A Fluorescence-Based Genetic Screen to Study Retinal Degeneration in Drosophila

December A Fluorescence-Based Genetic Screen to Study Retinal Degeneration in Drosophila Yu Huang 0 Jun Xie 0 Tao Wang 0 Bertrand Mollereau, Ecole Normale Supérieure, FRANCE 0 1 National Institute of Biological Sciences , Beijing , China , 2 College of Biological Sciences, China Agricultural University , Beijing , China The Drosophila visual system has been proved to be a powerful genetic model to study eye disease such as retinal degeneration. Here, we describe a genetic method termed “Rh1:: GFP ey-flp/hid” that is based on the fluorescence of GFP-tagged major rhodopsin Rh1 in the eyes of living flies and can be used to monitor the integrity of photoreceptor cells. Through combination of this method and ERG recording, we examined a collection of 667 mutants and identified 18 genes that are required for photoreceptor cell maintenance, photoresponse, and rhodopsin synthesis. Our findings demonstrate that this “Rh1::GFP eyflp/hid” method enables high-throughput F1 genetic screens to rapidly and precisely identify mutations of retinal degeneration. - OPEN ACCESS Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a ‘973’ grant (2014CB849700) to T.W. from the Chinese Ministry of Science. T.W. contributed to the study design, analysis, dicision to publish and preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. Introduction The Drosophila visual system has been shown to be proven to be a powerful genetic model for dissecting the molecular mechanisms underlying retinal degeneration and G-protein-coupled signaling cascades. Mutations in most genes that functions in phototransduction result in light-dependent photoreceptor cell death. Therefore, genetic screens in Drosophila could isolate mutations of many genes involved in retinal degeneration and could deepen our understanding of their counterpart genes in human diseases. Drosophila phototransduction offers the opportunity to combine classical and modern genetic approaches to identify genes and proteins that function in phototransduction and/or that are required for photoreceptor cell survival [ 1–4 ]. Electroretinogram recordings (ERGs) are among the analysis tools that have driven the progress of Drosophila phototransduction research; this technique is simple enough to be used to perform genetic screens [ 3 ]. However, due to the requirement of fixing animals, flies can not survive after ERG assay, which makes it less suitable for mutagenesis F1 screens. In addition, most retinal degeneration mutations in Drosophila were originally identified from photoresponse-based screens, which do not fully represent the complexity of retinal degeneration diseases in human. Moreover, classic screens of adult animals for aberrant phototransduction and eye morphology often cannot isolate essential genes involved in these pathways since such genes are often indispensable for organism viability. A few mosaic methods have been developed that make an entire eye homozygous for a mutation [ 5, 6 ]. Large scale screens for neurotransmission and phototransduction mutants have been conducted based on these methods [ 7, 8 ]. However, phototaxis in the F1 generation is not sensitive enough, and the ERG-based high throughput screening is time-consuming [ 8–10 ]. Given these nontrivial limitations, we were motivated to develop a fluorescence-based “Rh1::GFP ey-flp/hid” method for F1 screening of retinal degeneration mutants. This method is based on a modified EGUF/hid technique to generate eyes of homozygous mutations and uses GFP-tagged Rh1 (major rhodopsin) as a marker for photoreceptor cell integrity. Using this Rh1::GFP ey-flp/hid method, we screened the UCLA URCFG P-element recessive lethal collection, and identified several types of mutations affecting photoreceptor cell survival, phototransduction, and rhodopsin homeostasis. Results Development of the Rh1::GFP ey-flp/hid screening method To monitor the integrity of photoreceptor cells in live animals, we generated Rh1::GFP transgenic flies, which express a GFP-tagged major rhodopsin Rh1 protein in R1-6 photoreceptor cells under the control of the ninaE (rh1) promotor. Under a fluorescence microscope, regular Rh1::GFP compound eyes showed an intensely green fluorescing deep pseudopupil. This fluorescence signal was markedly reduced in flies raised on vitamin A-free food as well as in ninaA1 mutant flies with disrupted Rh1 biosynthesis. It was also reduced in the rdgABS12 mutant background which caused a rapid retinal degeneration (Fig 1A–1E). Since the fluorescing Rh1-GFP pseudopupil can be observed in living flies and because it represents the Rh1 levels and/or rhabdomere structures, it is ideally suited for a use in a high throughput genetic screen. Photoreceptor cell integrity, indicated by the number of Rh1 GFP-tagged rhabdomeres, was further visualized at (...truncated)