detected in the adult eyes of Sans63/Sans63 mutant flies (Fig. 4E), indicating that the observed staining was specific to Sans. In addition, we detected Cad99C immunoreactivity at what ap- peared to be the apical side of cone cells in control, but not in Cad99C57A/Cad99C57A mutant flies (Fig. 4F,G). Finally, Crinkled/ Myosin VIIa immunoreactivity was detected in cone cells in control, but not in ck13/ck13 mutant flies (Fig. 4H and data not shown). We conclude that Sans, Cad99C, and Crinkled/Myosin VIIa localize to cone cells in adult eyes. Sans, Cad99C, and Crinkled/Myosin VIIa localize to follicle cells Cad99C has previously been shown to localize to microvilli of follicle cells in stage 10 egg chambers [32,33]. We next determined the expression and localization of Crinkled/Myosin VIIa and Sans in ovaries. crinkled RNA was detected in nurse cells and follicle cells of stage 9–10 egg chambers (Fig. 5A). In stage 11, crinkled RNA was confined to the nurse cells (Fig. 5B). No staining was detected using a sense RNA probe to crinkled (Fig. 5C), indicating that the staining was specific. Crinkled/Myosin VIIa immunoreactivity was first detected during oogenesis at the apical side of follicle cells in stage 7 egg chambers (Fig. 5D,E). In stage 10 egg chambers, immunoreactivity was detected in the follicle cells surrounding the oocyte and in the germline (Fig. 5F), consistent with the expression of crinkled RNA. Similar to crinkled RNA, Crinkled/ Sans in Fly Development Myosin VIIa immunoreactivity was no longer detected in follicle cells of stage 11 egg chambers (data not shown). Higher magnification views of stage 10 egg chambers showed that Crinkled/Myosin VIIa immunoreactivity was present in punctate structures at the apical side of follicle cells and, in part, co-localized with Cad99C (Fig. 5H). Crinkled/Myosin VIIa immunoreactivity was not obviously altered in ovaries of Sans245/Sans245 mutant flies (Fig. 5G,I). Sans immunoreactivity was detected in the most anterior part of the germarium in what appeared to be the germline stem cells (Fig. 6A,B). Moreover, Sans immunoreactivity was observed in the follicular epithelium surrounding the oocyte of control stage 9–10A egg chambers (Fig. 6D,D9), consistent with the pattern of Sans mRNA expression. No immunoreactivity was detected in ovarioles of Sans245/Sans245 mutant flies (Fig. 6C, E,E9), indicating that the observed stainings were specific for Sans. We conclude that Sans, Cad99C, and Crinkled/Myosin VIIa localize to follicle cells. Sans co-localizes with the syntaxin Avalanche to early endocytic vesicles in follicle cells Next we analyzed in more detail the subcellular localization of Sans in follicle cells. Higher magnification views revealed that Sans localized to punctate structures within the follicle cell cytoplasm (Fig. 7A). Similarly, a YFP-Sans fusion protein, expressed using the GAL4-UAS-system, localized to intracellular punctate structures Figure 4. Localization of Sans, Cad99C, and Crinkled proteins in adult retinae. A: A scheme of a longitudinal section of a Drosophila adult ommatidium. B–C: Longitudinal optical sections of adult retinae from (B) wild-type and (C) Sans63/Sans63 mutant animals stained for Rhodamine- Phalloidin (red) to detect Filamentous (F-) actin. D–E: Longitudinal optical sections of adult retinae from (D) wild-type and (E) Sans63/Sans63 mutant animals stained for Sans (green) and F-actin (red). F–G: Longitudinal optical sections of adult retinae from (F) wild-type and (G) Cad99C57A/Cad99C57A mutant animals stained for Cad99C (green) and E-cadherin (red). E-cadherin labels the adherens junctions between photoreceptor cells. H: Longitudinal optical sections of wild-type adult retinae stained for Crinkled (green) and Cad99C (red). Sans, Cad99C, and Crinkled localize to cone cells. Scale bars: 10 mm. doi:10.1371/journal.pone.0004753.g004 PLoS ONE | www.plosone.org 5 March 2009 | Volume 4 | Issue 3 | e4753