The ability of epithelia to form and expand specialized lumina is a central architectural characteristic of tubular organs (e.g. kidney, intestine) and is indispensible to their function. These lumina are lined by a layer of intimately connected, polarized epithelial cells in which the apical membrane faces the lumen and the basolateral membrane faces the underlying basement membrane. Lumen formation is known to occur by at least two distinct mechanisms depending on apical-basal polarization rate, cellular proliferation rate, and cortical epithelial tension. Coordinated epithelial proliferation and epithelial barrier function are essential for subsequent lumen expansion and specialization, but how these forces produce a functional architecture is unknown. Lumen formation can be modeled through the use of 3D MDCK cyst cultures. Lumen expansion and specialization can be modeled through enteroids, 3D intestinal stem cell cultures that grow into complex structures containing key architectural features of intestinal epithelium, namely regenerative crypts and villus- domains displaying markers of differentiated epithelium. The presence of specialized structures within enteroids suggests that epithelial-intrinsic features are responsible for some morphogenetic processes. Previous studies have identified cellular machinery that is critical to each aspect of lumen formation; however, the machinery that intermediates the functional crosstalk between polarization, proliferation, and cortical tension is unknown. The scientific objective of this application is therefore to bridge this knowledge gap by studying the crosstalk between cellular proliferation, epithelial polarization, and actomyosin networks in MDCK cysts and enteroids. Specifically, these studies will build on my preliminary data that the tight junction protein ZO-1 controls actin organization, cellular proliferation, and initial epithelial polarization to investiate molecular interactions that promote initial lumen formation. The proposed studies will also translate these preliminary findings into an enteroid system to study this crosstalk during luminal expansion and specialization. Moreover, the proposed experiments will be the first to test epithelial-intrinsic forces during enteroid budding, which I hypothesize are also the result of polarized actomyosin organization and cellular proliferation. The rationale for this project is tha it will provide significant insight into the fundamental mechanisms of lumen formation and expansion, which are critical to function in many epithelial organs. The proposal is innovative because it will use simple 3D culture systems to i) investigate a previously unappreciated role for the tight junction protein ZO- 1 and ii) define the mechanisms by which epithelial-intrinsic forces drive lumen expansion during tissue growth. The proposed research is significant because it will broaden our understanding of tight junction biology to thinking of the tight junctin as a dynamic structure that orchestrates complex epithelial morphogenetic events. In doing so, these studies will bridge a fundamental knowledge gap of how actin organization, epithelial polarization, and cell proliferation are coordinated to build complex, functional epithelial organs

F30DK103511

2014-07-01

2017-06-30