Functional analysis of the Drosophila genome using systematic conditional RNAi-mediated gene knock-down – FunGenDroso

Objectives: An important goal in functional genomics is to systematically identify the genes underlying key biological functions at the organismal level. At present, genome-wide screens for genes regulating the development and normal physiology of multicellular organisms can only be efficiently performed in two model organisms, C. elegans et D. melanogaster. We propose here to set up a facility for the functional analysis of the Drosophila genome using high throughput conditional RNAi-mediated gene knock-down. Methodology: RNAi is a well-established method for gene inactivation in Drosophila. Combining RNAi gene inactivation with the GAL4/UAS expression system allows for RNAi to be made conditional in Drosophila. Systematic knock-down of all 14 000 predicted genes of the Drosophila genome will be achieved by screening a collection of UAS-RNAi transgenic flies that is being generated by Dr. Ryu Ueda (National Institute of Genetics, Mishima, Japon) with whom the coordinator of this application has been collaborating for the past four years. Four specific screens (see below) will be carried out by this facility during the period of funding. Each of four these screens aims at identifying novel genes in key biological pathways for which Drosophila serves as an outstanding model system. Expected results: On one hand, the results generated by the various screens performed on this facility will open new avenues of research in various domains of biological research. We expect that some of the genes identified in these screens will be of clear medical relevance. On another hand, we expect that this facility will play a very significant role in promoting scientific interactions within the French Drosophila community and will strengthen its competitiveness and leadership in Europe. Indeed, this technological facility will be open to all projects submitted by academic and non-academic teams of the French scientific community. Proposed screens: Screen 1: We will perform a pan-genomic RNAi screen for Drosophila host genes involved in the systemic (mediated by the fat body) and gut immune response to Gram-negative bacterial infection. In addition, we will screen for host factors that participate in gut tolerance to bacterial commensals. Screen 2: Non-coding micro-RNAs (miRNAs) are key regulators of gene expression in eukaryotes. Despite the importance of miRNA function, little is known about the regulation of miRNA activity. Using a fluorescent-based transgenic read-out, we will screen for genes required for miRNA biogenesis and silencing. This screen will help unraveling the complexity of the different miRNA silencing pathways in Drosophila. Screen 3: Sensory bristles have been extensively used as a model system to study how multipotential progenitor cells divide asymmetrically to generate distinct differentiated cells. We will perform a rapid and simple visual phenotypic screen in the aim of identifying new genes involved in cell polarity, asymmetric division and Notch-mediated binary fate decisions. Screen 4: The circadian clock controls rest-activity rhythms. We will screen for genes involved in the entrainment and free running of the circadian clock in Drosophila. Flies expressing UAS-RNAi in the well-characterized clock neurons will be behaviorally tested in two different environmental conditions. Constant light renders wild-type flies arrhythmic and will be used to isolate rhythmic mutants that stop the light signalization pathway to the clock (entrainment mutants). Constant darkness will be used to find mutants that do not display 24 h free running rhythms (clock mutants).