on amphioxus, small marine animals that occupy a pivotal position in chordate evolution and in which ParaHox gene clustering was first reported., Results: Using transcription activator-like effector nucleases (TALENs), we engineer frameshift mutations in the Pdx and Cdx genes of the amphioxus Branchiostoma floridae and establish mutant lines. Homozygous Pdx mutants have a defect in amphioxus endoderm, manifest as loss of a midgut region expressing endogenous GFP. The anus fails to open in homozygous Cdx mutants, which also have defects in posterior body extension and epidermal tail fin development. Treatment with an inverse agonist of retinoic acid (RA) signalling partially rescues the axial and tail fin phenotypes indicating they are caused by increased RA signalling. Gene expression analyses and luciferase assays suggest that posterior RA levels are kept low in wild type animals by a likely direct transcriptional regulation of a Cyp26 gene by Cdx. Transcriptome analysis reveals extensive gene expression changes in mutants, with a disproportionate effect of Pdx and Cdx on gut-enriched genes and a colinear-like effect of Cdx on Hox genes., Conclusions: These data reveal that amphioxus Pdx and Cdx have roles in specifying middle and posterior cell fates in the endoderm of the gut, roles that likely date to the origin of Bilateria. This conclusion is consistent with these two ParaHox genes playing a role in the origin of the bilaterian through-gut with a distinct anus, morphological innovations that contributed to ecological change in the Cambrian. In addition, we find that amphioxus Cdx promotes body axis extension through a molecular mechanism conserved with vertebrates. The axial extension role for Cdx dates back at least to the origin of Chordata and may have facilitated the evolution of the post-anal tail and active locomotion in chordates.

Harmful algal blooms (HAB) have become a major health concern worldwide, not just to humans that consume and recreate on contaminated waters, but also to the fauna that inhabit the environments surrounding affected areas. HABs contain heterotrophic bacteria, cyanobacterial lipopolysaccharide, and cyanobacterial toxins such as microcystins, that can cause severe toxicity in many aquatic species as well as bioaccumulation within various organs. Thus, the possibility of trophic transference of this toxin through the food chain has potentially important health implications for other organisms in the related food web. While some species have developed adaptions to attenuate the toxic effects of HAB toxins, there are still numerous species that remain vulnerable, including Lithobates catesbeiana (American bullfrog) tadpoles. In the current study we demonstrate that acute, short-term exposure of tadpoles to HAB toxins containing 1 µg/L (1 nmol/L) of total microcystins for only 7 days results in significant liver and intestinal toxicity within tadpoles. Exposed tadpoles had increased intestinal diameter, decreased intestinal fold heights, and a constant number of intestinal folds, indicating pathological intestinal distension, similar to what is seen in various disease processes, such as toxic megacolon. HAB-toxin-exposed tadpoles also demonstrated hepatocyte hypertrophy with increased hepatocyte binucleation consistent with carcinogenic and oxidative processes within the liver. Both livers and intestines of HAB-toxin-exposed tadpoles demonstrated significant increases in protein carbonylation consistent with oxidative stress and damage. These findings demonstrate that short-term exposure to HAB toxins, including microcystins, can have significant adverse effects in amphibian populations. This acute, short-term toxicity highlights the need to evaluate the influence HAB toxins may have on other vulnerable species within the food web and how those may ultimately also impact human health.

In the present study, the morphological development of the Brycon amazonicus digestive tract is described to provide basic knowledge for nutritional studies and, therefore, increase the survival of this species during larviculture. Samples were collected from hatching up to 25 days of age, measured, processed and observed under a stereomicroscope and light microscopy. Newly hatched larvae presented their digestive tract as a straight tube, dorsal to the yolk sac, lined with a single layer of undifferentiated cells. At 24 h post-hatching (hPH), the buccopharyngeal cavity was open, but the posterior region of the digestive tube remained closed. At 25 hPH, the digestive tube was completely open and could be divided into buccopharyngeal cavity, oesophagus and intestine. At 35 hPH, the intestine presented a dilatation in the proximal region, which had the function of storing food. Differentiation of the stomach started at 83 hPH, and mucous cells were observed in the epithelium. These cells are important in the production of mucus, whose function is to protect the organ against acidity, although the gastric glands began developing only from 171 hPH, when three stomach regions were observed: cardiac, fundic and pyloric. The gastric glands were observed in the cardiac region, indicating that this organ already had digestive functionality. From 243 hPH, the absorption and assimilation of nutrients were already possible but, only from 412 hPH, the digestive tract was completely developed and functional.

Background: Fetal swallowing of human amniotic fluid (hAF) containing trophic factors (TFs) promotes gastrointestinal tract (GIT) development. Preterm birth interrupts hAF swallowing, which may increase the risk of necrotizing enterocolitis (NEC). Postnatally, it is difficult to replicate fetal swallowing of hAF due to volume. We aimed to evaluate whether hAF lyophilization is feasible and its effect on hAF-borne TFs., Methods: We collected hAF (n = 16) from uncomplicated pregnancies. hAF was divided into three groups: unprocessed control (C), concentration by microfiltration (F), and by dialysis and lyophilization (L). EGF, HGF, GM-CSF, and TGF-α were measured in each group by multiplex assay. Bioavailability of TFs was measured by proliferation and LPS-induced IL-8 production by intestinal epithelial cells FHs74., Results: After dialysis/lyophilization, GM-CSF and TGF-α were preserved with partial loss of EGF and HGF. hAF increased cell proliferation and reduced LPS-induced IL-8 production compared to medium alone. Compared to control, dialysis/lyophilization and filtration of hAF increased FHs74 cell proliferation (p < 0.001) and decreased LPS-induced IL-8 production (p < 0.01)., Conclusions: Lyophilization and filtration of hAF is feasible with partial loss of TFs but maintains and even improves bioavailability of TFs measured by proliferation and LPS-induced IL-8 production by FHs74.

In the anterior foregut (AFG) of mouse embryos, the transcription factor SOX2 is expressed in the epithelia of the esophagus and proximal branches of respiratory organs comprising the trachea and bronchi, whereas NKX2.1 is expressed only in the epithelia of respiratory organs. Previous studies using hypomorphic Sox2 alleles have indicated that reduced SOX2 expression causes the esophageal epithelium to display some respiratory organ characteristics. In the present study, we produced mouse embryos with AFG-specific SOX2 deficiency. In the absence of SOX2 expression, a single NKX2.1-expressing epithelial tube connected the pharynx and the stomach, and a pair of bronchi developed in the middle of the tube. Expression patterns of NKX2.1 and SOX9 revealed that the anterior and posterior halves of SOX2-deficient AFG epithelial tubes assumed the characteristics of the trachea and bronchus, respectively. In addition, we found that mesenchymal tissues surrounding the SOX2-deficient NKX2.1-expressing epithelial tube changed to those surrounding the trachea and bronchi in the anterior and posterior halves, as indicated by the arrangement of smooth muscle cells and SOX9-expressing cells and by the expression of Wnt4 (esophagus specific), Tbx4 (respiratory organ specific), and Hoxb6 (distal bronchus specific). The impact of mesenchyme-derived signaling on the early stage of AFG epithelial specification has been indicated. Our study demonstrated an opposite trend where epithelial tissue specification causes concordant changes in mesenchymal tissues, indicating a reciprocity of epithelial-mesenchymal interactions., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2020. Published by The Company of Biologists Ltd.)

The poly(C)-binding protein (PCBP) family members belong to a subtype of RNA-binding proteins that are ubiquitously expressed with diverse functions. In mammals, PCBP family, also known as hnRNP E family, is composed of four proteins, namely PCBP1, PCBP2, PCBP3 and PCBP4. So far, no study has been documented on the physiological roles of each member in vertebrate development. Here we analysed the spatiotemporal expression patterns of zebrafish (Danio rerio) pcbp2 (identical to pcbp1 and pcbp2 in mammals), pcbp3 and pcbp4 at various stages of zebrafish embryonic development by whole-mount in situ hybridization. Our results revealed that all pcbp genes are maternally expressed, especially pcbp2, which is strongly expressed from the embryogenetic stage to larva. The expression patterns of PCBP members are similar to each other at the very early developmental stage sharing with common strong expression in the intestine, otic vesicle, retina and brain of zebrafish. Subsequently, the messenger RNAs of PCBP members are gradually constrained and highly expressed in intestines of the larvae. Collectively, our study figured out the expression pattern of each PCBP member in diverse organogenesis during embryo development, indicating that PCBP members may play predominant roles in the development of neural and digestive systems to maintain their normal physiological functions. Moreover, the similar expression patterns at the developmental stages and organ types among this family suggest that the aberrant expression of these genes would lead to the neural or intestinal diseases., Competing Interests: Declaration of competing interest The authors declare no conflicting interests., (Copyright © 2020. Published by Elsevier B.V.)

Background: Rab proteins are GTPases responsible for intracellular vesicular trafficking regulation. Rab11 proteins, members of the Rab GTPase family, are known to regulate vesicular recycling during embryonic development. In zebrafish, there are 3 rab11 paralogues, known as rab11a, rab11ba and rab11bb, sharing high identity with each other. However, the expression analysis of rab11 is so far lacking., Results: Here, by phylogeny analysis, we found the three rab11 genes are highly conserved especially for their GTPase domains. We examined the expression patterns of rab11a, rab11ba and rab11bb using RT-PCR and in situ hybridization. We found that all the three genes were highly enriched in the central nervous system, but in different areas of the brain. Apart from brain, rab11a was also expressed in caudal vein, pronephric duct, proctodeum, pharyngeal arches and digestive duct, rab11ba was detected to express in muscle, and rab11bb was expressed in kidney, fin and spinal cord. Different from rab11a and rab11ba, which both have maternal expressions in embryos, rab11bb only expresses during 24hpf to 96hpf., Conclusions: Our results suggest that rab11 genes play important but distinct roles in the development of the nervous system in zebrafish. The findings could provide new evidences for better understanding the functions of rab11 in the development of zebrafish embryos.

Macroautophagy/autophagy has been demonstrated to play an essential role in embryonic development. However, the role of autophagy during human fetal digestive tract development has not been investigated. Here, by using over 5,000 human embryonic digestive tract cells ranging from 6 weeks to 25 weeks, we explored the dynamic expression of autophagy-related genes at single-cell resolution, and found that the transcriptional activity of autophagy-related genes boosted remarkably and specifically in the early (between 6 and 9 weeks) stages. Interestingly, the small intestine cells at 9 weeks showed the most significant enrichment of autophagy-related genes than any other stages. In summary, our results for the first time revealed that autophagy may play an essential role in the development of the digestive tract, especially for the small intestine, in early human embryos. Abbreviations: GI: gastrointestinal; S-Intes: small intestine; t-SNE: t-distributed stochastic neighbor embedding.

In this study we examined the ontogenic development of the digestive tract of Vimba bream (Vimba vimba, Family: Cyprinidae) during the first 60 days of life (hatching to 60 days after hatching [DAH]). Samples of developing Vimba bream were randomly selected at various stages of development: 1-8, 10, 15, 20, 25, 30, 40, 50, and 60 DAH. For the histological and histochemical studies on the development of the alimentary canal, tissue sections prepared from the sampled hatchlings were stained with hematoxylin-eosin and periodic acid-Schiff and observed under a light microscope. The histological structures of both the mouth and esophagus were fully developed at 5 and 7 DAH, respectively. Intestinal differentiation was observed at 2 DAH, while mucosal folds appeared on the intestinal bulb at 7 DAH. At 5 DAH, with the appearance of goblet cells in the epithelium of the mouth, pharynx, and esophagus, the larvae showed secretion activity in these organs. At 6 DAH, secretion was observed in the intestine; at this stage of development, the surface of the gastrointestinal tract was covered in a neutral mucous-like layer of polysaccharide. The histological observations indicate that the early development of the digestive tract in Vimba vimba enables larvae to efficiently ingest and digest exogenous feed very quickly after hatching.

The embryonic gut tube is a cylindrical structure from which the respiratory and gastrointestinal tracts develop 1 . Although the early emergence of the endoderm as an epithelial sheet 2,3 and later morphogenesis of the definitive digestive and respiratory organs 4-6 have been investigated, the intervening process of gut tube formation remains relatively understudied 7,8 . Here we investigate the molecular control of macroscopic forces underlying early morphogenesis of the gut tube in the chick embryo. The gut tube has been described as forming from two endodermal invaginations-the anterior intestinal portal (AIP) towards the rostral end of the embryo and the caudal intestinal portal (CIP) at the caudal end-that migrate towards one another, internalizing the endoderm until they meet at the yolk stalk (umbilicus in mammals) 1,6 . Migration of the AIP to form foregut has been descriptively characterized 8,9 , but the hindgut is likely to form by a distinct mechanism that has not been fully explained 10 . We find that the hindgut is formed by collective cell movements through a stationary CIP, rather than by movement of the CIP itself. Further, combining in vivo imaging, biophysics and mathematical modelling with molecular and embryological approaches, we identify a contractile force gradient that drives cell movements in the hindgut-forming endoderm, enabling tissue-scale posterior extension of the forming hindgut tube. The force gradient, in turn, is established in response to a morphogenic gradient of fibroblast growth factor signalling. As a result, we propose that an important positive feedback arises, whereby contracting cells draw passive cells from low to high fibroblast growth factor levels, recruiting them to contract and pull more cells into the elongating hindgut. In addition to providing insight into the early gut development, these findings illustrate how large-scale tissue level forces can be traced to developmental signals during vertebrate morphogenesis.

The gastrointestinal tract has recently come to the forefront of multiple research fields. It is now recognized as a major source of signals modulating food intake, insulin secretion and energy balance. It is also a key player in immunity and, through its interaction with microbiota, can shape our physiology and behavior in complex and sometimes unexpected ways. The insect intestine had remained, by comparison, relatively unexplored until the identification of adult somatic stem cells in the Drosophila intestine over a decade ago. Since then, a growing scientific community has exploited the genetic amenability of this insect organ in powerful and creative ways. By doing so, we have shed light on a broad range of biological questions revolving around stem cells and their niches, interorgan signaling and immunity. Despite their relatively recent discovery, some of the mechanisms active in the intestine of flies have already been shown to be more widely applicable to other gastrointestinal systems, and may therefore become relevant in the context of human pathologies such as gastrointestinal cancers, aging, or obesity. This review summarizes our current knowledge of both the formation and function of the Drosophila melanogaster digestive tract, with a major focus on its main digestive/absorptive portion: the strikingly adaptable adult midgut., (Copyright © 2018 Miguel-Aliaga et al.)

Through the course of evolution, the gastrointestinal (GI) tract has been modified to maximize nutrient absorption, forming specialized segments that are morphologically and functionally distinct. Here we show that the GI tract of the Mexican tetra, Astyanax mexicanus, has distinct regions, exhibiting differences in morphology, motility, and absorption. We found that A. mexicanus populations adapted for life in subterranean caves exhibit differences in the GI segments compared to those adapted to surface rivers. Cave-adapted fish exhibit bi-directional churning motility in the stomach region that is largely absent in river-adapted fish. We investigated how this motility pattern influences intestinal transit of powdered food and live prey. We found that powdered food is more readily emptied from the cavefish GI tract. In contrast, the transit of live rotifers from the stomach region to the midgut occurs more slowly in cavefish compared to surface fish, consistent with the presence of churning motility. Differences in intestinal motility and transit likely reflect adaptation to unique food sources available to post-larval A. mexicanus in the cave and river environments. We found that cavefish grow more quickly than surface fish when fed ad libitum, suggesting that altered GI function may aid in nutrient consumption or absorption. We did not observe differences in enteric neuron density or smooth muscle organization between cavefish and surface fish. Altered intestinal motility in cavefish could instead be due to changes in the activity or patterning of the enteric nervous system. Exploring this avenue will lead to a better understanding of how the GI tract evolves to maximize energy assimilation from novel food sources., (Copyright © 2018. Published by Elsevier Inc.)

The emerging field of transcriptional regulation of cell shape changes aims to address the critical question of how gene expression programs produce a change in cell shape. Together with cell growth, division, and death, changes in cell shape are essential for organ morphogenesis. Whereas most studies of cell shape focus on posttranslational events involved in protein organization and distribution, cell shape changes can be genetically programmed. This review highlights the essential role of transcriptional regulation of cell shape during morphogenesis of the heart, lungs, gastrointestinal tract, and kidneys. We emphasize the evolutionary conservation of these processes across different model organisms and discuss perspectives on open questions and research avenues that may provide mechanistic insights toward understanding birth defects., (© 2018 Sivakumar and Kurpios.)

The gastrointestinal tract contains its own set of intrinsic neuroglial circuits - the enteric nervous system (ENS) - which detects and responds to diverse signals from the environment. Here, we address recent advances in the understanding of ENS development, including how neural-crest-derived progenitors migrate into and colonize the bowel, the formation of ganglionated plexuses and the molecular mechanisms of enteric neuronal and glial diversification. Modern lineage tracing and transcription-profiling technologies have produced observations that simultaneously challenge and affirm long-held beliefs about ENS development. We review many genetic and environmental factors that can alter ENS development and exert long-lasting effects on gastrointestinal function, and discuss how developmental defects in the ENS might account for some of the large burden of digestive disease.

Early prenatal stress disrupts maternal-to-offspring microbiota transmission and has lasting effects on metabolism, physiology, cognition, and behavior in male mice. Here we show that transplantation of maternal vaginal microbiota from stressed dams into naive pups delivered by cesarean section had effects that partly resembled those seen in prenatally stressed males. However, transplantation of control maternal vaginal microbiota into prenatally stressed pups delivered by cesarean section did not rescue the prenatal-stress phenotype. Prenatal stress was associated with alterations in the fetal intestinal transcriptome and niche, as well as with changes in the adult gut that were altered by additional stress exposure in adulthood. Further, maternal vaginal transfer also partially mediated the effects of prenatal stress on hypothalamic gene expression, as observed after chronic stress in adulthood. These findings suggest that the maternal vaginal microbiota contribute to the lasting effects of prenatal stress on gut and hypothalamus in male mice.

The development of the digestive tract is critical for proper food digestion and nutrient absorption. Here, we analyse the main organs of the digestive tract, including the oesophagus, stomach, small intestine and large intestine, from human embryos between 6 and 25 weeks of gestation as well as the large intestine from adults using single-cell RNA-seq analyses. In total, 5,227 individual cells are analysed and 40 cell types clearly identified. Their crucial biological features, including developmental processes, signalling pathways, cell cycle, nutrient digestion and absorption metabolism, and transcription factor networks, are systematically revealed. Moreover, the differentiation and maturation processes of the large intestine are thoroughly investigated by comparing the corresponding transcriptome profiles between embryonic and adult stages. Our work offers a rich resource for investigating the gene regulation networks of the human fetal digestive tract and adult large intestine at single-cell resolution.

During embryonic development, most organs are in a state of mechanical compression because they grow in a confined and limited amount of space within the embryo's body; the early gut is an exception because it physiologically herniates out of the coelom. We demonstrate here that physiological hernia is caused by a tensile force transmitted by the vitelline duct on the early gut loop at its attachment point at the umbilicus. We quantify this tensile force and show that applying tension for 48 h induces stress-dependent elongational growth of the embryonic gut in culture, with an average 90% length increase (max: 200%), 65% volume increase (max: 160%), 50% dry mass increase (max: 100%), and 165% cell number increase (max: 300%); this mechanical cue is required for organ growth as guts not subject to tension do not grow. We demonstrate that growth results from increased cell proliferation when tension is applied. These results outline the essential role played by mechanical forces in shaping and driving the proliferation of embryonic organs.

BackgroundExcess vernix caseosa produced by the fetal skin appears as particles suspended in the amniotic fluid in late gestation, is swallowed by the fetus, and is found throughout the newborn gastrointestinal tract as the first organisms are arriving to colonize the gut. Lipid-rich vernix contains an unusually high 29% branched chain fatty acids (BCFA). BCFAs reduce the incidence of necrotizing enterocolitis in an animal model, and were recently found predominantly in the sn-2 position of human milk triacylglycerols. Nothing is known about the influence of vernix BCFA on proinflammatory markers in human enterocytes.MethodsWe investigated the effect of vernix-monoacylglycerides (MAGs) (enriched with 30% BCFA) on interleukin (IL)-8 and NF-κB production in a human intestinal epithelial cell line (Caco-2). Caco-2 cells were pretreated with vernix-MAG or vernix-free fatty acid (FFA) prior to lipopolysaccharide (LPS) activation.ResultsBoth vernix-MAG and vernix-FFA increased cell BCFA and eliminated an LPS-induced 20% reduction in cell viability. In stimulated Caco-2 cells, vernix-MAG was more effective than vernix-FFA in suppressing IL-8 and NF-κB. Activated vernix-MAG-treated cells expressed less of the cell-surface Toll-like receptor4 (TLR-4) compared with controls.ConclusionThis is the first study to show the reduction of proinflammatory markers in human cells mediated by BCFA-MAG.

The gastrointestinal (GI) tract, in simplest terms, can be described as an epithelial-lined muscular tube extending along the cephalocaudal axis from the oral cavity to the anus. Although the general architecture of the GI tract organs is conserved from end to end, the presence of different epithelial tissue structures and unique epithelial cell types within each organ enables each to perform the distinct digestive functions required for efficient nutrient assimilation. Spatiotemporal regulation of signaling pathways and downstream transcription factors controls GI epithelial morphogenesis during development to confer essential regional-specific epithelial structures and functions. Here, we discuss the fundamental functions of each GI tract organ and summarize the diversity of epithelial structures present along the cephalocaudal axis of the GI tract. Next, we discuss findings, primarily from genetic mouse models, that have defined the roles of key transcription factors during epithelial morphogenesis, including p63, SOX2, SOX15, GATA4, GATA6, HNF4A, and HNF4G. Additionally, we examine how the Hedgehog, WNT, and BMP signaling pathways contribute to defining unique epithelial features along the cephalocaudal axis of the GI tract. Lastly, we examine the molecular mechanisms controlling regionalized cytodifferentiation of organ-specific epithelial cell types within the GI tract, concentrating on the stomach and small intestine. The delineation of GI epithelial patterning mechanisms in mice has provided fundamental knowledge to guide the development and refinement of three-dimensional GI organotypic culture models such as those derived from directed differentiation of human pluripotent stem cells and those derived directly from human tissue samples. Continued examination of these pathways will undoubtedly provide vital insights into the mechanisms of GI development and disease and may afford new avenues for innovative tissue engineering and personalized medicine approaches to treating GI diseases., (Copyright © 2018. Published by Elsevier Inc.)

The evolutionary fate of humans is intimately linked with that of our microbiome. Medical and technological advances have caused large-scale changes in the composition and maturation of human-associated microbial communities, increasing our susceptibility to infectious and developmental diseases. Restoration of the human microbiome must become a priority for biomedicine., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Congenital anorectal malformation is the most common digestive tract malformation in newborns. It has been reported that FOXD3/FOXD4, a forkhead transcription factor, regulates the generation, migration, and differentiation of neural crest cells. However, whether FOXD3/FOXD4 takes part in anorectal malformation remains unclear. In the present study, we used ethylene thiourea to induce the animal models of anorectal malformation in rat embryos and to interrogate the role of FOXD3/FOXD4 in anorectal malformation pathogenesis. Hindgut samples of the animal models were collected at E15, E17, E19, and E21 days of age. The expression of FOXD3/FOXD4 was detected by immunohistochemistry, western blot, and quantitative real-time fluorescence PCR. By immunohistochemical staining, FOXD3/FOXD4 was observed in epithelial cells of the rectum and the anus both in normal and rat embryos with anorectal malformation. Expression level analysis by western blot indicated that FOXD3/FOXD4 expression increased in ethylene thiourea-induced anorectal malformation groups. mRNA expression as determined by quantitative real-time fluorescence PCR analysis was consistent with the western blot results. Tentative conclusions were drawn that FOXD3/FOXD4 is expressed in the hindgut in rat embryos and is upregulated in anorectal malformation. FOXD3/FOXD4 is required for the development of the hindgut, and its aberrant expression may be an important factor leading to the incidence of anorectal malformation. Impact statement Congenital anorectal malformation (ARM) is the most common digestive tract malformation in newborns. The pathophysiological ground remains unclear. In this study, we used animal models of ARM for the first time to interrogate the role of FOXD3/FOXD4 in ARM pathogenesis. The animal models of ARM were successfully induced by ethylene thiourea (ETU) in rat embryos providing a strong basis for pathogenesis study of this disease. Expression analysis of FOXD3/FOXD4 was carried out in these models, and the results shape a deeper understanding of FOXD3/FOXD4 being required for the normal development of the hindgut. The aberrant expression of FOXD3/FOXD4 may be an important factor leading to ARM incidence.

Ribosome biogenesis is essential for the cell growth and division. Disruptions in ribosome biogenesis result in developmental defects and a group of diseases, known as ribosomopathies. Here, we report a mutation in zebrafish urb1, which encodes an essential ribosome biogenesis protein. The urb1 cq31 mutant exhibits hypoplastic digestive organs, which is caused by impaired cell proliferation with the differentiation of digestive organ progenitors unaffected. Knockdown of mtor or raptor leads to similar hypoplastic phenotypes and reduced expression of urb1 in the digestive organs. Overexpression of Urb1 results in overgrowth of digestive organs, and can efficiently rescue the hypoplastic liver and pancreas in the mtor and raptor morphants. Reduced syntheses of free ribosomal subunits and impaired assembly of polysomes are observed in the urb1 mutant as well as in the mtor and raptor morphants, which can be rescued by the Urb1 overexpression. These data demonstrate that Urb1 plays an important role in governing ribosome biogenesis and protein synthesis downstream of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), thus regulating the development of digestive organs. Our study indicates the requirement of hyperactive protein synthesis for the digestive organ development., (Copyright © 2017 Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, and Genetics Society of China. Published by Elsevier Ltd. All rights reserved.)

The gastrointestinal tract plays a vital role in nutrient supply, digestion, and absorption, and has a crucial impact on the entire organism. Much attention is being paid to utilize animal models to study the pathogenesis of gastrointestinal diseases in response to intestinal development and health. The piglet has a body size similar to that of the human and is an omnivorous animal with comparable anatomy, nutritional requirements, and digestive and associated inflammatory processes, and displays similarities to the human intestinal microbial ecosystem, which make piglets more appropriate as an animal model for human than other non-primate animals. Therefore, the objective of this review is to summarize key attributes of the piglet model with which to study human intestinal development and intestinal health through probing into the etiology of several gastrointestinal diseases, thus providing a theoretical and hopefully practical, basis for further studies on mammalian nutrition, health, and disease, and therapeutics. Given the comparable nutritional requirements and strikingly similar brain developmental patterns between young piglets and humans, the piglet has been used as an important translational model for studying neurodevelopmental outcomes influenced by pediatric nutrition. Because of similarities in anatomy and physiology between pigs and mankind, more emphasises are put on how to use the piglet model for human organ transplantation research.

The autonomic nervous system consists of sympathetic and parasympathetic nerves, which functionally antagonize each other to control physiology and homeostasis of organs. However, it is largely unexplored how the autonomic nervous system is established during development. In particular, early formation of parasympathetic network remains elusive because of its complex anatomical structure. To distinguish between parasympathetic (cholinergic) and sympathetic (adrenergic) ganglia, vesicular acetylcholine transporter (VAChT) and choline O-acetyltransferase (ChAT), proteins associated with acetylcholine synthesis, are known to be useful markers. Whereas commercially available antibodies against these proteins are widely used for mammalian specimens including mice and rats, these antibodies do not work satisfactorily in chickens, although chicken is an excellent model for the study of autonomic nervous system. Here, we newly raised antibodies against chicken VAChT and ChAT proteins. One monoclonal and three polyclonal antibodies for VAChT, and one polyclonal antibody for ChAT were obtained, which were available for Western blotting analyses and immunohistochemistry. Using these verified antibodies, we detected cholinergic cells in Remak ganglia of autonomic nervous system, which form in the dorsal aspect of the digestive tract of chicken E13 embryos. The antibodies obtained in this study are useful for visualization of cholinergic neurons including parasympathetic ganglia., (© 2017 Japanese Society of Developmental Biologists.)

Autophagy is essential for maintaining cellular homeostasis and survival under various stress conditions. Autophagy-related gene 9 (Atg9) encodes a multipass transmembrane protein thought to act as a membrane carrier for forming autophagosomes. However, the molecular regulation and physiological importance of Atg9 in animal development remain largely unclear. Here, we generated Atg9 null mutant flies and found that loss of Atg9 led to shortened lifespan, locomotor defects, and increased susceptibility to stress. Atg9 loss also resulted in aberrant adult midgut morphology with dramatically enlarged enterocytes. Interestingly, inhibiting the TOR signaling pathway rescued the midgut defects of the Atg9 mutants. In addition, Atg9 interacted with PALS1-associated tight junction protein (Patj), which associates with TSC2 to regulate TOR activity. Depletion of Atg9 caused a marked decrease in TSC2 levels. Our findings revealed an antagonistic relationship between Atg9 and TOR signaling in the regulation of cell growth and tissue homeostasis.

Most of the enteric nervous system derives from the "vagal" neural crest, lying at the level of somites 1-7, which invades the digestive tract rostro-caudally from the foregut to the hindgut. Little is known about the initial phase of this colonization, which brings enteric precursors into the foregut. Here we show that the "vagal crest" subsumes two populations of enteric precursors with contrasted origins, initial modes of migration, and destinations. Crest cells adjacent to somites 1 and 2 produce Schwann cell precursors that colonize the vagus nerve, which in turn guides them into the esophagus and stomach. Crest cells adjacent to somites 3-7 belong to the crest streams contributing to sympathetic chains: they migrate ventrally, seed the sympathetic chains, and colonize the entire digestive tract thence. Accordingly, enteric ganglia, like sympathetic ones, are atrophic when deprived of signaling through the tyrosine kinase receptor ErbB3, while half of the esophageal ganglia require, like parasympathetic ones, the nerve-associated form of the ErbB3 ligand, Neuregulin-1. These dependencies might bear relevance to Hirschsprung disease, with which alleles of Neuregulin-1 are associated., Competing Interests: The authors declare no conflict of interest., (Published under the PNAS license.)

Mutations in the human N- glycanase 1 ( NGLY1 ) cause a rare, multisystem congenital disorder with global developmental delay. However, the mechanisms by which NGLY1 and its homologs regulate embryonic development are not known. Here we show that Drosophila Pngl encodes an N -glycanase and exhibits a high degree of functional conservation with human NGLY1. Loss of Pngl results in developmental midgut defects reminiscent of midgut-specific loss of BMP signaling. Pngl mutant larvae also exhibit a severe midgut clearance defect, which cannot be fully explained by impaired BMP signaling. Genetic experiments indicate that Pngl is primarily required in the mesoderm during Drosophila development. Loss of Pngl results in a severe decrease in the level of Dpp homodimers and abolishes BMP autoregulation in the visceral mesoderm mediated by Dpp and Tkv homodimers. Thus, our studies uncover a novel mechanism for the tissue-specific regulation of an evolutionarily conserved signaling pathway by an N -glycanase enzyme.

The cardiovascular system is fundamental to life. Its vessels are the conduits for delivery of nutrients and oxygen to organs and the removal of wastes. During embryonic development, the vascular system is instrumental in the formation of organs. It contributes to the form and pattern of organs as diverse as the limbs and the gonads. Recent advances in molecular biology and genomics have afforded great insight to the control of vascular development at subcellular levels of organization. Nevertheless, there is little assembled information concerning the vascular development of the organ systems of the body. This paper begins by reviewing the modes of formation of embryonic blood vessels. This is followed by summaries of the ontogeny of the vasculature that supplies selected major thoracic and abdominal organs (heart, gut, liver, gonads, and kidney). The paper concludes with a description of the arterial development of the upper and lower extremities., (Copyright © 2016 The Author. Published by Elsevier Inc. All rights reserved.)

It has long been assumed that establishment of the fetal microbiome commences with the birthing process. However, recent studies have found bacterial DNA in umbilical cord blood, placenta, amniotic fluid, meconium, and fetal membranes in healthy normal pregnancies, leading to suggestions that the seeding of the fetal microbiome may commence in utero long before delivery. The origins of the microbiota of the fetal gastrointestinal (GI) tract have not yet been conclusively determined, although bacterial translocation from the maternal circulation, or ascension from the vagina, are both likely to be contributing pathways. Mother-to-child efflux of bacteria during pregnancy has the potential to markedly influence postnatal health, as the composition of gut microbiota determines production of important metabolites which are absorbed systemically and which modify immune function and development. Hence, the importance of understanding the colonization of the fetal GI microbiome is becoming clear, although few studies have investigated the origins, dynamics, and timing of the fetal microbiome. This is the topic of this review. By gaining a deeper understanding of the mechanisms underpinning fetal microbiome seeding, strategies may be developed to optimize fetal immune development and reduce the risk of adverse health and developmental outcomes.

L1 cell adhesion molecule (L1CAM) is a transmembrane molecule belonging to the L1 protein family. It has shown to be a key player in axonal guidance in the course of neuronal development. Furthermore, L1CAM is also crucial for the establishment of the enteric and urogenital organs and is aberrantly expressed in cancer originating in these organs. Carcinogenesis and embryogenesis follow a lot of similar molecular pathways, but unfortunately, comprehensive data on L1CAM expression and localization in human developing organs are lacking so far. In the present study we, therefore, examined the spatiotemporal distribution of L1CAM in the early human fetal period (weeks 8-12 of gestation) by means of immunohistochemistry and in situ hybridization (ISH). In the epithelia of the gastrointestinal organs, L1CAM localization cannot be observed in the examined stages most likely due to their advanced polarization and differentiation. Despite these results, our ISH data indicate weak L1CAM expression, but only in few epithelial cells. The genital tracts, however, are distinctly L1CAM positive throughout the entire fetal period. We, therefore, conclude that in embryogenesis L1CAM is crucial for further differentiation of epithelia., Competing Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Objectives: The purpose of this study was to describe the characteristics and outcomes of fetuses with a diagnosis of nonobstructive diffuse dilated bowel loops., Methods: We conducted a retrospective study of all pregnancies with fetal diagnosis of nonobstructive diffuse dilated bowel loops over 14 years in a large tertiary referral center. Fetomaternal and neonatal characteristics and outcomes were assessed., Results: Seven fetuses had sonograms showing diffuse dilated bowel loops; none of them had intestinal obstruction after labor. The median gestational age at diagnosis was 33 weeks 1 day (range, 27 weeks-34 weeks 1 day). The median gestational age at delivery was 34 weeks 1 day (range, 32 weeks 4 days-39 weeks 1 day). Four cases had premature rupture of membranes beyond 32 weeks. Four among the 7 had gastrointestinal manifestations. Three cases presented with hematochezia, which resolved with conservative treatment. One fetus had intractable diarrhea, had a diagnosis of rare microvillus inclusion disease, and died of sepsis after 92 days. Not a single case of Hirschsprung disease was observed in our group., Conclusions: Nonobstructive diffuse dilated bowel loops diagnosed in the second half of pregnancy are associated with premature rupture of membranes and premature labor. As neonatal gastrointestinal complications may be anticipated, prenatal parental counseling with a neonatologist and pediatric gastroenterologist should be conducted., (© 2016 by the American Institute of Ultrasound in Medicine.)

The architecture of the outer body wall cuticle is fundamental to protect arthropods against invading pathogens and numerous other harmful stresses. Such robust cuticles are formed by parallel running chitin microfibrils. Molting and also local wounding leads to dynamic assembly and disassembly of the chitin-matrix throughout development. However, the underlying molecular mechanisms that organize proper chitin-matrix formation are poorly known. Recently we identified a key region for cuticle thickening at the apical cell surface, the cuticle assembly zone, where Obstructor-A (Obst-A) coordinates the formation of the chitin-matrix. Obst-A binds chitin and the deacetylase Serpentine (Serp) in a core complex, which is required for chitin-matrix maturation and preservation. Here we present evidence that Chitinase 2 (Cht2) could be essential for this molecular machinery. We show that Cht2 is expressed in the chitin-matrix of epidermis, trachea, and the digestive system. There, Cht2 is enriched at the apical cell surface and the dense chitin-matrix. We further show that in Cht2 knockdown larvae the assembly zone is rudimentary, preventing normal cuticle formation and pore canal organization. As sequence similarities of Cht2 and the core complex proteins indicate evolutionarily conserved molecular mechanisms, our findings suggest that Cht2 is involved in chitin formation also in other insects., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Although the arrangement of internal organs in most metazoans is profoundly left-right (L/R) asymmetric with a predominant handedness, rare individuals show full (mirror-symmetric) or partial (heterotaxy) reversals. While the nematode Caenorhabditis elegans is known for its highly determinate development, including stereotyped L/R organ handedness, we found that L/R asymmetry of the major organs, the gut and gonad, varies among natural isolates of the species in both males and hermaphrodites. In hermaphrodites, heterotaxy can involve one or both bilaterally asymmetric gonad arms. Male heterotaxy is probably not attributable to relaxed selection in this hermaphroditic species, as it is also seen in gonochoristic Caenorhabditis species. Heterotaxy increases in many isolates at elevated temperature, with one showing a pregastrulation temperature-sensitive period, suggesting a very early embryonic or germline effect on this much later developmental outcome. A genome-wide association study of 100 isolates showed that male heterotaxy is associated with three genomic regions. Analysis of recombinant inbred lines suggests that a small number of loci are responsible for the observed variation. These findings reveal that heterotaxy is a widely varying quantitative trait in an animal with an otherwise highly stereotyped anatomy, demonstrating unexpected plasticity in an L/R arrangement of the major organs even in a simple animal.This article is part of the themed issue 'Provocative questions in left-right asymmetry'., (© 2016 The Authors.)

In this work we analyzed protein expression in the Aedes aegypti midgut during the larval (fourth instar, L4), pupal, and adult stages [including newly emerged (NE), sugar-fed (SF) and blood-fed (BF) females]. Two-dimensional electrophoresis showed 13 spots in the midgut of larvae, 95 in the midgut of pupae, 90 in the midgut of NE, and 76 in the midgut of SF or BF females. In the larval midguts, high serpin expression was noted, while in the pupae, protein abundance was lower than in the NE, SF, and BF females. The spots related to proteins linked to energy production, protein metabolism, signaling, and transport were highly expressed in the NE stage, while spots related proteins involved in translation were abundant in SF and BF females. The differential abundance of proteins in the midgut of A. aegypti at different developmental stages supports the necessity for midgut development during immature stage followed by the necessity of proteins related to digestion in adults., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

Common sequence variants in cis-regulatory elements (CREs) are suspected etiological causes of complex disorders. We previously identified an intronic enhancer variant in the RET gene disrupting SOX10 binding and increasing Hirschsprung disease (HSCR) risk 4-fold. We now show that two other functionally independent CRE variants, one binding Gata2 and the other binding Rarb, also reduce Ret expression and increase risk 2- and 1.7-fold. By studying human and mouse fetal gut tissues and cell lines, we demonstrate that reduced RET expression propagates throughout its gene regulatory network, exerting effects on both its positive and negative feedback components. We also provide evidence that the presence of a combination of CRE variants synergistically reduces RET expression and its effects throughout the GRN. These studies show how the effects of functionally independent non-coding variants in a coordinated gene regulatory network amplify their individually small effects, providing a model for complex disorders., (Copyright © 2016 Elsevier Inc. All rights reserved.)

The gastrointestinal (GI) tract is innervated by intrinsic enteric neurons and by extrinsic efferent and afferent nerves. The enteric (intrinsic) nervous system (ENS) in most regions of the gut consists of two main ganglionated layers; myenteric and submucosal ganglia, containing numerous types of enteric neurons and glial cells. Axons arising from the ENS and from extrinsic neurons innervate most layers of the gut wall and regulate many gut functions. The majority of ENS cells are derived from vagal neural crest cells (NCCs), which proliferate, colonize the entire gut, and first populate the myenteric region. After gut colonization by vagal NCCs, the extrinsic nerve fibers reach the GI tract, and Schwann cell precursors (SCPs) enter the gut along the extrinsic nerves. Furthermore, a subpopulation of cells in myenteric ganglia undergoes a radial (inward) migration to form the submucosal plexus, and the intrinsic and extrinsic innervation to the mucosal region develops. Here, we focus on recent progress in understanding the developmental processes that occur after the gut is colonized by vagal ENS precursors, and provide an up-to-date overview of molecular mechanisms regulating the development of the intrinsic and extrinsic innervation of the GI tract., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Hirschsprung disease (HSCR, OMIM 142623) is a neurocristopathy caused by a failure of the enteric nervous system (ENS) progenitors derived from neural crest cells (NCCs), to migrate, proliferate, differentiate or survive to and within the gastrointestinal tract, resulting in aganglionosis in the distal colon. The formation of the ENS is a complex process, which is regulated by a large range of molecules and signalling pathways involving both the NCCs and the intestinal environment. This tightly regulated process needs correct regulation of the expression of ENS specific genes. Alterations in the expression of these genes can have dramatic consequences. Several mechanisms that control the expression of genes have been described, such as DNA modification (epigenetic mechanisms), regulation of transcription (transcription factor, enhancers, repressors and silencers), post-transcriptional regulation (3'UTR and miRNAs) and regulation of translation. In this review, we focus on the epigenetic DNA modifications that have been described so far in the context of the ENS development. Moreover we describe the changes that are found in relation to the onset of HSCR., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Co-ordinated gastrointestinal function is the result of integrated communication between the enteric nervous system (ENS) and "effector" cells in the gastrointestinal tract. Unlike smooth muscle cells, interstitial cells, and the vast majority of cell types residing in the mucosa, enteric neurons and glia are not generated within the gut. Instead, they arise from neural crest cells that migrate into and colonise the developing gastrointestinal tract. Although they are "later" arrivals into the developing gut, enteric neural crest-derived cells (ENCCs) respond to many of the same secreted signalling molecules as the "resident" epithelial and mesenchymal cells, and several factors that control the development of smooth muscle cells, interstitial cells and epithelial cells also regulate ENCCs. Much progress has been made towards understanding the migration of ENCCs along the gastrointestinal tract and their differentiation into neurons and glia. However, our understanding of how enteric neurons begin to communicate with each other and extend their neurites out of the developing plexus layers to innervate the various cell types lining the concentric layers of the gastrointestinal tract is only beginning. It is critical for postpartum survival that the gastrointestinal tract and its enteric circuitry are sufficiently mature to cope with the influx of nutrients and their absorption that occurs shortly after birth. Subsequently, colonisation of the gut by immune cells and microbiota during postnatal development has an important impact that determines the ultimate outline of the intrinsic neural networks of the gut. In this review, we describe the integrated development of the ENS and its target cells., (Copyright © 2016 Elsevier Inc. All rights reserved.)