The Roles of the Basement Membrane and Semaphorin Signaling in Drosophila Egg Chamber Elongation

The morphogenesis of epithelial tissues is a highly dynamic process involving complex interactions between the cells in the tissue and between the cells and their physical environment. Basement membranes are specialized extracellular matrices that line the basal surfaces of epithelia and provide structural and mechanical integrity to the tissue. During morphogenesis, basement membranes can be structurally and mechanically patterned such that they can sculpt epithelia into complex 3D shapes, but the mechanical roles of basement membranes have only recently begun to be understood. The Drosophila melanogaster egg chamber is a genetically, experimentally, and visually tractable system to ask how an epithelium patterns its basement membrane through various mechanisms that allows the matrix to guide tissue shape changes. Egg chambers start as small spherical structures and grow nearly 1000 times in size, through which they become progressively elongated. During egg chamber morphogenesis, follicular epithelial cells synthesize and secrete their own basement membrane proteins and create a complex ultrastructural architecture by synergizing this secretion with a collective migration of the cells that results in a polarized array of fibrils. While these fibrils are known to be required for egg chamber elongation, whether they have a role in mechanically patterning the basement membrane or have some other secondary role to support tissue elongation has been debated. Additionally, much work has gone into uncovering how the collective migration of the follicle cells is governed. A set of planar polarized transmembrane proteins form two signaling axes: Fat2 and Lar, and Sema5c and PlexA. While there is increasing mechanistic insight into Fat2/Lar signaling, relatively little is known about how Sema5c and PlexA function. During my doctoral work, I have investigated the role of these basement membrane fibrils in promoting egg chamber elongation as well as elucidating the function of Sema5c in follicle cell migration. I have organized my work into three data chapters (2-4). In Chapter 2 I show that the elongation defect associated with Sema5c egg chambers is due to a reduction in basement membrane fibrils, and I also identify a genetic condition that suggests that our current understanding of the requirements of specific basement membrane architectures and mechanical properties is not sufficient to explain why an egg chamber that lacks these can properly elongate. In Chapters 3 and 4, I describe candidate-based and unbiased approaches in identifying new proteins that work with Sema5c. In Chapter 3, I describe my work investigating two candidates to act downstream of Sema5c, the F-actin disassembly factor Mical and the small GTPase Rap1, which are known effectors of PlexA signaling in Drosophila. I find evidence that Mical functions as a part of the Sema5c signaling axis but that it may have a second, noncanonical function unrelated to actin disassembly. I also show that Rap1 is involved in follicle cell migration and egg chamber elongation, but it is unclear what its molecular function is and whether it is part of the Sema5c signaling axis. In Chapter 4, I describe the results of a dominant enhancer/suppressor screen of Sema5c that was successful in identifying new genetic interactors. I provide initial characterization of these genes while also providing a guide for future work on mapping unidentified genes in genomic regions known to interact with Sema5c.While the specific molecular function of Sema5c will require further investigation, altogether, my work suggests that the role of the basement membrane in guiding egg chamber elongation is likely more complex than previously understood.