Cell-cell interactions are important in establishing cellular diversity in the development of multicellular organisms. In Drosophila melanogaster, it has become apparent that inductive interactions between the germline-derived oocyte and the overlying somatic posterior follicle cells establish the major body axes of the egg chamber and embryo. Posterior follicle cell-to-oocyte signaling also establishes a specialized region of cytoplasm within the posterior of the oocyte that is indispensable for germ cell determination and abdominal segmentation of embryos (the germ plasm). Although these inductive interactions are critical for defining the axes and determination of the germline, thus far, few components that function within the oocyte to regulate this important signal transduction pathway have been identified. Investigating these processes will haw important implications for understanding human developmental defects and cancers. Our long-tern objectives are to elucidate the molecular mechanisms that define the major body axes and assembly of germ plasm components in Drosophila. The mago nashi (mago) gene has been demonstrated to encode a protein that localizes within the posterior pole of the oocyte to regulate the response of the oocyte to the posterior follicle cell-to-oocyte signal. Utilizing a yeast two-hybrid screen the Tsunagi protein, an RNA-binding protein, was identified as a Mago interactor. The Mago:Tsunagi complex is required to define the major body axes and is indispensable for localizing components of the germ plasm. The objectives in this proposal are as follows: (1) Germline clonal analysis, immunological techniques and cytological methods will be used to investigate whether mago nashi and tsunagi interact for the determination and/or differentiation the oocyte. (2) To identify factors that interact with Mago protein localized within the posterior pole, immunoprecipitation and biochemical methods will be employed. (3) Two approaches will enable identification and purification of RNAs bound in vivo to Mago:Tsunagi complexes. In the first approach, RNAs transcribed from a germarial and/or ovarian cDNA iterative affinity selection library will be co-immunoprecipitated with Mago:Tsunagi complexes from ovarian extracts. In the second approach, immunoprecipitations of ovarian extracts will be used to purify endogenous RNAs that form a complex with Mago and Tsunagi. ? ?
