View Program - 63rd Annual Drosophila Research Conference

The death and clearance of nurse cells marks an important developmental milestone in Drosophila melanogaster oogenesis. A subset of the Main Body Follicle Cells (MBFCs) that encapsulate the oocyte and the nurse cells within the egg chamber undergo marked cytoskeletal remodeling to become Stretch Follicle Cells (SFCs). Towards the final stages of oogenesis, the nurse cells dump their contents into the oocyte and are subsequently induced to die by the phagocytosis machinery of SFCs. This non-autonomous, phagocyte-dependent form of cell death is accomplished by the SFCs migrating into the spaces between the nurse cells and acidifying them externally by employing proton-pumping Vacuolar-ATPases. In this study, we characterize the co-transcriptional events that precipitate the transition of columnar, non-phagocytic MBFCs into squamous, phagocytic SFCs, including those that orchestrate the engulfment and processing of nurse cells. We focus on differential expression at the transcript level arising from alternative splicing, which significantly amplifies the transcriptomic repertoire, and thereby the proteomic diversity of tissues.
To capture the incidence and diversity of splicing events between MBFCs and SFCs at the genome-level, we sequenced the mRNAs undergoing translation by immunoprecipitating GFP-tagged ribosomes using Translating Ribosome Affinity Purification (TRAP-seq). We analyzed the short-read RNA-seq data using HITindex to identify terminal exons that could act as hybrid internal exons, and LeafCutter to identify intron excision events and novel splice sites.
We observed that hundreds of terminal exons in MBFCs were classified as hybrid internal exons in SFCs. Gene ontology analysis revealed that a plurality of genes with hybrid first exons had important physiological functions such as cytoskeletal, trans-membrane, or secreted proteins and were involved in major signaling pathways, including the immune-related Imd and TNF-alpha pathways. Specifically, we discovered that the first exons of Vacuolar-ATPases subunit Vha100-2 and kinase Src42A in MBFCs were used as internal exons in SFCs. Additionally, we found statistically significant exon skipping events in the calcium-binding protein Mlc1 and in Sec23, which is involved in vesicle trafficking in autophagy.
In summary, we have identified novel regulatory events orchestrating nurse cell clearance that are not captured at the gene-level, indicating that co-transcriptional regulation of gene expression could play a more prominent role in cell death and clearance than was previously appreciated.

Characterizing the landscape of alternative splicing events regulating the clearance of nurse cells by non-professional phagocytes in Drosophila melanogaster oogenesis