Endocytic regulation of intestinal development The intestinal mucosa is a highly differentiated epithelial barrier that is fundamental for normal function of the intestine. Vesicular trafficking is now recognized as an essential regulator of epithelial polarity, integrity and cell differentiation, and studies in model systems have shown that endocytosis and membrane trafficking are essential for normal ontogenesis of the intestine. During development, the epithelial cells of the intestine go through an intensely endocytic stage that precedes the acquisition of mature intestinal architecture. We identified endotubin (EDTB) as a highly conserved integral membrane protein in the endocytic complex of the developing intestine. In vitro, EDTB regulates tight junction assembly and contact-mediated inhibition of proliferation. In addition, it binds the small GTPase Rab14, which regulates trafficking between endosomes and the apical plasma membrane. However the role of EDTB during development is completely unknown. We have generated an intestinal epithelial cell-specific EDTB knockout mouse and, in Preliminary Data, show that EDTB knockout early in development results in aberrant ontogenesis of the neonatal intestine, including loss of the apical endocytic complex, intracellular accumulation of apical plasma membrane proteins, and an aberrant brush border. We will use this EDTB conditional knockout mouse, together with enteroid cultures and cell lines, to identify the mechanistic basis for these changes in epithelial differentiation. We will define the mechanisms for interaction with tight junction proteins and define the domains of EDTB that mediate its effects. Also, we will analyze the role of Rab14 using intestinal enteroid cultures and human intestinal epithelial cell lines. Finally, we will examine the signaling pathways known to impact intestinal differentiation and define their interaction with EDTB and apical endosomes. Collectively, these studies will provide insight into the cellular mechanisms of intestinal development, which is essential for our understanding of intestinal function. When completed, the experiments outlined in this proposal will define the role of vesicular trafficking in normal growth, development, and homeostasis of the intestinal epithelium. This fundamental knowledge will have implications for our understanding of intestinal disease, both in neonates and adults, and could lay the groundwork for therapeutic targets.
to human disease: Our understanding of the developing gut and how interference with normal development can result in disease is incomplete. Vesicular trafficking is now recognized as an essential regulator of epithelial polarity, integrity and cell differentiation. Elucidating the role of membrane trafficking in tight junction integrity, signaling, and differentiation is essential to our understanding of the development of the mucosal barrier.
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