Characterization of regulators of blood cell number identified in a Drosophila genome-wide RNAi screen.
The control of cell number depends on the balance between pro- and anti-death signals. The Drosophila PDGF/VEGF Receptor (PVR) provides an anti-apoptotic signal that is necessary for the survival of both embryonic hemocytes and the hemocyte-like Kc cell line, thus providing a model system for uncovering other mammalian PDGF/VEGF receptors regulated signaling pathways for hematopoietic cell survival. RNAi silencing of PVR in Kc cell leads to inactivation of both Ras/Erk and Akt/Tor, and reactivation of one of these pathways or inhibiting a pro-death regulator, leads to restoration of cell numbers. To identify other pathways, which involved in hemocyte survival, a genome-wide RNAi screen to identify suppressors and enhancers of the PVR RNAi-mediated cell number reduction had been conducted previously. The screen provided the basis for this thesis project, as we subsequently characterized the biological and molecular roles of genes identified in the screen. Among the suppressors identified are many known tumor suppressors and negative regulators of the Akt/Tor and Ras/Erk pathways, and as well as heterodimeric nuclear hormone receptor- ecdysone receptor (EcR) and ultraspiracle (usp). Loss of EcR or usp suppresses cell death in a PVR-silenced background, and expression of dominant negative EcR rescues hemocyte survival in PVR null mutant embryos. Ecdysone stimulation in Kc cells triggers cell death, possibly by the induction of reaper and E93. When trying to assess the relationship of EcR/usp or ecdysone signaling with the PVR signaling pathway, biochemistry data suggest that both function in a parallel fashion. Interestingly, PVR is required for ecdysone-induced upregulation of EcR, suggesting a regulatory balance between pro-survival PVR- and pro-death ecdysone signaling that stabilizes cell lifespan. These studies suggest a novel role of ecdysone signaling in Drosophila embryonic development and blood cell survival, and emphasize parallels with the homologous vertebrate rexinoid receptor (RXR) that acts in pro-death signaling and tumor suppression. In addition, novel PVR suppressors identified in the screen, and their mammalian counterparts, may play a role in cell number control and tumor suppression.