Tracing colonic embryonic transcriptional profiles and their reactivation upon intestinal damage.

We lack a holistic understanding of the genetic programs orchestrating embryonic colon morphogenesis and governing damage response in the adult. A window into these programs is the transcriptomes of the epithelial and mesenchymal cell populations in the colon. Performing unbiased single-cell transcriptomic analyses of the developing mouse colon at different embryonic stages (embryonic day 14.5 [E14.5], E15.5, and E18.5), we capture cellular and molecular profiles of the stages before, during, and after the appearance of crypt structures, as well as in a model of adult colitis. The data suggest most adult lineages are established by E18.5. We find embryonic-specific gene expression profiles and cell populations that reappear in response to tissue damage. Comparison of the datasets from mice and human colitis suggests the processes are conserved. In this study, we provide a comprehensive single-cell atlas of the developing mouse colon and evidence for the reactivation of embryonic genes in disease.

Embryonic macrophages function during early life to determine invariant natural killer T cell levels at barrier surfaces.

It is increasingly recognized that immune development within mucosal tissues is under the control of environmental factors during early life. However, the cellular mechanisms that underlie such temporally and regionally restrictive governance of these processes are unclear. Here, we uncover an extrathymic pathway of immune development within the colon that is controlled by embryonic but not bone marrow-derived macrophages, which determines the ability of these organs to receive invariant natural killer T (iNKT) cells and allow them to establish local residency. Consequently, early-life perturbations of fetal-derived macrophages result in persistent decreases of mucosal iNKT cells and is associated with later-life susceptibility or resistance to iNKT cell-associated mucosal disorders. These studies uncover a host developmental program orchestrated by ontogenically distinct macrophages that is regulated by microbiota, and they reveal an important postnatal function of macrophages that emerge in fetal life.

Bioactive fractions of Eucalyptus camaldulensis inhibit important foodborne pathogens, reduce listeriolysin O-induced haemolysis, and ameliorate hydrogen peroxide-induced oxidative stress on human embryonic colon cells.

Extract of E. camaldulensis was partitioned into aqueous and ethanol fractions by a precipitation and sedimentation-based technique and profiled for phytochemical components. Antimicrobial evaluation yielded inhibitory concentrations of 16-64 and 158-316 µg/mL, and bactericidal concentrations of 32-64 and 316->2528 µg/mL for ethanol and aqueous fractions, respectively. Antioxidant activities evaluated using 2,2-diphenyl-1-picrylhydrazyl and 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic) acid assays showed IC50 values of 7.07 and 65.67 µg/mL, and 17.96 and 201.3 µg/mL for aqueous and ethanol fractions. Total phenolic content of 9.04 ± 0.26 and 3.58 ± 0.04 GAE/mg fraction, and flavonoid content of 2.07 ± 0.02 and 3.37 ± 0.05 QE/mg fraction were recorded for aqueous and ethanol fractions. At subinhibitory concentrations fractions significantly reduced listeriolysin O-induced haemolysis (p < 0.05), and ameliorated H2O2-induced toxicity by 8-23 and 15-83%. Nitrite production reduced by 4-17 and 3-14 µM following fractions treatment. The fractions showed bioactive properties, with oxidative stress amelioratory effects, and could be a potentials source of preservatives and functional food additives.

A dorsal-ventral gradient of Wnt3a/ß-catenin signals controls mouse hindgut extension and colon formation.

Despite the importance of Wnt signaling for adult intestinal stem cell homeostasis and colorectal cancer, relatively little is known about its role in colon formation during embryogenesis. The development of the colon starts with the formation and extension of the hindgut. We show that Wnt3a is expressed in the caudal embryo in a dorsal-ventral (DV) gradient across all three germ layers, including the hindgut. Using genetic and lineage-tracing approaches, we describe novel dorsal and ventral hindgut domains, and show that ventrolateral hindgut cells populate the majority of the colonic epithelium. A Wnt3a-ß-catenin-Sp5/8 pathway, which is active in the dorsal hindgut endoderm, is required for hindgut extension and colon formation. Interestingly, the absence of Wnt activity in the ventral hindgut is crucial for proper hindgut morphogenesis, as ectopic stabilization of ß-catenin in the ventral hindgut via gain- or loss-of-function mutations in Ctnnb1 or Apc, respectively, leads to severe colonic hyperplasia. Thus, the DV Wnt gradient is required to coordinate growth between dorsal and ventral hindgut domains to regulate the extension of the hindgut that leads to colon formation.

Neuronal Development and Onset of Electrical Activity in the Human Enteric Nervous System.

BACKGROUND & AIMS: The enteric nervous system (ENS) is the largest branch of the peripheral nervous system, comprising complex networks of neurons and glia, which are present throughout the gastrointestinal tract. Although development of a fully functional ENS is required for gastrointestinal motility, little is known about the ontogeny of ENS function in humans. We studied the development of neuronal subtypes and the emergence of evoked electrical activity in the developing human ENS. METHODS: Human fetal gut samples (obtained via the MRC-Wellcome Trust Human Developmental Biology Resource-UK) were characterized by immunohistochemistry, calcium imaging, RNA sequencing, and quantitative real-time polymerase chain reaction analyses. RESULTS: Human fetal colon samples have dense neuronal networks at the level of the myenteric plexus by embryonic week (EW) 12, with expression of excitatory neurotransmitter and synaptic markers. By contrast, markers of inhibitory neurotransmitters were not observed until EW14. Electrical train stimulation of internodal strands did not evoke activity in the ENS of EW12 or EW14 tissues. However, compound calcium activation was observed at EW16, which was blocked by the addition of 1 µmol/L tetrodotoxin. Expression analyses showed that this activity was coincident with increases in expression of genes encoding proteins involved in neurotransmission and action potential generation. CONCLUSIONS: In analyses of human fetal intestinal samples, we followed development of neuronal diversity, electrical excitability, and network formation in the ENS. These processes are required to establish the functional enteric circuitry. Further studies could increase our understanding of the pathogenesis of a range of congenital enteric neuropathies.

Complete mesocolic excision for colonic cancer.

INTRODUCTION: Complete mesocolic excision for colonic cancer is similar in concept to total mesorectal excision for rectal cancer. This review aims to provide the embryological and anatomical rationale behind CME, and to review the current literature on CME, relative to the feasibility via laparoscopy, the oncological adequacy and outcomes. EVIDENCE ACQUISITION: A literature search was performed at the end of 2017 according the PRISMA guidelines for systematic reviews. Of 3980 articles found, we analyzed 96 articles. Of note, many case series had overlapping populations; there were five review articles, two consensus conference proceedings, six comparative but only one randomized trial. EVIDENCE SYNTHESIS: The embryonic and anatomical rationale is well described. CME is feasible via laparoscopy, which may facilitate dissection and anatomic precision: no statistically significant differences were found when compared to open CME regarding overall survival. However, morbidity may be higher in the hands of non-expert laparoscopic surgeons. CONCLUSIONS: Oncological adequacy can be obtained with laparoscopic CME, with increased lymph node retrieval. However, until now, there is no formal proof that CME improves local recurrence or survival.

The relation of fetal colon and rectum diameters with labor in healthy late-third trimester pregnancies.

OBJECTIVE: To investigate the associations of fetal colon-rectum diameters with labor and fetal distress or meconium passage in healthy pregnancies in the late 3rd trimester. STUDY DESIGN: A total of 162 healthy, singleton pregnant women at ≥360/7 weeks who were in the latent-phase of labor (n = 69) or those not in labor (n = 93, controls) at the time of ultrasound examination were enrolled. Fetal colon (ascending, transverse, descending, sigmoid) and rectum diameters, Doppler indices of materno-fetal vessels were measured. Data were analyzed according to the mode of delivery. RESULTS: Fetal colon-rectum diameters were smaller in women in labor compared to controls (p = 0.001). Positive correlations were observed between fetal colon-rectum diameters and interval between ultrasound and labor onset in the control group except for those who had scheduled cesarean sections (C/S) (p = 0.001). Similar colon-rectum measurements were obtained in fetuses delivered via cesarean section due to fetal distress or to other indications (p > 0.05). In women who had uterine contractions during ultrasound examination; later delivered by vaginal route, no association was observed between Apgar scores and colon-rectum diameters during latent-phase (p > 0.05), and also there were significant positive correlations between different segments of colon-rectum diameters and duration of neonatal meconium passage (p < 0.05). CONCLUSION: Fetal colon and rectum diameters are smaller during labor and the measurements tend to diminish as the labor approaches, but do not indicate fetal distress.

Fetal MRI in management of complicated meconium ileus: Prenatal and surgical imaging.

OBJECTIVE: To review fetal MRI cases surgically proven to have meconium ileus (MI) and obstruction, describe the common fetal MRI findings that distinguish cases of complicated MI, and to compare these findings with surgical images and perinatal outcomes. METHOD: We performed a retrospective review of all fetal MRI examinations and the corresponding medical record from our tertiary care children's hospital over an 18-month period. Postnatal management and outcomes were reviewed for these patients, and those patients with surgical or postmortem diagnosis of complicated MI were included in the study. RESULTS: Our analysis revealed 7 cases. In this cohort, 3 imaging features of the fetal bowel were repeatedly seen: gradient appearance of intraluminal bowel contents, abnormally localized meconium signal, and collapsed appearance of the colon on MRI. Surgical diagnoses confirmed MI. All live-born infants underwent surgical repair. CONCLUSION: Fetal MRI should be included in the diagnostic algorithm of any pregnancy where fetal bowel obstruction is suspected to better risk stratify patients.

Colonic mesenchyme differentiates into smooth muscle before its colonization by vagal enteric neural crest-derived cells in the chick embryo.

During development, the gastrointestinal (GI) tract arises from a primary tube composed of mesoderm and endoderm. The mesoderm gives rise to the digestive mesenchyme, which in turn differentiates into multiple tissues, namely the submucosa, the interstitial cells of Cajal and the smooth muscle cells (SMCs). Concomitant with these early patterning events, the primitive GI tract is colonized by vagal enteric neural crest-derived cells (vENCDCs), a population of cells that gives rise to the enteric nervous system, the intrinsic innervation of the GI tract. Reciprocal neuro-mesenchymal interactions are essential for the coordinated development of GI musculature. The aim of this study is to examine and compare the kinetics of mesenchymal cell differentiation into SMCs along the anterior-posterior axis to the pattern of vENCDCs migration using whole-mount in situ hybridization and paraffin section immunofluorescence analyses on chick embryonic GI tracts from E4-Stage 23 to E7-Stages 30-31. We confirmed that gastric and pre-umbilical intestine mesenchyme differentiation into SMCs occurs after vENCDCs colonization. However, we found that colonic and post-umbilical intestine mesenchyme differentiation occurs before vENCDCs colonization. These findings suggest that regional-specific mechanisms are involved in the mesenchyme differentiation into SMCs along the GI anterior-posterior axis.

Organotypical Tissue Cultures from Fetal and Neonatal Murine Colon.

The complex functions of the gastrointestinal tract rely on the coordinated interplay of several cell and tissue types involving epithelium, connective tissue, smooth muscles as well as cells of the immune and nervous system. It is therefore obvious, that these functions can hardly be investigated sufficiently using cell lines or two-dimensional cell cultures.Here, we describe an easy to produce three-dimensional organotypical explants culture from fetal and neonatal murine colon. This model is suitable for in vitro testing of intestinal function or the evaluation of developmental or pathological processes.

Spatiotemporal-specific lncRNAs in the brain, colon, liver and lung of macaque during development.

Genome-wide expression profiling during development provides useful information to uncover the potential molecular mechanisms of development in mammals. Recent studies have revealed that a subset of lncRNAs can regulate major biological processes during development. Here, we sequenced four tissues, including brain, colon, liver and lung, using RNA-seq across three developmental stages (early, middle and late stage), and then constructed genome-wide expression profiles during macaque development. In each tissue, we identified developmental time-specific lncRNA and mRNA clusters that displayed diverse expression alteration patterns, including a gradual increase, a gradual decrease, or a reversal of expression. These lncRNAs showed more specific functional associations with their corresponding tissues relative to the developmental time-specific mRNAs. Furthermore, we identified 20 spatiotemporal-specific co-modules including 101 lncRNAs and 609 mRNAs distributed at different developmental stages in different tissues. Our findings suggested that lncRNAs could play critical roles in the development of macaques through close cooperation with mRNAs. Finally, we predicted the functions of the spatiotemporal-specific lncRNAs by their spatiotemporal cooperation with mRNAs and further validated our findings using gene knockdown data of mouse. Our study reveals the spatiotemporal characteristics of lncRNAs and provides a functional map of the spatiotemporal-specific lncRNAs during the development of macaques.

From anatomy to laparoscopic surgery, or how to reconcile surgeons to embryology.

PURPOSE: With the development of laparoscopy, new surgical techniques for colon resection were required. New anatomic plans of dissection were described for laparoscopic technique (medial to lateral approach) and the surgeons had to learn a complete different anatomy known as "laparoscopic anatomy". To help the surgeon through the milestones of laparoscopic colon resection, we propose an embryological and anatomical analysis of the changes of the colon and peritoneum during the foetal period to highlight the laparoscopic approach and surgical landmarks. METHODS: Seventeen human foetuses, age ranged from 7½ to 33 weeks were studied by dissections and histology. Three adult cadavers underwent laparoscopic colon surgery. RESULTS: Photographic representations of surgical views are displayed, and detailed descriptions applicable to anatomical structures are presented. CONCLUSION: Understanding the changes in the colon and peritoneum morphology leads to a clarification of the surgical technique for laparoscopic colon surgery.

Development and entrainment of the colonic circadian clock during ontogenesis.

Colonic morphology and function change significantly during ontogenesis. In mammals, many colonic physiological functions are temporally controlled by the circadian clock in the colon, which is entrained by the central circadian clock in the suprachiasmatic nuclei (SCN). The aim of this present study was to ascertain when and how the circadian clock in the colon develops during the perinatal period and whether maternal cues and/or the developing pup SCN may influence the ontogenesis of the colonic clock. Daily profiles of clock genes Per1, Per2, Cry1, Cry2, Rev-erbα, Bmal1, and Clock expression in the colon underwent significant modifications since embryonic day 20 (E20) through postnatal days (P) 2, 10, 20, and 30 via changes in the mutual phasing among the individual clock gene expression rhythms, their relative phasing to the light-dark regime, and their amplitudes. An adult-like state was achieved around P20. The foster study revealed that during the prenatal period, the maternal circadian phase may partially modulate development of the colonic clock. Postnatally, the absence and/or presence of rhythmic maternal care affected the phasing of the clock gene expression profiles in pups at P10 and P20. A reversal in the colonic clock phase between P10 and P20 occurred in the absence of rhythmic signals from the pup SCN. The data demonstrate ontogenetic maturation of the colonic clock and stress the importance of prenatal and postnatal maternal rhythmic signals for its development. These data may contribute to the understanding of colonic function-related diseases in newborn children.

Expression pattern of the homeotic gene Bapx1 during early chick gastrointestinal tract development.

Regulation of the Bone Morphogenetic Protein (BMP) signaling pathway is essential for the normal development of vertebrate gastrointestinal (GI) tract, but also for the differentiation of the digestive mesenchymal layer into smooth muscles and submucosal layer. Different studies demonstrated that Bapx1 (for bagpipe homeobox homolog 1) negatively regulates the BMP pathway, but its precise expression pattern during the development and the differentiation of the GI tract mesenchyme actually remains to be examined. Here, we present the spatio-temporal expression profile of Bapx1 in the chick GI tract. We show that Bapx1 is first expressed in the undifferentiated mesenchyme of the gizzard and the colon. After the differentiation of the digestive mesenchyme, we found Bapx1 strongly expressed in the gizzard smooth muscle and in the submucosa layer of the colon. This expression pattern provides new insights into the roles of Bapx1 during the regionalization of the GI tract and the differentiation of the digestive mesenchyme of the colon and the stomach.

A study of calretinin in Hirschsprung pathology, particularly in total colonic aganglionosis.

INTRODUCTION: Calretinin, a calcium-binding protein, has been reported to be an important new marker in Hirschsprung's disease (HD). The aim is to study the diagnostic value of Calretinin in total colonic aganglionosis (TA), prematurity, and superficial biopsy when nerve hyperplasia may not be accessed by ACE activity. METHODS: Records of patients diagnosed with HD at our institution from 1985 to 2010 were studied and patients with TA identified. We examined tissue samples from those TA, partial colectomies for HD, biopsies for suspicion of HD, and rectal tissue from aborted fetuses. Immunohistochemical analysis of Calretinin was compared with ACE gold standard method in all cases. RESULTS: In the majority of the cases, the diagnosis was ascertained by ACE activity and Calretinin staining. However, in 9 cases, the diagnosis was possible with Calretinin staining but not with ACE: in 4 TA because of the absence of nerve hyperplasia, and in 5 cases because the biopsies were too superficial to examine the nerve hyperplasia. In addition, Calretinin was expressed in the gut as early as 22 gestational weeks. CONCLUSION: The use of Calretinin staining may be superior to ACE activity, particularly in the context of TA, superficial biopsies, and prematurity, allowing earlier diagnosis.

Tcof1 acts as a modifier of Pax3 during enteric nervous system development and in the pathogenesis of colonic aganglionosis.

Hirschsprung disease (HSCR) is a human congenital disorder, defined by the absence of ganglia from variable lengths of the colon. These ganglia comprise the enteric nervous system (ENS) and are derived from migratory neural crest cells (NCCs). The inheritance of HSCR is complex, often non-Mendelian and characterized by variable penetrance. Although extensive research has identified many key players in the pathogenesis of Hirschsprung disease, a large number of cases remain genetically undefined. Therefore, additional unidentified genes or modifiers must contribute to the etiology and pathogenesis of Hirschsprung disease. We have discovered that Tcof1 may be one such modifier. Haploinsufficiency of Tcof1 in mice results in a reduction of vagal NCCs and their delayed migration along the length of the gut during early development. This alone, however, is not sufficient to cause colonic aganglionosis as alterations in the balance of NCC proliferation and differentiation ensures NCC colonize the entire length of the gut of Tcof1(+/-) mice by E18.5. In contrast, Tcof1 haploinsufficiency is able to sensitize Pax3(+/-) mice to colonic aganglionosis. Although, Pax3 heterozygous mice do not show ENS defects, compound Pax3;Tcof1 heterozygous mice exhibit cumulative apoptosis which severely reduces the NCC population that migrates into the foregut. In addition, the proliferative capacity of these NCC is also diminished. Taken together with the opposing effects of Pax3 and Tcof1 on NCC differentiation, the synergistic haploinsufficiency of Tcof1 and Pax3 results in colonic aganglionosis in mice and may contribute to the pathogenesis of Hirschsprung disease.