370 Effect of Azithromycin on Esophageal Hypomotility (EH) and Prediction of Response by Esophageal Stimulations Tests During High Resolution Manometry

Abstract

of villus formation at embryonic day (E)14.5. This analysis revealed crack-like extensions of the lumen from the apical surface into the epithelium, which lengthen over time. The tips of these cracks are associated with dividing cells. Because of this association and a precedent for lumen-forming cell divisions in the zebrafish neural keel, we speculated that these cells might generate cracks by trafficking apical proteins to the cytokinetic plane. Using intestinal explant cultures in concert with nocodazole-mediated synchronization of the cell cycle, we examined the trafficking of the apical protein Ezrin during cell division. We found that Ezrin is deposited along the cytokinetic plane of divisions at crack tips, defining new villus domains and segregating daughter cells onto adjacent villi. We call these specialized cell divisions e-divisions (lumen extending). We also noted a second type of cell division, which functions in intestinal growth, but is not associated with apical cracks and does not define new villus domains. We have named these divisions g-divisions (growth building). Eand g-divisions differ cell biologically in several ways: localization of apical proteins, segregation of daughter cells, midbody morphology, and angle of the mitotic spindle relative to the nearest overlying apical surface. Ezrin null mice have fused villi with underlying isolated secondary lumens. Such a phenotype has been thought to support the secondary lumen model of normal villus morphogenesis, because it was believed that the loss of Ezrin perturbs their fusion with the main lumen. However, we have now determined that dividing cells in Ezrin null mice have mis-oriented mitotic spindles; we speculate that mis-orientation of e-divisions may in fact cause ectopic lumens and fused villi in this model. Indeed, the characteristic phenotype of Ezrin null mice can be recapitulated in wild-type intestines after treatment with blebbistatin, a myosin II inhibitor that results in mis-oriented spindles both in vitro and in intestinal explant cultures. This indicates that correct spindle orientation is indispensible for proper villus morphogenesis. Further understanding of this process will open up new avenues of in vitro bioengineering of intestinal surface, potentially providing life-saving therapies for those with intestinal failure.