| Drosophila ventral furrow formation has frequently been used as a model to study developmentally-regulated cell-shape changes. However, a technique to follow all cellular changes during this process within a single living embryo has been lacking. We describe a novel technique, called "end-on imaging", to collect time-lapse images of transversely-mounted living embryos. End-on imaging revealed several new features of dorsoventral development. First, we observed a wave of syncytial nuclear divisions predicting the location of the ventral furrow. Second, we determined that there is a five-minute gap between the end of cellularization and the start of ventral furrow formation, suggesting that the two processes may share the same pool of cytoskeletal components. Lastly, we show that apical-membrane flattening, the first step in ventral furrow formation, is due to the ventral cells pushing against the vitelline membrane, rather than flattening the dome-shaped, apical surfaces of these cells by a pulling or constriction motion. To determine the role of myosin II during the cell shape changes, a Sqh-KillerRed construct was generated and tested in embryos in a null background. KillerRed did not produce noticeable phototoxic effect in early Drosophila embryos when strongly excited during cellularization, ventral furrow formation or cytokinesis. Therefore, to study the role of myosin II during the cell-shape changes of VFF, timed injections of Y-27632 a Rho kinase inhibitor that blocks myosin II activation, were performed. Embryos were injected during cellularization, after cellularization but before apicobasal nuclear migration, after cellularization but before or during early apical constrictions, during late apical constrictions, or after the initial infolding of the furrow. It appears that myosin II is not required for nuclear migration, early constriction events or after the infolding of the furrow, but is required for the late apical constriction events, suggesting a "critical period" for myosin II requirement during the transition from positive to negative curvature. |
