Adapting the auxin-inducible degron system to investigate the roles of germ granule proteins, Aubergine and Tudor, after primordial germ cell formation in Drosophila

Abstract

Across the animal kingdom, germ cells contain ribonucleoprotein (RNP) condensates necessary for proper germline development called germ granules. In some organisms like the Drosophila melanogaster, germ granules form as early as oogenesis, and in other organisms, like the mouse, they form much later, after primordial germ cell (PGC) specification. Despite this difference in developmental timeline, germ granules across species share many components, like a Tudor domain scaffolding protein and piRNA binding proteins like Aubergine. Although hypothesized to be hubs of post-transcriptional regulation, the role of germ granules in PGCs, especially after their specification, remains largely unknown. Investigating the role of conserved proteins, like Tudor and Aubergine, after PGC specification in Drosophila may provide further insight into the more conserved functions germ granules have across species. Here, I show that Tudor and Aubergine persist in Drosophila germ granules until PGCs have reached the gonads, indicating they may be needed after PGC specification. I then lay the groundwork for investigating the roles of Tudor and Aubergine in pole cells and germ granules after PGC specification. Using CRISPR Cas9 Scarless editing methods and classical Drosophila genetics, I generate the necessary CRISPR lines needed to deplete Tudor and Aubergine in the PGCs after their specification using an auxin inducible degradation system. These Drosophila lines can be used for future experimentation to help determine the roles of Aubergine and Tudor past PGC specification, potentially pointing to conserved roles they play in the germ granules of other organisms.