Endocytic regulation of intestinal developmentThe intestinal mucosa is a highly differentiated epithelial barrier that is fundamental for normal function of theintestine. Vesicular trafficking is now recognized as an essential regulator of epithelial polarity integrity and celldifferentiation and studies in model systems have shown that endocytosis and membrane trafficking areessential for normal ontogenesis of the intestine. During development the epithelial cells of the intestine gothrough an intensely endocytic stage that precedes the acquisition of mature intestinal architecture. Weidentified endotubin (EDTB) as a highly conserved integral membrane protein in the endocytic complex ofthe developing intestine. In vitro EDTB regulates tight junction assembly and contact-mediated inhibitionof proliferation. In addition it binds the small GTPase Rab14 which regulates trafficking betweenendosomes and the apical plasma membrane. However the role of EDTB during development iscompletely unknown. We have generated an intestinal epithelial cell-specific EDTB knockout mouse andin Preliminary Data show that EDTB knockout early in development results in aberrant ontogenesis ofthe neonatal intestine including loss of the apical endocytic complex intracellular accumulation of apicalplasma membrane proteins and an aberrant brush border. We will use this EDTB conditional knockoutmouse together with enteroid cultures and cell lines to identify the mechanistic basis for these changesin epithelial differentiation. We will define the mechanisms for interaction with tight junction proteins anddefine the domains of EDTB that mediate its effects. Also we will analyze the role of Rab14 usingintestinal enteroid cultures and human intestinal epithelial cell lines. Finally we will examine the signalingpathways known to impact intestinal differentiation and define their interaction with EDTB and apicalendosomes. Collectively these studies will provide insight into the cellular mechanisms of intestinaldevelopment which is essential for our understanding of intestinal function. When completed the experimentsoutlined in this proposal will define the role of vesicular trafficking in normal growth development and homeostasisof the intestinal epithelium. This fundamental knowledge will have implications for our understanding of intestinaldisease both in neonates and adults and could lay the groundwork for therapeutic targets.