Figure 3. Genomic organization of Sans and characterization of Sans mutant alleles. A: Genomic organization in the region of Sans and extent of the deletions in Sans mutant alleles. The position of the EP-element GE10371 which was used to generate the Sans mutants is indicated. B: Exon-intron structure of Sans. The scale and genomic position is the same as for the extent of the deletions shown in (A). The two Sans transcripts differ by an addition of seven nucleotides to the 59 end of transcript A. C,D: Stage 10 (C) wild-type and (D) Sans245/Sans245 mutant egg chambers hybridized with a Sans-specific RNA probe. The hybridization signal is undetectable in the mutant. E: Western blot analysis of ovaries with an anti-Sans antiserum. Genotypes of the flies are indicated. The anti-Sans antiserum detects a protein of ,75 kDa (arrow), the expected molecular weight for Sans, in extracts of wild-type (lane 1) and heterozygous (lanes 3,5,7), but not homozygous (lanes Sans in Fly Development 2,4,6,8) Sans mutant ovaries. Subsequent blotting with an anti a- Tubulin antibody (lower panel) shows the loading of similar amounts of protein in each lane. Molecular weights are indicated to the left (in kDa). doi:10.1371/journal.pone.0004753.g003 Drosophila ovary is composed of chains of egg chambers proceeding through 14 stages from the germarium to the oviduct [34]. Each egg chamber consists of 16 germline cells, one oocyte and 15 nurse cells, encapsulated by a monolayer of somatic, epithelial follicle cells. Sans expression was confined to follicle cells contacting the oocyte in stage 9–10A egg chambers (Fig. 2F,H), a time when follicle cell microvilli are remodeled and elongate. Sans RNA localized to the apical side of follicle cells (Fig. 2F). No staining was detected using a sense RNA probe to Sans (Fig. 2G), indicating that the staining was specific. Sans is dispensable for fly development and fertility To functionally analyze Sans, we generated four different mutant alleles of Sans by imprecise excision of EP-element GE10371 inserted in the first intron of the Sans gene (Fig. 3A,B). All four mutations deleted parts of the predicted coding sequence for Sans, but did not obviously alter neighboring genes. These mutations, however, affect the putative gene CG30487, whose predicted coding sequence lies on the complementary DNA strand within an intron of Sans. Analysis by in situ hybridization with a Sans specific RNA probe showed that Sans transcript could no longer be detected in Sans245/Sans245 mutant egg chambers (Fig. 3C,D), suggesting that expression of Sans was impaired from this mutant allele. To test this further, we performed Western-blot analysis of control and Sans mutant ovaries using a polyclonal anti- Sans antiserum raised against the full-length protein (see Materials and Methods). A protein of approximately 75 kDa was detected in wild-type or control heterozygous mutant ovaries (Fig. 3E). Proteins of approximately 50 kDa, possibly corresponding to truncate forms of Sans, were detected in Sans38/Sans38 mutant flies, suggesting that this allele is hypomorphic (Fig. 3E). However, no Sans protein was detected in Sans63/Sans63, Sans245/Sans245, and Sans254/Sans254 homozygous mutant flies (Fig. 3E), suggesting that these alleles are amorphic. Flies homozygous mutant for any of the four Sans alleles were adult viable, male and female fertile, and had no gross morphological defects (data not shown), indicating that Sans is not essential for fly development and fertility. Sans, Cad99C, and Crinkled/Myosin VIIa localize to cone cells in the adult eye In the vertebrate eye, a number of USH1 proteins have been localized to the ciliary and periciliary regions of photoreceptor cells (reviewed in [35]). In addition, myosin VIIa is expressed in the retina pigmented epithelium [36], a multifunctional epithelium placed between the photoreceptors and the vascular meshwork of the choroid layer. To determine the localization of the three USH1 orthologs Sans, Cad99C, and Crinkled/Myosin VIIa in the Drosophila eye, we stained adult eyes with antisera specific to these proteins. The adult compound eye is a regular hexagonal array of approximately 750 ommatidia. Each ommatidium consists of 8 photoreceptors, 4 cone cells, and pigment cells (Fig. 4A). The cone cells overlie the photoreceptors and secrete lens material from their apical side. Photoreceptors display rod-like stacks of photosensitive microvilli at their apical surfaces, called rhabdo- meres. The overall morphology of rhabdomeres, as visualized by staining for F-actin, was indistinguishable between control and Sans63/Sans63 mutant flies (Fig. 4B,C). Sans immunoreactivity was strongly detected in cone cells (Fig. 4D). No immunoreactivity was PLoS ONE | www.plosone.org 4 March 2009 | Volume 4 | Issue 3 | e4753