Embryonic cycle 14 in Drosophila is characterized by morphological changes that transform the syncytial embryo into a cellular blastoderm, and then into a gastrula with defined cell types organized in a three dimensional pattern. Our work is focused on the cellular mechanisms that underlie these transitions and the genes that control them. We propose to investigate the mechanisms that subdivide the syncytial embryo into a cellular blastoderm and confer on those cells the properties of a polarized epithelium. We will use immunostaining and live imaging to investigate the dynamic organization of the actin cytoskeleton and its relationship to junctional stability and membrane insertion. We will analyze junctional morphology in mosaic embryos that ectopically express Nullo at the cellular blastoderm stage and characterize the effects of both loss of Nullo and its prolonged expression on membrane insertion and cell polarity. The second part of our proposed experiments investigates the changes in cell shape that occur in the ventral furrow during the initial stages of gastrulation. We will use two-photon microscopy to visualize the changing distribution of actin and myosin and correlate these changes with the extent of apical constriction in individual cells. We will use fluorescent recovery after photobleaching (FRAP) to measure the dynamic turnover of actin and myosin in the ventral furrow and compare the behavior of mutant and wild type embryos. We will analyze the role of membrane targeting in the ventral furrow, focusing on the apical accumulation of Folded Gastrulation secretory vesicles, as well as mesoderm specific accumulation patterns of Notch and Slam proteins. We will also extend our analysis of morphological change at embryonic cycle 14 to a more global characterization of gene activity at that stage. These experiments combine our previous genetic strategies to generate embryos that lack specific chromosomes and chromosomal regions with microarrays to distinguish maternal and zygotic transcripts in cellularization. We will identify RNAs whose presence requires zygotic transcription and investigate the mechanisms that control their early expression. We will also define regions of the genome required for cycle 14 degradation of specific maternal RNA. We will relate the observed changes in RNA to the nuclear/cytoplasmic ratio that controls cell cycle at the Drosophila midblastula transition, and to the morphological changes that occur at that time. ? ? ?
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