The goal of this application is to study the molecular mechanisms involved in germ cell differentiation in Drosophila. The process of oogenesis in this organism begins when a germ line stem cell divides to give rise to another stem cell and a cystoblast. The cystoblast divides four times with incomplete cytokinesis producing a cyst of 16 cells connected by ring canals. One of these cells will become the oocyte and the rest will form nurse cells. Three different genes involved in different aspects of this process will be considered in this application. A second gene that will be studied in this application is stonewall (stwl), which seems to encode a nuclear protein with homology to some transcription factors that could serve as an oocyte determination and/or differentiation factor. Mutations in this gene give rise to egg chambers that contain 16 nurse cells and no oocyte. This gene is expressed only in germ cells starting at the 16-cell cyst stage, when oocyte differentiation begins. The third gene under study is orb, which encodes a cytoplasmic protein member of the RRM family of RNA- binding proteins. Orb is required for germ cell viability in the 16-cell cyst, and for proper localization of anterior/posterior and dorso/ventral determinants in the oocyte. The first part of the application deals with studies of the bag-of- marbles (bam) gene. bam encodes a protein present in the fusome, a germ cell-specific organelle that matures from a large spherical dot in stem cells to a highly branched structure that penetrates each cystocyte through the ring canals. Bam protein is found in the cytoplasm of mitotically active cystocyte and as a component of the fusome. The bam protein appears to act as a cystoblast differentiation factor, since bam mutants accumulate tumorous masses of germ cells that divide by stem cell-like divisions and do not differentiate into cystoblasts. Bab protein disappears in post-mitotic cystocytes and Dr. McKearin proposes to find out if a PEST sequence present in the bam protein is responsible for this degradation and if deletion of the PEST sequence alters cyst development. The question of whether bam plays an instructive or permissive role in cystoblast differentiation will be addressed by transforming flies with the bam gene under the control of promoters that target its expression to germ line or somatic cells. Dr. McKearin also proposes to isolate other proteins that interact with bam and are necessary for its normal function. This will be accomplished by co- immunoprecipitation experiments, by genetic screens to identify dominant suppressors of the bam phenotype, and by the use of the yeast two-hybrid system. The second part of the proposal deals with the characterization of the stonewall gene. The phenotype of mutations in this gene will be studied at the EM level. Since stwl mutants fail to differentiate an oocyte, and the future oocyte in the cystocyte forms stable synaptonemal complexes, these studies will concentrate on the examination of the synaptonemal complex in stwl germ cells. Since the stwl protein contains an HTH domain present in transcription factors such as Adf1, Dr. McKearin will explore the possibility that stwl is a DNA binding protein by gel shift experiments using bulk DNA and 35S-labeled stwl protein made in vitro. The functional significance of the HTH domain will be tested by making in vitro deletions and point mutations followed by germ line transformation. Hypomorphic alleles of stwl will be made with the ultimate aim of isolating enhancers and suppressors of the stwl phenotype as a means of identifying target genes. The last part of the proposal will rely on the enhancement of the mutant phenotype of D/V genes by mutations in orb as a means to isolate other componenets of a putative multi-protein complex, of which orb is a member, responsible for targeting RNAs involved in establishment of D/V polarity. These other genes will be identified by screening through existing collections of P insertions, by screening through collections of defined deficiencies, and by inducing new mutations with EMS.
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