Sans in Fly Development Figure 5. Expression and localization of Crinkled/Myosin VIIa in ovaries. A–C: Wild-type ovaries of the indicated stages hybridized with (A,B) a crinkled-specific antisense RNA probe or (C) a sense RNA probe. crinkled is expressed in follicle cells surrounding the oocyte in stage 10 egg chambers. D–G: Ovaries of (D–F) wild-type and (G) Sans245/Sans245 mutant flies stained for Crinkled/Myosin VIIa (green) and F-actin (red). In (F,G) stage 10 egg chambers are shown. H,I: High magnification views of follicle cells of stage 10 egg chambers of (H) wild-type and (I) Sans245/Sans245 mutant flies stained for Crinkled/Myosin VIIa (green), F-actin (red) and Cad99C (white). Crinkled/Myosin VIIa localizes to punctate structures at the apical side of follicle cells and, in part, to Cad99C-stained microvilli in wild-type and Sans245/Sans245 mutant flies. Scale bars: D–G: 100 mm; H,I: 20 mm. doi:10.1371/journal.pone.0004753.g005 (Fig. 7B). Sans, as well as the YFP-Sans fusion protein, were enriched beneath the apical membrane of follicle cells (Fig. 7C–E). The syntaxin Avalanche (Avl) has been previously shown to localize in follicle cells to punctate structures within the cytoplasm below the apical plasma membrane [37], similar to Sans. Avl is present in an early compartment of the endocytic pathway and is required for endocytosis of the Notch and Crumbs transmembrane proteins [37]. To test whether Sans and Avl localize to the same punctate structures, we stained follicle cells for Sans and Avl. Sans- labelled punctate structures frequently co-localized with Avl (Fig. 7E,F). Image analysis revealed that 78% (n = 319) of the Sans-labelled punctate structures were also co-labeled by the Avl antibody. We conclude that Sans, at least in part, localizes to Avl-associated early-endocytic vesicles. Sans is apparently not required for the normal morphogenesis of follicle cell microvilli Mutations in Cad99C, the ortholog of the human Usher syndrome type 1F protein protocadherin 15, result in an abnormal morphology of follicle cell microvilli [32,33]. Similar to Cad99C, Sans is expressed in stage 9–10A follicle cells, prompting us to test whether Sans is also required for normal microvilli morphogenesis. To this end, we immunostained stage 10 egg chambers of control and Sans245/Sans245 mutant flies for F-actin, which identifies microvilli. As shown in Fig. 8 (A and C), the morphology of follicle cell microvilli, as revealed by this analysis, was indistinguishable between control and Sans245/Sans245 mutant flies. Moreover, follicle cell microvilli also appeared to be normal in mutants for crinkled (ck13)/myosin VIIA (Fig. 8B). Cad99C, a marker for microvilli [32,33], appeared to localize normally on microvilli of ck13/ck13 mutant follicle cells, as analyzed by immunofluorescence (Fig. 8A,B). Cad99C also localized to microvilli in Sans245/Sans245 mutant follicle cells, even though Cad99C staining on microvilli appeared, in some instances, to be less pronounced compared to controls (Fig. 8C,D). The total amount of Cad99C in ovaries, as analyzed by Western blotting, was similar in control and Sans mutant flies (Fig. 8E). Thus, unlike Cad99C, Sans does not appear to play an essential role during microvilli morphogenesis in the follicle epithelium. PLoS ONE | www.plosone.org 6 March 2009 | Volume 4 | Issue 3 | e4753