Sans in Fly Development Figure 8. Follicle cell microvilli display an apparently normal morphology in Sans and ck mutant flies. A–C: Cross-sectional views of (A) wild-type, (B) ck13/ck13 mutant, and (C) Sans245/Sans245 mutant follicle cells of stage 10-egg chambers stained for F-actin (red), Cad99C (green), and a- Tubulin (blue). At this level of resolution, microvilli appear to be normal in the mutants. D: Clones of Sans245/Sans245 mutant follicle cells marked by the absence of GFP (blue) of stage 10-egg chambers stained for F-actin (red) and Cad99C (green). A cross-sectional view is shown (apical is to the top). D9 displays a heat-map of the staining shown in D. Cad99C is reduced on microvilli of Sans245/Sans245 mutant follicle cells. E: Western blot analysis of ovaries with an anti-Cad99C antiserum. Genotypes of the flies are indicated. Similar amounts of Cad99C (arrow) are detected in extracts of wild-type (lane 1), heterozygous (lanes 3,5,7), and homozygous (lanes 2,4,6,8) Sans mutant ovaries. Subsequent blotting with an anti a-Tubulin antibody (lower panel) shows the loading of similar amounts of proteins in each lane. Molecular weights are indicated to the left (in kDa). Scale bars: A–C: 10 mm; D: 20 mm. doi:10.1371/journal.pone.0004753.g008 revealed by the exclusion of the dye neutral red (data not shown). In contrast to the vertebrate hair cells, microvilli morphogenesis in the follicle epithelium appears to be more robust towards single gene perturbations. We find that, although single mutants for Sans or crinkled are adult viable [29] (this study), animals double mutant for these genes do not develop to adulthood (data not shown), underscoring the developmental role of Usher syndrome orthologs in Drosophila. Future work will be needed to analyze microvillar morphogenesis in cells double mutant for Sans and crinkled and to determine the roles of the two thus far uncharacterized Usher type 1 orthologs Cad88C and CG5921 during Drosophila development. A role for Sans in vesicle trafficking? Based on the vesicle-like localization of sans in mammalian hair cells, it has been speculated that sans might be involved in vesicle trafficking and, in particular, the trafficking of cadherin 23 or protocadherin 15 to stereocilia [12]. However, the identity of the vesicle to which sans associated had not been revealed. We now show that, in Drosophila follicle cells, Sans to a large extent co- localizes with the syntaxin Avalanche to endocytic vesicles beneath the apical plasma membrane. These results are consistent with an evolutionary conserved role of Sans in vesicle trafficking. The decreased amount of Cad99C on microvilli, which we detected with low penetrance in Sans245/Sans245 mutant follicle cells is, furthermore, suggestive of a function of Sans in the trafficking of Cad99C. Our work provides a basis for the further analysis of Usher syndrome-related genes in Drosophila. Materials and Methods Bioinformatic analysis BLAST searches [40] were performed against the non- redundant protein National Center for Biotechnology Information (NCBI) database. The Simple Modular Architecture Research Tool (SMART) [41] was used to predict the domain organization of sans-related molecules across evolution. Phylogenetic analysis was performed as previously described [42]. Accession numbers of the sequences used in this study are: Hs BAC85629, Hs CAH18298, Hs SANS (NP_775748), Mm SANS (NP_789817), Hs Harp (NP_665872), Mm Harp (NP_082361) [43], Dm AAQ21582 (DSANS), Ag XP_308746, Dr NP_001002188, Dr XP_696799. Molecular cloning To clone the CG13320/Sans coding sequence together with its 59 and 39 untranslated regions, we first isolated total RNA from third instar y w larvae using the RNeasy mini kit (Qiagen). cDNA was synthesized with the SMART RACE cDNA Amplification kit (Clontech) and used as template in a PCR reaction using the primers 59-CCGGAATTCGCTTCCGGGTAACGGCTTTCG- GAACG-39 and 59-CGGGGTACCGGAGTGATAATTGT- TAGGCTTTAATTTAG-39 (the underlined sequences are EcoRI and KpnI restriction sites used for cloning), corresponding to nucleotides 1–27 and 1833–1861 of the EST sequence LD20463. The sequencing of distinct PCR products revealed the presence of two different isoforms of CG13320 transcripts, one corresponding to the LD20463 sequence (+7nn isoform) and the other lacking 7 nucleotides in the 59 untranslated region (nucleotides 163–169 of LD20463, 27nn isoform). The translation initiation site was identified at position 249 in the sequence NM_176160 (position 216 in the sequence LD20463) on the basis of the presence of a Kozak consensus sequence (cgaatATG), partially corresponding to the consensus sequence previously reported [44]. The translation initiation site was preceded by Stop codons in all three reading frames. The CG13320 gene encodes a protein of 516 amino acids that differs at positions 405 (T405S) from the sequence deposited for CG13320 (AAQ21582), probably due to polymorphisms between the Drosophila strains used for genome sequencing and the y w strain used in this study. To generate the UAS-YFP-Sans transgene, the CG13320 coding sequence, together with the 39 untranslated region, was amplified by PCR using the primers 59-CCGGAATTCATGTCATCG- GATCGG TTTCACAAAGC-39 and 59-CGGGGTACCGGAGT- GATAATTGTTAGGCTTT AATTTAG-39 (the underlined sequences are EcoRI and KpnI restriction sites used for cloning), corresponding to nucleotides 216–241 and 1833–1861 of LD20463. The resulting PCR product was cloned in the pUAST vector [45]. Enhanced yellow fluorescent protein (EYFP) was PCR amplified from pEYFP-N1 (Clontech), using the primers 59- CCGGAATTCGTCGCCACCATGGTGAGCAA GG-39 and 59-CCGGAATTCCTTGTACAGCTCGTCCATGCC-39 (the un- derlined sequences are EcoRI restriction sites used for cloning). The resulting PCR product was cloned 59 to and in frame with the Sans coding sequence in the pUAST vector. The correct nucleotide sequence of the cloned PCR products were confirmed by sequencing prior to injection into y w embryos to obtain transgenic flies. Fly stocks Mutant alleles for CG13320 were generated by imprecise excision of EP-elements [46] using standard procedures. The starting EP-element line was GE10371 (GenExel, Inc.), in which the EP-element is oriented with the UAS sites facing away from the CG13320 coding sequence. The deficiency line used was Df(2R)CX1, wg12 b1 pr1/SM1 (Bloomington Drosophila stock center at Indiana University). The allele Sans38 contains a genomic deletion of 2135 bp, spanning from nucleotide 121224 to 123357 of the genomic sequence AE003820. The allele Sans63 contains a genomic deletion of 965 bp from nucleotide 122363 to nucleotide 123328. The allele Sans245 contains a genomic deletion of 1331 bp, spanning from nucleotide 122367 to nucleotide 123696. The allele Sans254 contains a genomic deletion of 1593 bp, spanning from nucleotide 121561 to nucleotide 123152. The four Sans alleles described here are homozygous viable, indicating that CG13320 (Sans) is not essential for viability. This conclusion is consistent with previous independently conducted genetic studies which did not uncover any vital gene between CG3845 (l(2)01424) and CG3886 (Psc) [47,48], an interval including Sans. Additional fly stocks used were FRT42D Sans245 (this study), UAS-YFP-Sans (this study), PLoS ONE | www.plosone.org 10 March 2009 | Volume 4 | Issue 3 | e4753