Genetic screen and functional analysis of steroid-triggered cell death in Drosophila.

摘要

The steroid hormone ecdysone triggers the stage-specific destruction of Drosophila larval tissues through transcriptional cascades that induce two distinct forms of programmed cell death: caspase-dependent apoptosis and lysosome-mediated autophagy. In this thesis, I describe the use of genetic screens to further our understanding of the steroid-triggered programmed cell death response. Pupal lethal mutants were screened for specific defects in the destruction of larval salivary glands. A pilot screen using existing collections of P-element insertions resulted in the identification of known cell death regulators as well as mutations in novel regulators of cell death, such as dCBP and dTrf2. A large-scale EMS mutagenesis screen on the third chromosome identified 48 mutants, which include seven multiallelic complementation groups. At least five of these groups represent novel regulators of cell death, three of which were mapped to specific genes: CG5146, Med24, and CG7998. I further characterized two of these genes, Med24 and CG7998, for their roles in salivary gland cell death. I showed that mutations in CG7998 and Med24 selectively block caspase activation without affecting autophagy in persistent mutant salivary glands. CG7998 encodes a malate dehydrogenase that localizes to the mitochondria. Although mitochondrial number and morphology appear normal in CG7998 mutant salivary glands, these animals have significantly lower levels of ATP and accumulate late-stage citric acid cycle intermediates, suggesting that the cell death defects arise from a deficit in cellular energy production. Med24 encodes a component of the Mediator transcriptional coactivator complex. Unexpectedly, however, the ecdysone-induction of key death regulator genes occurs normally in Med24 mutant salivary glands. This study identifies novel mechanisms for regulating the destruction of larval tissues during Drosophila metamorphosis, and provides new directions for defining the molecular mechanisms of steroid-triggered programmed cell death during development.