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.
Highlights
In multicellular organisms, the production of functional gametes depends on the activity of specialized stem cells called “germline stem cells” (GSCs) located in the gonads
Autophagy is necessary during animal development and impaired autophagy has been implicated in several diseases including cancer, neurodegenerative diseases, infectious diseases, cardiopathy and autoimmunity (Jiang and Mizushima, 2014; Schneider and Cuervo, 2014)
Genetic and pharmacological experiments in Drosophila demonstrate that reduction of autophagy activity leads to an accumulation of Ref(2)P-positive protein aggregates, suggesting that it can be used as a marker of autophagic activity (Nezis et al, 2008; Bartlett et al, 2011; Devorkin and Gorski, 2014)
Summary
The production of functional gametes depends on the activity of specialized stem cells called “germline stem cells” (GSCs) located in the gonads (reviewed in Fuller and Spradling, 2007; Dansereau and Lasko, 2008). Cellular damage caused by genotoxic agents and reactive oxygen species can damage organelles, proteins, and DNA within the stem cells. The prolonged sustenance of GSCs, require efficient homeostasis mechanisms to be operational constitutively within these cells. Such homeostasis mechanism(s) must function to significantly reduce cellular damage. Macroautophagy (autophagy) is the cellular mechanism that is involved in removing toxic protein aggregates and damaged organelles such as mitochondria from within the cytoplasm (Takeshige et al, 1992; Mizushima, 2007; Yu et al, 2018). The involvement of autophagy during gametogenesis, reproduction and germline aging is not extensively studied
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