RFX6 regulates human intestinal patterning and function upstream of PDX1.

The gastrointestinal (GI) tract is complex and consists of multiple organs with unique functions. Rare gene variants can cause congenital malformations of the human GI tract, although the molecular basis of these has been poorly studied. We identified a patient with compound-heterozygous variants in RFX6 presenting with duodenal malrotation and atresia, implicating RFX6 in development of the proximal intestine. To identify how mutations in RFX6 impact intestinal patterning and function, we derived induced pluripotent stem cells from this patient to generate human intestinal organoids (HIOs). We identified that the duodenal HIOs and human tissues had mixed regional identity, with gastric and ileal features. CRISPR-mediated correction of RFX6 restored duodenal identity. We then used gain- and loss-of-function and transcriptomic approaches in HIOs and Xenopus embryos to identify that PDX1 is a downstream transcriptional target of RFX6 required for duodenal development. However, RFX6 had additional PDX1-independent transcriptional targets involving multiple components of signaling pathways that are required for establishing early regional identity in the GI tract. In summary, we have identified RFX6 as a key regulator in intestinal patterning that acts by regulating transcriptional and signaling pathways.

Z-scores of fetal bladder size for antenatal differential diagnosis between posterior urethral valves and urethral atresia.

OBJECTIVE: To construct reference values for fetal urinary bladder distension in pregnancy and use Z-scores as a diagnostic tool to differentiate posterior urethral valves (PUV) from urethral atresia (UA). METHODS: This was a prospective cross-sectional study in healthy singleton pregnancies aimed at constructing nomograms of fetal urinary bladder diameter and volume between 15 and 35 weeks' gestation. Z-scores of longitudinal bladder diameter (LBD) were calculated and validated in a cohort of fetuses with megacystis with ascertained postnatal or postmortem diagnosis, collected from a retrospective, multicenter study. Correlations between anatomopathological findings, based on medical examination of the infant or postmortem examination, and fetal megacystis were established. The accuracy of the Z-scores was evaluated by receiver-operating-characteristics (ROC)-curve analysis. RESULTS: Nomograms of fetal urinary bladder diameter and volume were produced from three-dimensional ultrasound volumes in 225 pregnant women between 15 and 35 weeks of gestation. A total of 1238 urinary bladder measurements were obtained. Z-scores, derived from the fetal nomograms, were calculated in 106 cases with suspected lower urinary tract obstruction (LUTO), including 76 (72%) cases with PUV, 22 (21%) cases with UA, four (4%) cases with urethral stenosis and four (4%) cases with megacystis-microcolon-intestinal hypoperistalsis syndrome. Fetuses with PUV showed a significantly lower LBD Z-score compared to those with UA (3.95 vs 8.83, P < 0.01). On ROC-curve analysis, we identified 5.2 as the optimal Z-score cut-off to differentiate fetuses with PUV from the rest of the study population (area under the curve, 0.84 (95% CI, 0.748-0.936); P < 0.01; sensitivity, 74%; specificity, 86%). CONCLUSIONS: Z-scores of LBD can distinguish reliably fetuses with LUTO caused by PUV from those with other subtypes of LUTO, with an optimal cut-off of 5.2. This information should be useful for prenatal counseling and management of LUTO. © 2021 The Authors. Ultrasound in Obstetrics & Gynecology published by John Wiley & Sons Ltd on behalf of International Society of Ultrasound in Obstetrics and Gynecology.

Histomorphometric analysis of the epithelial lumen, mesenchyme, smooth muscle cell layers, and mesentery of the mouse developing duodenum in relation with the macroscopic morphogenesis.

Integral analysis of the development of the epithelium, mesenchyme, and smooth muscle cell (SMC) layers, i.e., the inner circular (IC) and outer longitudinal layers, as well as their relation with the mesentery is necessary to understand macroscopic gut development. We here focused on the proximal duodenum with the characteristic "C"-shaped loop and analyzed the duodenum down to the duodenojejunal flexure in C57BL/6J mouse embryos at embryonic days (E) 13.5, 15.5, and 17.5 by histomorphometric analysis. We examined the angle of the axis of the epithelial lumen, which was oval at E13.5 against the mesentery, along with the epithelial cell nuclear shape, the adjacent mesenchymal cell density in relation to the epithelial lumen axis, and the development of SMC layers. The luminal axis of the oval epithelial lumen at E13.5 rotated clockwise against the mesentery in the proximal duodenum. The shape of epithelial nuclei was longer and thinner at the long axis but shorter and broader at the short axis, whereas mesenchymal density was significantly lower in the area on the luminal long axis than that on the short axis. The number of SMC layers in the IC at E13.5, E15.5, and E17.5 showed a regional difference in relation to the mesentery, but no regional difference along the long axis of the duodenum. These findings suggest that epithelial lumen winding against the mesentery and the corresponding changes in the epithelial cell shape and surrounding mesenchymal density may be involved in the formation of the "C" loop of the proximal duodenum.

Abnormal Intestinal Anatomy in Late-stage Human Fetuses: Three Case Series.

OBJECTIVE: We reported three cases of fetuses with abnormal intestinal anatomy found during our recent study of the transverse mesocolon using 20 late-stage fetuses. CASES: The first case (CRL: 328 mm) appeared to have a duodenum and transverse colon trapped in Winslow's foramen (foramen epiploicum) and the duodenum superior portion elongated rightward. The second case (CRL: 264 mm) had a transverse colon inserted deeply into a space between the right kidney and duodenum. The third case (CRL: 276 mm) had a descending colon that ran inferiorly through a deep space between the left kidney and duodenum. Each case had a greater omentum that was shifted leftward, but this is usual. These 3 abnormalities were not evident in the anterior view during dissection of the liver, stomach, jejunum, and ileum. With underdeveloped pancreatic ducts due to unknown reason other than the internal hernia, the first case seemed to be fatal after birth. However, the second and third cases could have recovered after birth because there was no evidence of definite malrotation and because of loose attachments of the intestines to surrounding structures. CONCLUSIONS: The intestinal morphologies described here could cause some sort of symptoms, such as abdominal pain, whose cause might be difficult to determine.

Dual embryonic origin of the mammalian enteric nervous system.

The enteric nervous system is thought to originate solely from the neural crest. Transgenic lineage tracing revealed a novel population of clonal pancreatic duodenal homeobox-1 (Pdx1)-Cre lineage progenitor cells in the tunica muscularis of the gut that produced pancreatic descendants as well as neurons upon differentiation in vitro. Additionally, an in vivo subpopulation of endoderm lineage enteric neurons, but not glial cells, was seen especially in the proximal gut. Analysis of early transgenic embryos revealed Pdx1-Cre progeny (as well as Sox-17-Cre and Foxa2-Cre progeny) migrating from the developing pancreas and duodenum at E11.5 and contributing to the enteric nervous system. These results show that the mammalian enteric nervous system arises from both the neural crest and the endoderm. Moreover, in adult mice there are separate Wnt1-Cre neural crest stem cells and Pdx1-Cre pancreatic progenitors within the muscle layer of the gut.

Diagnosis of fetal megacystis with chromosomal abnormality by 2D prenatal ultrasound: A case report.

RATIONALE: The pathogenesis of fetal megacystis is divided into obstructive and nonobstructive. Megacystis combined with chromosomal abnormalities is rare and most of the cases are nonobstructive. PATIENT CONCERNS: The fetus showed posterior urethral obstructive megacystis with features of bladder enlargement, "keyhole" feature, and thick bladder wall. DIAGNOSES: Here, we present a case of fetal megacystis diagnosed by ultrasound at pregnancy week 15+2 and with multisystem abnormalities. OUTCOMES: Moreover, the fetus showed edema, umbilical cord cyst, cardiac dysplasia, hook-shaped hand, and strephenopodia. These abnormalities strongly suggested chromosomal abnormalities. The fetus was diagnosed with trisomy 18 by amniocentesis. Posterior urethral obstructive megacystis was confirmed by pathology. LESSONS: In conclusion, this case suggests that in the presence of fetal megacystis and multisystem abnormalities, causes should be investigated and the possibility of chromosomal abnormalities should be considered in the presence of multisystem developmental abnormalities.

Use of RNA-seq to determine variation in canine cytochrome P450 mRNA expression between blood, liver, lung, kidney and duodenum in healthy beagles.

RNA sequencing (RNA-seq) is a powerful tool for the evaluation and quantification of transcriptomes and expression patterns in animals, tissues, or pathological conditions. The purpose of this study was to determine the physiologic expression of cytochrome P450 (CYP) mRNA transcripts in whole blood, kidney, duodenum, liver, and lung in healthy, adult male (n = 4) and female (n = 4) beagles via RNA-seq. mRNA expression was above background (transcripts per million) for 45 canine CYPs, with liver, duodenum, and lung expressing a high number of xenobiotic metabolizing CYPs, while prominent endogenous metabolizing CYP expression was present in blood and kidney. The relative expression pattern of CYP2A13, 2B11, 2C21, 2D15, 2E1, 3A12, and 27A1 in liver, lung, and duodenum was verified through qPCR. This is the first global profiling of physiologic CYP mRNA expression in multiple canine tissues, providing a platform for further studies characterizing canine CYPs and changes in gene expression in disease states.

Fetal megacystis: A systematic review.

Fetal megacystis is variably defined and understood. The literature on fetal megacystis was systematically reviewed, focusing on prenatal diagnosis, associations and outcomes. This yielded a total of 18 primary references and eight secondary references. Fetal megacystis has an estimated first-trimester prevalence of between 1:330 and 1:1670, with a male to female ratio of 8:1. In the first trimester, megacystis is most commonly defined as a longitudinal bladder dimension of ≥7 mm. Later in pregnancy, a sagittal dimension (in mm) greater than gestational age (in weeks) + 12 is often accepted. Megacystis can be associated with a thickened bladder wall, which has been objectively defined as >3 mm. Oligohydramnios is present in approximately half of all cases. The most common underlying diagnosis is posterior urethral valves (57%), followed by urethral atresia/stenosis (7%), prune belly syndrome (4%), megacystis-microcolon-intestinal-hypoperistalsis syndrome (MMIHS) (1%), and cloacal anomalies (0.7%). Karyotype anomalies are found in 15%, and include trisomy 18, trisomy 13 and trisomy 21. Ultrasound imaging alone is often insufficient to enable a definitive diagnosis, although it may indicate that a specific diagnosis is more likely. Overall, about 50% of reported fetuses with megacystis are terminated, but this proportion varies considerably between countries and over time. Prognostic stratification is evolving, with the most important factors being oligohydramnios, gestational age at diagnosis, degree of bladder enlargement, renal hyperechogenicity, karyotype, and sex. CONCLUSIONS: This review demonstrated some consensus on the ultrasound criteria for defining fetal megacystis, and illustrated the spectrum of pathologies and their relative frequencies that can cause this condition. It also underlined important associated karyotype anomalies. To progress understanding of the natural history of enlarged fetal bladders, more accurate diagnostics are required, and risk stratification needs to be refined to facilitate prenatal counseling.

Spatiotemporal distribution of extracellular matrix changes during mouse duodenojejunal flexure formation.

Although gut flexures characterize gut morphology, the mechanisms underlying flexure formation remain obscure. Previously, we analyzed the mouse duodenojejunal flexure (DJF) as a model for its formation and reported asymmetric morphologies between the inner and outer bending sides of the fetal mouse DJF, implying their contribution to DJF formation. We now present the extracellular matrix (ECM) as an important factor for gut morphogenesis. We investigate ECM distribution during mouse DJF formation by histological techniques. In the intercellular space of the gut wall, high Alcian-Blue positivity for proteoglycans shifted from the outer to the inner side of the gut wall during DJF formation. Immunopositivity for fibronectin, collagen I, or pan-tenascin was higher at the inner than at the outer side. Collagen IV and laminins localized to the epithelial basement membrane. Beneath the mesothelium at the pre-formation stage, collagen IV and laminin immunopositivity showed inverse results, corresponding to the different cellular characteristics at this site. At the post-formation stage, however, laminin positivity beneath the mesothelium was the reverse of that observed during the pre-formation stage. High immunopositivity for collagen IV and laminins at the inner gut wall mesenchyme of the post-formation DJF implied a different blood vessel distribution. We conclude that ECM distribution changes spatiotemporally during mouse DJF formation, indicating ECM association with the establishment of asymmetric morphologies during this process.

Imaging Evaluation of Fetal Megacystis: How Can Magnetic Resonance Imaging Help?

Evaluation of the kidneys, bladder, and amniotic fluid volume forms part of any standard obstetrical ultrasound. When a fetal genitourinary anomaly is suspected, a more detailed evaluation is necessary. This detailed imaging can be challenging in the setting of decreased or absent amniotic fluid or large maternal body habitus, and in complex malformations. In these situations, magnetic resonance imaging can help to better define the fetal anatomy and provide a more confident and specific prenatal diagnosis.

Asymmetric morphology of the cells comprising the inner and outer bending sides of the murine duodenojejunal flexure.

The asymmetric shape of component cells determines the asymmetric features of developing organs. Here, we focused on the murine duodenojejunal flexure (DJF), which bends without affecting the mesentery, and analyzed the morphological asymmetries of the mucosal epithelium and gut wall cells between the inner and outer bending sides at embryonic days 10.75-11.75. In the mucosal epithelium, the cell shape and the expression of epithelial markers (Cdx2, E-cadherin) showed no differences between the two DJF sides. In contrast, the gut wall cells comprising the inner and outer sides of the DJF were elongated along the inner-outer axis and perpendicular to this axis, respectively. Furthermore, the gut wall cells in the outer side possessed cytoplasmic processes connecting cells via adherens junctions, but those in the inner side were attached via adherens junctions of juxtaposed cell bodies and were relatively more crowded. In immunohistochemistry experiments, there was no remarkable difference in the positive reactions of markers for mesenchyme (vimentin), smooth muscle cells (αSMA), endothelial cells (LYVE-1, CD34), and undifferentiated neurons (Sox10) between the DJF sides. Interestingly, Tuj1-positive cells, indicating differentiated neurons, were observed in the middle layer of the gut wall, and these cells were significantly more abundant and tended to be larger in the inner side than in the outer side of the DJF. In conclusion, we clarified the asymmetries of gut wall cell morphology and neural differentiation between the inner and outer sides of the DJF. These characteristics of the developing murine DJF indicate its asymmetric formation.

Superior mesenteric artery syndrome and its associated gastrointestinal implications.

The superior mesenteric artery (SMA) syndrome is a rare but potentially life-threatening gastrointestinal condition. Over the years, it has been referenced by several names, the most common of which is Wilkie's syndrome. These numerous terminologies have made it difficult to estimate its true frequency in the general population. Common symptoms associated with this syndrome include intermittent postprandial abdominal pain, nausea, and bilious vomiting. Our review revealed that although it is currently well-defined in the literature, the diagnosis of SMA syndrome remains challenging as other disorders can mimic its presentation. However, CT angiography is currently favored in the literature for diagnosis as it can not only show the narrowed aorto-mesenteric angle and distance, but also the extent of duodenal obstruction. In addition, we found no consensus on the preferred mode of therapy once SMA syndrome is diagnosed. The agreement among authors is that the treatment options should be based on severity of the disease, using conservative measures as the first line of therapy in mild SMA syndrome. Duodenojejunostomy is the preferred surgical approach when conservative management fails, or in severe cases.

Analysis of duodenojejunal flexure formation in mice: implications for understanding the genetic basis for gastrointestinal morphology in mammals.

The mammalian gut undergoes morphological changes during development. We studied the developing mouse duodenojejunal flexure (DJF) to elucidate the mechanism of formation. During embryonic days 10.75-13.75, DJF formation was morphologically classified into three stages: the expansion stage, flexure formation stage, and flexure elongation stage. From the expansion to the flexure formation stages, the DJF wall showed asymmetric morphology and proliferation along the left-right intestinal axis. From the flexure formation to the flexure elongation stage, the DJF started to bend dorsally with counterclockwise rotation along the antero-caudal intestinal axis, indicating that the original right side of the duodenum was rotated towards the dorsal body wall during development of the DJF. The direction of attachment of the dorsal mesentery to the DJF did not correspond to the bending direction of the DJF during flexure formation, and this finding indicated that the dorsal mesentery contributed very little to DJF formation. During DJF formation, Aldh1a2 and hedgehog mRNAs were detected at the DJF, and their expression levels differed along the bending axis. In conclusion, DJF formation might be triggered by asymmetric morphology and proliferation along the left-right intestinal axis under the control of retinoic acid and hedgehog signaling.

Anisotropic growth shapes intestinal tissues during embryogenesis.

Embryogenesis offers a real laboratory for pattern formation, buckling, and postbuckling induced by growth of soft tissues. Each part of our body is structured in multiple adjacent layers: the skin, the brain, and the interior of organs. Each layer has a complex biological composition presenting different elasticity. Generated during fetal life, these layers will experience growth and remodeling in the early postfertilization stages. Here, we focus on a herringbone pattern occurring in fetal intestinal tissues. Common to many mammalians, this instability is a precursor of the villi, finger-like projections into the lumen. For avians (chicks' and turkeys' embryos), it has been shown that, a few days after fertilization, the mucosal epithelium of the duodenum is smooth, and then folds emerge, which present 2 d later a pronounced zigzag instability. Many debates and biological studies are devoted to this specific morphology, which regulates the cell renewal in the intestine. After reviewing experimental results about duodenum morphogenesis, we show that a model based on simplified hypothesis for the growth of the mesenchyme can explain buckling and postbuckling instabilities. Being completely analytical, it is based on biaxial compressive stresses due to differential growth between layers and it predicts quantitatively the morphological changes. The growth anisotropy increasing with time, the competition between folds and zigzags, is proved to occur as a secondary instability. The model is compared with available experimental data on chick's duodenum and can be applied to other intestinal tissues, the zigzag being a common and spectacular microstructural pattern of intestine embryogenesis.

Formation of duodenal atresias in fibroblast growth factor receptor 2IIIb-/- mouse embryos occurs in the absence of an endodermal plug.

PURPOSE: Duodenal atresia in humans has been hypothesized to arise from a failure of the duodenal lumen to recanalize after formation of an endodermal plug. Recently, mutations in the fibroblast growth factor receptor 2 gene (Fgfr2IIIb) have been shown to cause atretic defects of the duodenum in mice. However, work in rats suggests that murine species do not form an endodermal plug during normal duodenal development. These lines of data led us to hypothesize that mice are able to form a duodenal atresia in the absence of an endodermal plug. To test this hypothesis, we examined duodenal development in wild-type and Fgfr2IIIb-/- embryos. METHODS: Paraffin sections were generated for H&E, E-cadherin, or terminal deoxynucleotidyl transferase-mediated X-dUTP nick end labeling staining from Fgfr2IIIb-/- and wild-type embryos between embryonic days (E) 10.5 and E14.5. Sections were photographed and reconstructed into 3-dimensional display using Adobe Photoshop and Amira Visage software. RESULTS: Normal mouse duodenum does not form an endodermal plug, although a plug does form in the pyloric region of the stomach at E14.5. Fgfr2IIIb-/- embryos experience significant apoptosis in the duodenal region at E10.5, followed by the disappearance of the endoderm in the atretic precursor by E11.5. Thereafter, the mesoderm of the atretic precursor involutes over the next 2 days in the absence of further apoptosis. Interestingly, an endodermal plug was not observed at any point during the formation of a duodenal atresia. CONCLUSIONS: These results suggest that duodenal atresia in the Fgfr2IIIb-/- model does not arise from persistence of an epithelial plug. Rather it appears to result from the loss of the endoderm because of apoptosis very early in development.

Two patterns of development of interstitial cells of Cajal in the human duodenum.

At the end of the embryonic period of human development, c-kit immunoreactive (c-kit IR) cells identifiable as interstitial cells of Cajal (ICC) are present in the oesophagus and stomach wall. In the small and large bowel, c-kit-IR cells appear later (in the small bowel at 9 weeks, and in the colon at 10-12 weeks), also in the MP region. The object of this study was to determine the timing of appearance and distribution of c-kit IR cells in the human embryonic and foetal duodenum. I used immunohistochemistry to examine the embryonic and foetal duodenum for cells expressing CD117 (Kit), expressed by mature ICC and ICC progenitor cells and CD34 to identify presumed ICC progenitors. Enteric plexuses were examined by way of antineuron-specific enolase and the differentiation of smooth muscle cells was studied using antidesmin antibodies. At the end of the embryonic period of development, c-kit IR cells were solely present in the proximal duodenum in the form of a wide belt of densely packed cells around the inception of the myenteric plexus (MP) ganglia. In the distal duodenum, c-kit IR cells emerged at the beginning of the foetal period in the form of thin rows of pleomorphic cells at the level of the MP. From the beginning of the fourth month, the differences in the distribution of ICC in the different portions of the duodenum were established, and this relationship was still present in later developmental stages. In fact, in the proximal duodenum, ICC of the MP (ICC-MP), ICC of the circular muscle (ICC-CM) and ICC of the septa (ICC-SEP) were present, and in the distal duodenum ICC-MP and ICC-SEP only. In conclusion, in the humans there is a difference in the timing and patterns of development of ICC in the proximal duodenum compared to the distal duodenum.

Retinaldehyde dehydrogenase 2 is down-regulated during duodenal atresia formation in Fgfr2IIIb-/- mice.

BACKGROUND: Homozygous null mutation of fibroblast growth factor receptor 2 (Fgfr2IIIb) or its ligand fibroblast growth factor 10 (Fgf10) results in duodenal atresia in mice. Mutations of either of these genes in humans cause Matthew-Wood syndrome and associated duodenal stenosis. Recently, mutations in the retinol-binding protein receptor gene STRA6 were reported to be implicated in this syndrome as well. This suggests that the retinoic acid (RA) signaling pathway interacts with the Fgf10-Fgfr2IIIb signaling pathway during duodenal development. Accordingly, we hypothesized that Fgfr2IIIb-/- mouse embryos would exhibit disruptions in expression of Raldh2, the gene for the enzyme that regulates the final step in the conversion of vitamin A to the active form RA, during duodenal atresia formation. MATERIALS AND METHODS: Fgfr2III -/- mice were generated from heterozygous breedings. Embryos were harvested between embryonic day (E) 11.0 to E 13.5 and genotyped by polymerase chain reaction (PCR). Duodenums were dissected out, fixed and photographed. Whole mount and section in situs were performed for Raldh2. RESULTS: Fgfr2IIIb-/- embryos demonstrate subtle changes in the duodenal morphology by E11.5 with complete involution of the atretic precursor by E 13.5. Raldh2 appears to be down-regulated as early as E 11.5 in the atretic precursor a full 2 days before this segment disappears. CONCLUSIONS: In Fgfr2IIIb-/- mouse embryos, a reduction of Raldh2 expression is observed within the region that is forming the atresia. This is the first demonstration of such an event in this model. As in humans, these results implicate disruptions between Fgfr2IIIb receptor function and RA signaling in the formation of this defect and indicate that Fgfr2IIIb-/- mouse embryos are a valid model for the study of the atretic spectrum of defects in human duodenal development.

Fetal intrahepatic gallbladder and topographical anatomy of the liver hilar region and hepatocystic triangle.

The fetal gallbladder (GB) is embedded in a deep fossa surrounded by the liver parenchyma. Using 15 specimens with intrahepatic GB (crown-rump length 45-92 mm; approximately 9-13 weeks of gestation), we assessed the fetal topographical anatomy of the hepatocystic triangle and the porta hepatis. The cystic duct displayed a long upward course (0.9-4.5 mm along the supero-inferior axis) from the GB, along the duodenum, to the common bile duct in the hepatoduodenal ligament, via an independent mesentery separated from liver parenchyma by a recess of the peritoneal cavity. Notably, the course varied in length among specimens, not among stages. At the porta hepatis, we were able to distinguish the supraportal course of the posterior right hepatic duct overriding a portal vein branch to segment 8 (6/15) from the other, infraportal course (9/15). In the latter type, the portal vein bifurcation was superior to the cystic duct course. Two margins of the hepatocyctic triangle were very long in fetuses because of the inferiorly located intrahepatic GB. Thus, the triangle seems to be difficult to identify in prenatal ultrasound. During changes in location after 9 weeks, the GB fundus remains attached to the liver because the cystic artery was often embedded in the liver parenchyma. A failure in the embedding and re-exposure process of the GB may result in anomalous peritoneal folds around the GB.

Boundaries, junctions and transitions in the gastrointestinal tract.

Contiguous regions along the mammalian gastrointestinal tract, from the esophagus to the rectum, serve distinct digestive functions. Some organs, such as the esophagus and glandular stomach or the small bowel and colon, are separated by sharp boundaries. The duodenal, jejunal and ileal segments of the small intestine, by contrast, have imprecise borders. Because human esophageal and gastric cancers frequently arise in a background of tissue metaplasia and some intestinal disorders are confined to discrete regions, it is useful to appreciate the molecular and cellular basis of boundary formation and preservation. Here we review the anatomy and determinants of boundaries and transitions in the alimentary canal with respect to tissue morphology, gene expression, and, especially, transcriptional control of epithelial identity. We discuss the evidence for established and candidate molecular mechanisms of boundary formation, including the solitary and combinatorial actions of tissue-restricted transcription factors. Although the understanding remains sparse, genetic studies in mice do provide insights into dominant mechanisms and point the way for future investigation.