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.

1 2Although gut flexures characterize the gut morphology, the mechanisms underlying 3 flexure formation remain obscure. Previously, we analyzed the mouse duodenojejunal flexure 4 (DJF) as a model for its formation, and reported asymmetric morphologies between the inner 5 and outer bending sides of the fetal mouse DJF, implicating their contribution to DJF formation. 6In this study, we present the extracellular matrix (ECM) as an important factor for gut 7 morphogenesis. We investigated the ECM distribution during mouse DJF formation using 8 histological techniques. In the intercellular space of the gut wall, high Alcian Blue-positivity, for 9 proteoglycans, shifted from the outer to the inner side of the gut wall during DJF formation. 10 However, the immunopositivity for fibronectin-, collagen I-, or pan-tenascin was higher at the 11 inner, than at the outer side. Further, collagen IV and laminins localized to the epithelial 12 basement membrane. Beneath the mesothelium at the pre-formation stage, collagen IV and 13 laminin immunopositivity showed inverse results, corresponding to the difference in cellular 14 characteristics at this site. At the post-formation stage, however, laminin-positivity beneath the 15 mesothelium was the reverse of that observed during the pre-formation stage. In addition, high 16 immunopositivity for collagen IV and laminins at the inner gut wall mesenchyme of 17 post-formation DJF implied different blood vessel distribution. Therefore, we concluded that 18 4 ECM distribution changes spatiotemporally during mouse DJF formation, thus indicating its 1 association with the establishment of asymmetric morphologies during DJF formation. 2 3

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.

The aim of the present study was to clarify roles of ATP-dependent potassium channels (KATP channels) in motility of the striated muscle portion in the esophagus. An isolated segment of the rat esophagus was placed in an organ bath and mechanical responses were recorded using a force transducer. Electrical stimulation of the vagus nerve evoked contractile response of striated muscle in the esophageal segment. Application of glibenclamide, an antagonist of KATP channels, increased amplitude of vagally mediated twitch contractions of the rat esophagus. On the other hand, minoxidil, an agonist of KATP channels, decreased amplitude of twitch contractions. RT-PCR revealed the expression of subunits of KATP channels in esophageal tissue. In addition, immunopositivity for subunits of KATP channels was observed in the striated muscle cells of the esophageal muscle layer. These findings indicate that KATP channels contribute to motor regulation of striated muscle in the rat esophagus.

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