ABSTRACT
Oogenesis is a fundamental process that forms the egg and, is crucial for the transmission of genetic information to the next generation. Drosophila oogenesis has been used extensively as a genetically tractable model to study organogenesis, niche-germline stem cell communication, and more recently reproductive aging including germline stem cell (GSC) aging. Autophagy, a lysosome-mediated degradation process, is implicated in gametogenesis and aging. However, there is a lack of genetic tools to study autophagy in the context of gametogenesis and GSC aging. Here we describe the generation of three transgenic lines mcherry-Atg8a, GFP-Ref(2)P and mito-roGFP2-Orp1 that are specifically expressed in the germline compartment including GSCs during Drosophila oogenesis. These transgenes are expressed from the nanos promoter and present a better alternative to UASp mediated overexpression of transgenes. These fluorescent reporters can be used to monitor and quantify autophagy, and the production of reactive oxygen species during oogenesis. These reporters provide a valuable tool that can be utilized in designing genetic screens to identify novel regulators of autophagy and redox homeostasis during oogenesis.
ABSTRACT
Mitochondrial reactive oxygen species (mROS), a major source of ROS within cells, functions as an important signaling molecule and has the ability to damage cellular macromolecules including DNA and proteins. Monitoring mROS levels is therefore essential to understand cell-cell communication and programmed cell death in all types of cell including stem cells. Here, we describe generation and characterization of a redox sensor for mROS that is specifically expressed in the germline stem cells (GSCs) in Drosophila. This redox sensor can be used to monitor the production of mROS and mitophagy in the GSCs during oogenesis.
