Utilizing the FLP-Out System for Clonal RNAi Analysis in the Adult Drosophila Ovary.

The ability to conduct spatially controlled RNA interference (RNAi) for gene knockdown using the UAS/Gal4 system is among the most appealing techniques available for analysis of gene function in the Drosophila ovary. While gene knockdown experiments in somatic cells in the developing organism (i.e., embryos and larvae) are effectively and commonly performed, the use of RNAi in adult ovarian cells can be hampered by the unintended deleterious effects of Gal4 expression in "off-target" developing tissues. Mosaic analysis overcomes these problems by imparting temporal and spatial control over gene manipulation, providing a useful tool to compare manipulated cells with wild-type cells in the same tissue. Here, we provide a method to utilize the UAS/Gal4 system in combination with the Flippase (FLP)-Flippase Recognition Target (FRT) system to generate positively labeled "FLP-Out" clones expressing a chosen RNAi in both the germline and the soma in the Drosophila ovary. This protocol outlines each step of the generation of clones and the selection of appropriate fly stocks and reagents, providing a guide to this powerful tool in the Drosophila genetic toolbox. These techniques allow for RNAi analysis within a specific cell type, providing an opportunity to study a variety of unique aspects of cell function that would not be possible in more traditional RNAi-based experiments.

ß-importin Tnpo-SR promotes germline stem cell maintenance and oocyte differentiation in female Drosophila.

Germ cell development requires interplay between factors that balance cell fate and division. Early in their development, germ cells in many organisms divide mitotically with incomplete cytokinesis. Key regulatory events then lead to the specification of mature gametes, marked by the switch to a meiotic cell cycle program. Though the regulation of germ cell proliferation and meiosis are well understood, how these events are coordinated during development remains incompletely described. Originally characterized in their role as nucleo-cytoplasmic shuttling proteins, ß-importins exhibit diverse functions during male and female gametogenesis. Here, we describe novel, distinct roles for the ß-importin, Transportin-Serine/Arginine rich (Tnpo-SR), as a regulator of the mitosis to meiosis transition in the Drosophila ovary. We find that Tnpo-SR is necessary for germline stem cell (GSC) establishment and self-renewal, likely by controlling the response of GSCs to bone morphogenetic proteins. Depletion of Tnpo-SR results in germ cell counting defects and loss of oocyte identity. We show that in the absence of Tnpo-SR, proteins typically suppressed in germ cells when they exit mitosis fail to be down-regulated, and oocyte-specific factors fail to accumulate. Together, these findings provide new insight into the balance between germ cell division and differentiation and identify novel roles for ß-importins in germ cell development.