Project Summary How epithelial sheets remodel themselves to adopt new tissue conformations through changes in neighbor relationships and cell shape dynamics has been a key question in development and disease. Interestingly, many of the pioneering studies performed in model systems have largely been confined to 2D analysis, and have often been challenged to image cell behaviors that occur in basal regions that lie deeper into the tissue. The intercalation movements that occur during tissue elongation in the Drosophila gastrula have been a classic system for understanding epithelial remodeling, and have been fundamental to informing the developmental paradigms that describe how cells can change position in an adherent epithelium. Nearly all of the studies in this system have been confined to 2D analysis of apical events in the early fly embryo, and no studies to date have systematically analyzed the full 3D behaviors that drive epithelial remodeling and tissue extension in the Drosophila embryonic epithelium. Thus, one of the biggest remaining questions in the field is how the volumetric nature of epithelial cells affects force propagation and remodeling of the cell surface along the entire apical-basal axis. Fundamental questions on where forces originate from as well as how far and fast forces propagate across different apical-basal layers have remained unanswered. In our preliminary analysis, we have been successful in completing the first full 4D segmentation of the intercalating Drosophila epithelium through the use of Lattice Light Sheet Microscopy (LLSM). We find that intercalation can be initiated at any position we have surveyed along the apical-basal axis. This is striking as previous studies have largely implicated apical force generation, and a single study has suggested that contractile forces can also originate from the basal surface of the epithelium. In the proposed project, we are developing the tools to perform the first comprehensive, quantitative 3D analysis of cell intercalation in the early Drosophila embryo. We will then determine the molecular mechanisms driving 3D force generation, and whether different mechanical regimes exist across the apical-basal axis. Preliminary data suggests highly novel dynamic Myosin II and F-actin populations that show rapid axial propagation in lateral and basal regions. The 3D distributions of these populations are being mapped and the relevant actin nucleating and Myosin regulatory networks will be determined. These results will provide the first comprehensive understanding of the cortical and contractile networks that determine the mechanical environment of a gastrulating epithelium. We will also use 3D data sets in wild-type and functionally compromised backgrounds to examine how epithelial forces propagate along apical-basal and planar dimensions using topological mapping metrics. We will determine how far, and at what velocities, contractile forces spread in an intact, developing epitheliu...