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Abstract

Organismal development is a complex process that involves cell growth and proliferation, specification of cell fates, and reorganization of tissues relative to one another. The generation of subcellular asymmetries is fundamental to many of these developmental events. For example, the building and contracting of actomyosin networks at precise positions within cells effects cell shape changes. Coupling these cell shape changes across a tissue drives tissue morphogenesis and reorganization of germ layers. Additionally, the polarization of cellular contents prior to cell division results in an asymmetric cell division that generates two daughter cells of distinct fates. Rho family GTPases, including Rho1/RhoA and Cdc42, play key roles in the formation of subcellular asymmetries. For example, Rho1 drives the formation of actomyosin structures that induce apical constriction and tissue invagination during ventral furrow formation in the Drosophila embryo, and Cdc42 promotes cortical polarity in Drosophila neuroblasts. However, the formation of these subcellular asymmetries is dynamic, and many questions about their formation and function remain. What protein activity is sufficient to initiate the formation of a subcellular structure? Does cell cycle progression regulate this inducer's activity? Once subcellular domains form, are they self-sustaining? Addressing these and related questions necessitates experimental approaches that afford acute control over protein activity in space and time. Toward this end, this thesis documents the development of optogenetic probes that acutely activate two Rho family GTPases, Rho1 and Cdc42, in Drosophila. I first describe the application of the Rho1 optogenetic probe to morphogenesis during Drosophila gastrulation. Using this tool, we discovered that Rho1 activity is sufficient to induce ectopic morphogenesis throughout the Drosophila embryo. However, the cell shape changes that underlie these ectopic morphogenetic events differ between cells in the dorsal and ventral epithelium. The response of ventral, but not dorsal, cells recapitulates cellular behaviors normally observed during ventral furrow formation, the first step in Drosophila gastrulation. Our work indicates that a Rho1-independent, ventral-specific activity exists and cooperates with Rho1 during ventral furrow formation. This challenges the common Rho1-centric view of this morphogenetic event. Additionally, I describe the development of a Cdc42 biosensor and a Cdc42 optogenetic probe for use in monitoring and effecting Cdc42 activity during Drosophila neuroblast polarization. The optogenetic tools developed here promise to be widely useful for furthering our understanding of the roles of Rho1 and Cdc42 in generating subcellular asymmetries during Drosophila development.

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