Sans in Fly Development Figure 6. Localization of Sans protein in ovaries. A–E: Ovaries of (A,B,D) wild-type and (C,E) Sans245/Sans245 mutant flies stained for Sans (green), a-Tubulin (blue), and F-actin (red). (A–C) show the germarium and (D,E) stage 10 egg chambers. In (D9,E9) only the Sans staining is shown. Sans is expressed in what appears to be the germline stem cells of the germarium (A,B) and in follicle cells contacting the oocyte (D). Sans staining is also detected at the cortex of the oocyte. Anterior is to the left. Scale bars: A: 50 mm; B,C: 10 mm; D,E: 100 mm. doi:10.1371/journal.pone.0004753.g006 Discussion In this study, we have identified and characterized a Drosophila ortholog of the Usher syndrome protein sans. Drosophila Sans is expressed in tubular organs of the embryo and in cone cells in the adult eye. In the ovary, Sans is expressed in germline stem cells and in follicle cells during stages 9–10A. In follicle cells, Sans colocalizes with Avalanche, a syntaxin associated with early endosomes. Orthologs of USH1 genes in Drosophila The Drosophila melanogaster genome encodes genes orthologous to the five known USH1 genes: crinkled/myosin VIIa (USH1B) [29], CG5921 (USH1C) (data not shown), Cad88C (USH1D) [38], Cad99C (USH1F) [32,33], and Sans (USH1G) (this study), providing an opportunity to study the cellular and developmental roles of these genes in this genetically tractable model organism. The analysis of crinkled has revealed a striking parallel in the role of this gene and USH1B for auditory perception. crinkled mutants fail to properly develop the flies auditory organs and they are deaf [30,31]. Measurements of auditory mechanics and nerve responses indicate that Sans mutants do not have auditory defects (Martin Go¨ pfert, personal communication). A further cell type in which orthologs of USH1 genes have been analyzed in Drosophila is the follicle epithelium [32,33]. Like other epithelia, the follicle cells display microvilli at their apical surface. Follicle cell microvilli are dynamic in that they extend in length during stages 9–10A of oogenesis and that they disappear during the later stages. Interestingly, all five Usher syndrome type 1 orthologs, Cad99C, Cad88C, CG5921, crinkled, and Sans, are expressed in follicle cells when they display long microvilli [32,33,39] (this study). At least three Usher syndrome type 1 proteins share similar subcellular localizations in Drosophila follicle cells and vertebrate hair cells. Cad99C/protocadherin 15 and Crinkled/myosin VIIa localize to microvilli/stereocilia. Sans localizes to vesicles beneath the apical surface of cells. Moreover, in the adult Drosophila eye, Sans, Cad99C, and Crinkled/Myosin VIIa localize to a single cell type, the lens-secreting cone cell. Based on their localization, we speculate that Sans, Cad99C, and Crinkled/Myosin VIIa might be part of a protein network, similar to what has been proposed for their vertebrate orthologs [12,16]. Sans and microvilli morphogenesis Mutations in Cad99C result in misorientation of follicle cell microvilli [32,33]. Normal microvilli orientation appears physio- logically important, since misorientation of microvilli in Cad99C mutants correlates with an impairment of eggshell deposition necessary to protect the egg and developing embryo from its environment [32,33]. In contrast to Cad99C mutants, follicle cell microvilli appear to be normal in both Sans and crinkled mutants, although we cannot exclude subtle defects. Sans mutant flies also deposit eggs surrounded by an apparently integer eggshell, as PLoS ONE | www.plosone.org 7 March 2009 | Volume 4 | Issue 3 | e4753