A Genetic Screen Identifies New Regulators of Steroid-Triggered Programmed Cell Death in Drosophila

Abstract

The steroid hormone ecdysone triggers the rapid and massive destruction of larval tissues through transcriptional cascades that culminate in rpr and hid expression and caspase activation. Here we describe the use of genetic screens to further our understanding of this steroid-triggered programmed cell death response. Pupal lethal mutants were screened for specific defects in larval salivary gland destruction. A pilot screen using existing P-element collections resulted in the identification of mutations in known cell death regulators, E74 and hid, as well as multiple alleles in CBP (nejire) and dTrf2. A large-scale EMS mutagenesis screen on the third chromosome resulted in the recovery of 48 mutants. These include seven multiallelic complementation groups, at least five of which do not map to regions or genes previously associated with cell death. Five mutants display defects in the transcriptional induction of rpr and hid, and all display a penetrant block in caspase activation. Three were mapped to specific genes: CG5146, which encodes a protein of unknown function, Med24, which encodes a component of the RNA polymerase II mediator complex, and CG7998, which encodes a putative mitochondrial malate dehydrogenase. These genetic screens provide new directions for understanding the regulation of programmed cell death during development.

Genetic Control of Specificity to Steroid-Triggered Responses in Drosophila

“…The UAS-Atg1 K38Q stock was generated in the Nuefeld laboratory (Scott et al 2007). All PSG mutations were generated in an EMS-mutagenesis screen on the third chromosome (Wang et al 2008). The allelic combinations used here for phenotypic and molecular analysis of each PSG locus are underlined in Table 1.…”

“…The PSG mutations identified in this manner map to the following 10 genes: belle (bel), brwd3/ ram, E93 (Eip93F), headcase (hdc), mdh2, med12, med24, pak, psg2, and reptin (rept) ( Table 1). Of these, the mapping of PSG mutations in belle (Ihry et al 2012) and in med24, mdh2, and psg2 (Wang et al 2008) have been reported. The remaining 19 PSG loci complemented all publicly available lethal mutations in their respective regions and were not pursued further.…”

“…belle is a DEAD box RNA-helicase required for translation of the ecdysone-induced transcription factor E74A (Ihry et al 2012); in turn, E74A is required for the ecdysone-triggered destruction of salivary glands (Jiang et al 2000;Wang et al 2008). brwd3/ram contains a bromodomain and several WD40 repeats and likely regulates chromatin function (D'Costa et al 2006).…”

“…To dissect the genetic control of specificity to steroidtriggered responses, we previously conducted a large-scale EMS-mutagenesis screen for mutations that disrupt the destruction of larval salivary glands (Wang et al 2008). We generated .8600 lethal mutations and identified among them 37 complementation groups that had a persistent salivary gland (PSG) phenotype in otherwise normal-looking animals (Wang et al 2008).…”

“…We generated .8600 lethal mutations and identified among them 37 complementation groups that had a persistent salivary gland (PSG) phenotype in otherwise normal-looking animals (Wang et al 2008). Here, we characterize 10 complementation groups mapped to genes from this screen.…”