Surface behaviors in development: pattern formation in the cell cortex and nuclear envelope

Abstract

The shaping of biological systems into the forms required for their function requires a complex interplay of force generation and response, genetics, biochemical signaling, and geometrical cues. Morphogenetic processes unfold across wide-ranging spatial and temporal scales, further requiring tightly regulated feedback mechanisms connecting diverse components at different organizational levels. This dissertation addresses some mechanisms through which biological surfaces generate and respond to forces during development of the fruit fly Drosophila melanogaster, as well as the role of regulatory proteins and feedback loops in one of the main force-generating signaling pathways in the actomyosin cortex. First, we characterized the shape of nuclear envelopes in female germline cells in the Drosophila melanogaster egg chamber, quantifying the appearance and progressive amplitude increase of fluc- tuating wrinkles in the envelope. We showed these amplitudes scale in a manner consistent with pre- dictions from a model of a thin elastic shell subject to fluctuating loads. We further showed wrinkle amplitude is reduced upon reduction in cytoplasmic fluctuations following microtubule disruption, and we found a decrease in Lamin C concentration in the nuclear envelope during development that accompanies the increase in wrinkle amplitude. Next, we focused on the dynamics of intercellular transport of contents from support germline cells to the oocyte in a process known as ‘nurse cell dumping’ that occurs during late oogenesis. We found that this large-scale fluid transport process, required to provide the oocyte with sufficient nu- trients to support early embryonic development, unfolds in two phases, with changes in actomyosin behavior only required for the latter phase. The first phase instead relies on a cell-size gradient in the support cells that sets up a pressure gradient to drive fluid between cells into the oocyte in a re- producible order. The order and timescale of transport match predictions from a model based on a modified form of the Young-Laplace law applied to the sixteen-cell network. The second phase of transport requires traveling waves of actomyosin contractility, with directionality and completeness of transport reduced upon perturbation of wave behavior induced by knockdown of RhoGAP15B, a negative regulator of the small GTPase RhoA. Finally, we investigated the connection between availability of RhoA regulators and the down- stream patterns of cortical actomyosin contractility in space and time. Building on results from the second phase of nurse cell dumping, we identified a RhoGEF, Pbl/Ect2, required for proper regu- lation of actomyosin waves and complete nurse cell dumping. We showed that, prior to wave onset, Ect2 and RhoGAP15B exit the nucleus and accumulate at the nurse cell cortex in an order match- ing the order of wave onset, suggesting simultaneous increase of both RhoGEF and RhoGAP might induce waves. We tested this hypothesis in the early Drosophila embryo, showing that overexpres- sion of a different RhoGEF/RhoGAP pair converts actomyosin from the pulsatile behavior that usually occurs in this system to traveling waves. This result further suggests increased RhoGEF and RhoGAP together induce wave onset in the actomyosin cortex, a finding consistent with recent work in the oocytes of starfish and Xenopus. Taken together, our results from the first chapter shed light on how properties of the nuclear membrane shape its response to external forces during development. The second and third chapters provide insight into how feedback loops in the RhoA system affect patterns of force production in individual cell cortices that must be coupled to produce tissue-level shape changes. Additionally, the results presented here provide further examples of how developmental processes depend on the interplay of biological and physical mechanisms.