The proposed research is the continuation of a study which started eight years ago and in general is concerned with the mechanisms underlying gene expression and pattern formation in development. More specifically our studies aim at the elucidation of the genetic circuitry which controls the first steps in the differentiation of the nervous system in Drosophila. Our studies with two phenotypically interacting genetic units involved in this circuitry, Notch and E (spl) led to the discovery that Notch encodes for a transmembrane protein with homology to the mammalian epidermal growth factor and that E (spl) codes for a protein which is homologous to mammalian a proteins. These findings led to the suggestion, that these genes are part of a cell interaction mechanism. We hypothesize further that this mechanism is responsible for the fine tuning of the differentiation pattern in certain tissues, among them the embryonic nervous system. We request funds to pursue these studies. The proposed experiments aim at examining the complexity of the genetic circuitry interacting with Notch and E (spl), analyze the molecular nature of phenotypic interaction, study the biochemical nature of the E (spl) gene product and attempt to gain insights into the function of Notch by examining its mammalian counterparts. In particular, we propose to carry out extensive genetic screens to isolate mutants that interact with specific, molecularly characterized, mutations in the Notch locus. We will use as our genetic markers certain Abruptex mutants which are caused by point mutations in the extracellular part of the Notch protein and notched, a point mutation mapping close to the c terminal intracellular part of the protein. Alleles identified via these screens will be analyzed genetically and molecularly. The beta transducing homologous E (spl) product will be studied in depth: We will produce specific polyclonal and monoclonal antibodies against that gene product and attempt to isolate and characterize it. We will analyze epigenetic relationships between the neurogenic loci by in situ and immunocytochemical analysis and biochemically try to identify protein components that may interact with Notch and E (spl). To that end both biochemical methods as well as anti autotype antibodies will be explored. The genetic circuitry controlling Notch will also be addressed by analyzing trans acting Notch transcription factors by in vitro transcription, footprint analysis and in vivo assays. We will finally proceed with the characterization of Notch homologous sequences in the mouse.
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