Cellularization in Drosophila melanogaster is disrupted by the inhibition of Rho activity and the activation of Cdc42 function

Regulation of cytoskeletal dynamics is essential for cell shape change and morphogenesis. Drosophila melanogaster embryos offer a well-defined system for observing alterations in the cytoskeleton during the process of cellula rization, a specialized form of cytokinesis. During cellularization, the ac tomyosin cytoskeleton forms a hexagonal array and drives invagination of th e plasma membrane between the nuclei located at the cortex of the syncytial blastoderm. Rho, Rac, and Cdc42 proteins are members of the Rho subfamily of Ras-related G proteins that are involved in the formation and maintenanc e of the actin cytoskeleton throughout phylogeny and in D. melanogaster. To investigate how Rho subfamily activity affects the cytoskeleton during cel lularization stages, embryos were microinjected with C3 exoenzyme from Clos tridium botulinum or with wild-type, constitutively active, or dominant neg ative versions of Rho, Rac, and Cdc42 proteins. C3 exoenzyme ADP-ribosylate s and inactivates Rho with high specificity, whereas constitutively active dominant mutations remain in the activated GTP-bound state to activate down stream effecters. Dominant negative mutations likely inhibit endogenous sma ll G protein activity by sequestering exchange factors. Of the 10 agents mi croinjected, C3 exoenzyme, constitutively active Cdc42, and dominant negati ve Rho have a specific and indistinguishable effect: the actomyosin cytoske leton is disrupted, cellularization halts, and embryogenesis arrests. Time- lapse video records of DIC imaged embryos show that nuclei in injected regi ons move away from the cortex of the embryo, thereby phenocopying injection s of cytochalasin or antimyosin. Rhodamine phalloidin staining reveals that the actin-based hexagonal array normally seen during cellularization is di srupted in a dose-dependent fashion. Additionally, DNA stain reveals that n uclei in the microinjected embryos aggregate in regions that correspond to actin disruption. These embryos halt in cellularization and do not proceed to gastrulation. We conclude that Rho activity and Cdc42 regulation are req uired for cytoskeletal function in actomyosin-driven furrow canal formation and nuclear positioning. (C) 1998 Academic Press.