Comparison of the translatome of follicle cells during developmental phagoptosis and starvation-induced apoptotic death of nurse cells in Drosophila melanogaster oogenesis.
The death and clearance of nurse cells is a consequential milestone in Drosophila melanogaster oogenesis. In preparation for oviposition, the germline-derived nurse cells bequeath to the developing oocyte all their cytoplasmic contents and undergo programmed cell death. The death of the nurse cells is controlled non-autonomously and is precipitated by epithelial follicle cells of somatic origin acquiring a squamous morphology and acidifying the nurse cells externally. Alternatively, stressors such as starvation can induce the death of nurse cells earlier in mid-oogenesis, manifesting apoptosis signatures, followed by their engulfment by epithelial follicle cells. To identify and contrast the molecular pathways underlying these morphologically and genetically distinct cell death paradigms, both mediated by follicle cells, we compared their genome-wide translational profiles before and after differentiating to acquire a phagocytic capability, as well as during well-fed and nutrient-deprived conditions. By coupling the GAL4-UAS system to Translating Ribosome Affinity Purification (TRAP-seq) we performed high-throughput screens to identify pathways selectively activated or repressed by follicle cells to employ nurse cell-clearance routines contextually and preferentially. Our analyses and in vivo follow-up studies identified several key genes and pathways that play vital roles during the making of a healthy egg, such as maintaining the oocyte reserve, ensuring structural integrity of the egg chamber, and regulating metabolic and signaling pathways. Overall design: To capture the translatome or the set of all mRNAs undergoing translation in follicle cells, we performed TRAP-seq by expressing GFP-tagged large ribosomal subunit 10a using the GAL4/UAS system. We then performed immunoprecipitation to capture ribosome-bound mRNAs and subsequently performed single-end RNA-sequencing of the transcripts. We used the GR1-Gal4 strain to express RpL10a-GFP in Main Body Follicle Cells (GR1>RpL10a-GFP) and PG150-Gal4 strain to express RpL10a-GFP in Stretch Follicle Cells (PG150>RpL10a-GFP). Flies were either fed or starved to obtain the profiles of follicle cells during developmental phagoptosis and starvation-induced apoptotic death of nurse cells.