Small lipophilic hormones, acting through their corresponding nuclear receptors, control a wide range of developmental and physiological responses in higher organisms. Although extensive studies have focused on the mechanisms by which nuclear receptors control target gene transcription, relatively little is known about how a hormonal signal is transduced into an appropriate biological response during development. We propose to define one such pathway in detail - steroid-triggered cell death - using Drosophila as a model system. It is well known that steroids play a central role in controlling cell death in higher organisms, including humans. Only in Drosophila, however, has a genetic cascade been identified that links the hormone to a death response - the destruction of the larval salivary glands in response to the steroid hormone ecdysone during metamorphosis. We propose to build off this foundation, using an open-ended genetic screen to identify key players in this pathway. By using GFP as a marker for salivary glands in living animals, we will identify mutants that show specific defects in the steroid-triggered death response. A pilot screen has demonstrated the feasibility of this approach. We identified known genes in the death pathway as well as several new players, including genes that encode the CBP transcriptional co-factor and the TBP-related factor, TRF2. We propose to characterize these two genes in detail, defining the mechanisms that link them to cell death. We also propose to expand our search for death regulators through saturation mutagenesis of approximately 40% of the genome. Mutations will be mapped to specific genes, and functions for these genes will be assigned. This work provides a basis for understanding the molecular mechanisms of hormone signal transduction - defining the players in a genetic cascade that link the hormone to a stage- and tissue-specific biological response during development. This work also represents the first attempt to use random mutagenesis to dissect an endogenous programmed cell death response in Drosophila, raising the possibility that we will uncover novel death regulators. Finally, our studies provide a foundation for determining how steroids control cell death in humans, with implications for understanding and treating human disease.
