Uncovering novel targets of Escargot-inhibited cell death in the Drosophila ovary by RNA-seq. Victoria Kathryn Jenkins, Kim McCall. Department of Biology, Boston University, Boston, MA.

   Cell death, required for proper homeostasis and development, is controlled by the interactions of a wide variety of pro- and anti-death genes. The Drosophila ovary is the site of two cell death events which illustrate aspects of apoptotic, autophagic, and potentially other non-canonical types of cell death. Starving the fly of protein can induce the death of entire pre-vitellogenic mid-stage egg chambers. All egg chambers, as they approach maturation, undergo the developmentally required late-stage death of non-oocyte germline cells (nurse cells). Overexpression of the anti-apoptotic protein DIAP-1 or the transcription factor Escargot causes a cytologically identical undead phenotype in starved fly ovaries, causing nurse cells to remain unkilled in both mid-stage and late-stage death events. DIAP-1 has long been known to block caspase (apoptotic protease) activation, but an anti-death function of Escargot (or another member of the Snail family of transcription factors) has not yet been described in Drosophila. It is not known how overexpression of escargot, normally required for gastrulation, cell cycle regulation, and the epithelial-to-mesenchymal transition, can mimic overexpression of diap-1. In order to identify targets of Escargot that may regulate cell death in the ovary, we have performed RNA-seq on ovaries of well-fed versus protein-starved flies overexpressing escargot or diap-1, both driven by the germline NGT;nanos-Gal4 promoter, compared to controls. Ten females of each genotype were provided either yeast paste or apple juice agar for 48 hours prior to sacrifice at 6 days post eclosion. Ovary mRNA was extracted and sequenced as 50 bp single reads by an Illumina HiSeq 2000, yielding 29.0 +/- 4.7 million reads per group. The resulting transcriptomes were compared to find differences in gene expression, splice isoform usage, and other mechanisms that Escargot may use to influence both known and potentially novel cell death genes, as well as to uncover the signaling events resulting in an undead phenotype similar to that seen in flies overexpressing DIAP-1.