This proposal focuses on the study of the molecular mechanisms underlaying the establishement of anterior-posterior polarity in the Drosophila embryo and the role of cell-cell communication between posterior follicle cells and the oocyte in this process. Preliminary work has shown that loss of Notch function causes hyperplasia of the follicle cells at the posterior end of the oocyte and relocalization of morphogenetic determinants along the A-P axis of the oocyte. Constitutive expression of a gain of function allele of Notch caused the oposite effect, with loss of polar cells and hyperplasia of stlk cells. These results suggest that Notch functions in cell fate determination of posterior follicle cells, and that subsequent signaling from these cells is required for the establishement of the A-P axis in the oocyte. In the first part of this application, Dr. Ruohola-Baker proposes to extend her previous work on the effect of Notch gain of function mutant on follicle cell fate. This cell fate change will be analyzed using cell specific markers from enhancer trap lines that show lacZ expression in specific groups of follicle cells. . Other markers specific for polar cells such as Fasciclin III and the neuralized gene will also be used. Activated Notch will be expressed using the GAL4 system and heat shock induced GAL4; in addition, enhancer traps that express GAL4 in specific follicle cells will also by utilized. To test the hypothesis that posterior follicle cells send a signal to the oocyte that determines its A-P polarity, the effect of polar cell depletion on the establishement of this axis will be analyzed using two alternative approaches. The first one will take advantage of the gain of function Notch phenotype that results in loss of polar cells and will make use of several markers such as bicoid and oskar mRNAs, staufen protein and a kinesin-beta gal fusion ptotein. . The second approach will use laser cell ablation to destroy the posterior polar cells. Dr. Ruohola- Baker will also analyze the effect of Notch mutations on the microtubule cytoskeleton of the oocyte using confocal microscopy. The second part of the application describes experiments aimed at studying the role of other neurogenic genes in oogenesis and characterizing new genes with a Notch-like phenotype. Preliminary results indicate that N, bib and neu are required for germ line cell division. Mosaic analysis will be used to determine whether they are required in the germ line or follicle cells. Dr. Ruohola-Baker has already identified three new mutations, named 11A4, 2A4, and 27B13, that show a phenotype similar to N during oogenesis. The 11A4 mutation is particularly interesting: it shows no deffects in the posterior follicle cells but it causes miss-localization of bicoid and kinesi- betagal, suggesting that it acts in follicle cell-oocyte signaling or inside the oocyte. The gene has been cloned and preliminary sequence data shows homology to exuperantia. Further genetic and molecular characterization of these mutatns will be carried out. This will include in situ hybridization to determine the cells that express these genes; antibody labeling to determine subcellular location of the protein; mosaic studies to define cells that require protein function; and ectopic expression using the GAL4 system . The last part of the proposal describes the isolation of new mutants involved in follicle cell determination and follicle cell-oocyte signaling by identifying suppressors of the 11A4 phenotype and examining enhancer trap lines with interesting patterns of expression.
