revealed that four of the five USH1 proteins are present on stereocilia tips: myosin VIIa, cadherin 23, protocadherin 15, and harmonin [13,17,18]. The in vitro binding and in situ co- localization of these four USH1 proteins suggest that they form functional complexes. Cadherin 23 and protocadherin 15, furthermore, have been recently shown to be part of the tip links that connect stereocilia, thereby providing cohesion between adjacent stereocilia [19,20]. Sans, in contrast to the other identified USH1 proteins, localizes to vesicular structures beneath the apical plasma membrane of hair cells [12] and has not been reported to reside on stereocilia. Based on its localization and physical interaction with myosin VIIa and harmonin, it has been proposed that sans may control the trafficking of USH1 proteins to stereocilia [12]. Animal models have been important in further understanding the etiology of Usher syndrome. Mice mutant for myosin VIIa (shaker-1), harmonin (deaf circler), cadherin 23 (waltzer), protocadherin 15 (Ames waltzer), or sans (Jackson shaker) display severe hearing impairment, vestibular dysfunction, and disorganized stereocilia in the inner ear [7,18,21,22,23,24,25]. Localization studies in mutant mice have shown that both myosin VIIa and sans are required to direct harmonin to the stereocilia tips [18]. Mutations in myosin VIIa, cadherin 23, and protocadherin 15a are also associated with disorganized hair bundles in zebrafish [26,27,28]. In Drosophila melanogaster, orthologs of USH1 genes have been identified. Mutations in crinkled, the fly ortholog of myosin VIIa [29], result in disorganization of Johnston’s organ, the flies’ auditory system, and in deafness [30,31]. Moreover, Cad99C, the ortholog of protocadherin 15, localizes to microvilli of the fly follicular epithelium, and mutations in this cadherin result in the misorientation of microvilli [32,33]. These findings indicate an evolutionary conserved function for this class of cadherins in organizing actin-containing cellular protrusions. Here, in order to further understand the role of sans during development and disease, we identified and characterized a Drosophila melanogaster ortholog of this gene. We show that Drosophila Sans is expressed in tubular organs in the embryo, lens-secreting cone cells in the adult eye, and microvilli-displaying follicle cells Sans in Fly Development during oogenesis. Similar to its vertebrate ortholog, Sans protein localizes to punctate structures we identified as early endosomes associated with the syntaxin Avalanche. Results The Drosophila genome encodes an ortholog of the human Usher syndrome type 1G protein sans To identify the Drosophila ortholog of the human sans protein, we used the human sans protein sequence in a BLASTp search against the Drosophila melanogaster genome. The product encoded by gene CG13320 was most similar to human sans. Back-Blast using CG13320 protein as a query sequence retrieved sans as the most related protein in vertebrates. Overall, human sans and Drosophila CG13320 are 32% identical (168/524 identical amino acid residues). A higher sequence identity was detected in the N- terminus (53%; 15/28), the ankyrin repeats (60%, 53/88), and the SAM domain (39% (26/66). Human sans and Drosophila CG13320 share a conserved C-terminal PDZ-domain binding site (data not shown) and display a similar domain organization including three ankyrin repeats and one SAM domain (Fig. 1). Moreover, in a phylogenetic tree, CG13320 clustered in the same clade as vertebrate sans and harp (Fig. 1). Harp is an ankyrin and SAM- domain containing protein related to sans. We conclude that CG13320 is the Drosophila ortholog of human sans and refer to it as Sans (see also FlyBase at www.flybase.org). Sans is expressed in tubular organs of the embryo and in ovarian follicle cells To further characterize Drosophila Sans, we analyzed its expression pattern in embryonic, larval, and adult tissues by RNA in situ hybridization using a Sans-specific RNA probe. In the embryo, Sans mRNA was first detectable at stage 11 in the hindgut (Fig. 2A). In later stages, Sans mRNA was detected in the foregut, the hindgut, the tracheal placodes, the tracheal primary branches, and the six mechanosensory Keilin’s organs (Fig. 2B–E9). During larval stages, Sans was expressed ubiquitously in eye-antennal, wing, leg, and haltere imaginal discs (data not shown). The adult Figure 1. Identification of a Drosophila melanogaster ortholog of human Usher syndrome type 1G protein sans. Phylogenetic tree. Drosophila melanogaster Sans is closely related to vertebrate sans/Harp proteins. Harp/sans proteins contain three ankyrin repeats (blue rectangles) and one SAM domain (green triangle). Hs: Homo sapiens; Ag: Anopheles gambiae; Dm Drosophila melanogaster; Dr: Danio rerio; Mm: Mus musculus. doi:10.1371/journal.pone.0004753.g001 PLoS ONE | www.plosone.org 2 March 2009 | Volume 4 | Issue 3 | e4753