Abstract
The insect steroid hormone ecdysone triggers programmed cell death of obsolete larval tissues during metamorphosis and provides a model system for understanding steroid hormone control of cell death and cell survival. Previous genome-wide expression studies of Drosophila larval salivary glands resulted in the identification of many genes associated with ecdysone-induced cell death and cell survival, but functional verification was lacking. In this study, we test functionally 460 of these genes using RNA interference in ecdysone-treated Drosophila l(2)mbn cells. Cell viability, cell morphology, cell proliferation, and apoptosis assays confirmed the effects of known genes and additionally resulted in the identification of six new pro-death related genes, including sorting nexin-like gene SH3PX1 and Sox box protein Sox14, and 18 new pro-survival genes. Identified genes were further characterized to determine their ecdysone dependency and potential function in cell death regulation. We found that the pro-survival function of five genes (Ras85D, Cp1, CG13784, CG32016, and CG33087), was dependent on ecdysone signaling. The TUNEL assay revealed an additional two genes (Kap-α3 and Smr) with an ecdysone-dependent cell survival function that was associated with reduced cell death. In vitro, Sox14 RNAi reduced the percentage of TUNEL-positive l(2)mbn cells (p<0.05) following ecdysone treatment, and Sox14 overexpression was sufficient to induce apoptosis. In vivo analyses of Sox14-RNAi animals revealed multiple phenotypes characteristic of aberrant or reduced ecdysone signaling, including defects in larval midgut and salivary gland destruction. These studies identify Sox14 as a positive regulator of ecdysone-mediated cell death and provide new insights into the molecular mechanisms underlying the ecdysone signaling network governing cell death and cell survival.
Highlights
Steroid hormones are small hydrophobic signaling molecules which bind to their receptors to control gene expression and initiate the regulation of growth, development, homeostasis and programmed cell death (PCD) [1]
Previous studies identified many genes and proteins associated with fruit fly salivary gland cell death, but functional verification was lacking
We have analyzed 460 of those genes using RNA interference (RNAi), a genetic approach to inhibit gene function, to assess their possible cell death or cell survival related function. This is the first large-scale functional screen for genes involved in steroid hormone regulated cell death and cell survival
Summary
Steroid hormones are small hydrophobic signaling molecules which bind to their receptors to control gene expression and initiate the regulation of growth, development, homeostasis and programmed cell death (PCD) [1]. Components of the steroidregulated PCD transcriptional regulatory cascades in insects and mammals have been well characterized. The transcriptional cascade induced by the steroid hormone 20hydroxyecdysone (ecdysone) has been implicated in the activation of PCD in larval intersegmental muscle [4,5,6], newly eclosed adult central nervous system [7,8], larval salivary glands [9], and larval midgut [10]. Given the functional conservation of many genes in humans and Drosophila, experiments to identify the genes required for hormonal control of Drosophila PCD will provide a better molecular understanding of the process itself, but may be valuable in the context of human disease treatment and diagnostics
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