This proposal addresses one of biology's most basic questions: how is a group of undifferentiated cells transformed into an ordered array of differentiated tissues? The focus is on the initial phase of this transformation in which a field of cells is divided into a few discrete domains. To examine this process, biochemical and genetic approaches will be combined to study the molecular interactions that control Drosophila embryogenesis. The dorsal morphogen is a promoter selective transcription factor that determines cell fate as a function of position along the dorsal/ventral axis of the Drosophila embryo. Early in development, this protein comes to be distributed in an activity gradient, with high activity on the ventral side and low activity on the dorsal side of the embryo. This graded distribution of Dorsal results in the spatially regulated expression of genes that are essential for germ layer establishment. For example, since Dorsal is an activator of the twist (twi) gene, twi is only transcribed in the ventrally- situated presumptive mesoderm where Dorsal activity is high. In contrast, Dorsal is a repressor of the decapentaplegic (dpp) and zerknllt (zen) genes, which are therefore only transcribed in the dorsally-situated presumptive ectoderm where Dorsal activity is low. The goal of this research is to determine how Dorsal interacts with other nuclear factors to activate some promoters and repress others. These factors include certain basic helix-loop- helix (BHLH) transcription factors, which interact with Dorsal to broaden and intensify domains of Dorsal-mediated activation, as well as co-repressor proteins, which assist in Dorsal-mediated transcriptional repression. By contributing to an understanding of animal development, these studies should help to elucidate the basis of human developmental disorders.
The specific aims are to 1) Analyze the synergistic interactions between Dorsal and bHLH transcription factors. In vitro transcription and transient transfection assays will be employed to examine the molecular interactions that allow Dorsal to synergize with the bHLH factors encoded by twi, daughterless, and the Achaete Scute complex. In addition, the protein domains responsible for the synergy will be mapped and analyzed to determine if they mediate direct contacts between the factors. Using information gained from this analysis, dominant negative alleles of the factors will be developed and introduced into the embryo to determine the developmental role of Dorsal/BHLH factor synergy. 2) Characterize the interactions between Dorsal and co- repressors to elucidate the mechanisms of ventral repression. First the germline transformation assay will be employed to map the domains in Dorsal that are required for repression and which may, therefore, directly contact co-repressor proteins. Second, through the use of genetic mosaics, NTF-1, a putative co-repressor, will be analyzed to determine if this factor is essential for normal dorsal/ventral pattern formation. Third, additional putative co-repressor proteins will be characterized. Fourth, the role in dorsal/ventral pattern formation of direct interactions between Dorsal and co- repressors will be assessed.
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