From Cell Shape to Body Shape: Epithelial Morphogenesis in Drosophila melanogaster

Embryonic development is associated with extensive rearrangements of tissues. The driving force for these rearrangements is generated by a dynamic reorganization of the Actin cytoskeleton of individual cells. In many instances, conserved Rho-family GTPases that are activated by Rho guanine nucleotide exchange factors (RhoGEFs) play an important role in this process. The present thesis investigates the mechanisms that control activation and specificity of the GTPase Rho1 by DRhoGEF2 during morphogenesis of the Drosophila embryonic epidermis. DRhoGEF2 is the Drosophila ortholog of the human RhoGEFs, LARG (Leukemia-associated RhoGEF), PDZ-RhoGEF and p115 RhoGEFs. In addition to a catalytic module, DRhoGEF2 encompasses an N-terminal PSD-95/Dlg/ZO-1 (PDZ) domain, a Regulator of G-protein Signaling (RGS) domain and a cysteine-rich diacylglycerol (DAG)-binding domain in the central region. Previously, DRhoGEF2 has been shown to play an important role in the constriction of Actin filaments during pole cell formation, blastoderm cellularization, and invagination of the germ layers. In this thesis, we describe a role for DRhoGEF2 in the morphogenesis of segmental grooves, which are segmentally repeated tissue infoldings that form in the epidermis during mid-embryogenesis. Groove formation is associated with up-regulation of the groove cell-markers odd-skipped, crumbs and enabled, and we demonstrate that Hh-signaling promotes their accumulation. In addition, we describe the cell shape changes that lead to the formation of segmental grooves, and take a genetic approach to characterize the role of DRhoGEF2 and the Rho1-effector Diaphanous (Dia) in this process. Both DRhoGEF2 and dia are required for groove formation. However, detailed analysis of their function suggests that DRhoGEF2 and Dia regulate different aspects of cytoskeletal reorganization. We find that Dia polymerizes Actin filaments whereas DRhoGEF2 regulates F-Actin contraction but not polymerization. In order to functionally dissect the roles of the different domains of DRhoGEF2, we have created several truncated forms of DRhoGEF2. Expression studies suggest that the PDZ, RGS and DAG domains are not essential for protein activity or constitutive membrane targeting, although the PDZ-domain may have a specific role in mediating DRhoGEF2 localization in the early embryo. Taken together our data suggest that both DRhoGEF2 and Dia are required for groove morphogenesis but that they are connected to the Actin cytoskeleton through distinct Rho1 effector pathways. We hypothesize that DRhoGEF2 provides specificity to Rho1-effector pathway activation during epithelial morphogenesis