Fig. 9. Flies carrying a hypomorphic or a null mutation in rtnl1 show only a mild phenotype in cyst maturation during oogenesis. (A) Scheme of potential Rtnl1 protein isoforms; red and orange boxes show protein-encoding exons. The green stars indicate the position where the GFP-exon is inserted and, thus, only the two largest protein isoforms are labeled in the Rtnl1-GFP line. The black bar indicates the deletion in the rtnl1^null allele that eliminates all of the reticulon-homology domain encoding the membrane-association domain of the protein (Wakefield and Tear, 2006). (B) The rtnl1¹ allele, generated by F1-EMS mutagenesis screen (see Materials and Methods) appears to result in expression of a truncated protein. (Left) Western blot of Rtnl1-GFP exon-trap ovaries in the left panel; (right) immunoprecipitation with anti-GFP antibody followed by western blotting of Rtnl1-GFP exon-trap and rtnl1¹ ovaries. Red dots indicate the main Rtnl1-GFP protein band. (C) Compared with control germaria, germaria of both homozygous rtnl1^null and rtnl1¹ mutant females show a less tight concentration of Orb on the fusome, and also sometimes even cysts completely lacking detectable concentration of Orb (arrows). Nonetheless, budding egg chambers in region 3 always have Orb concentrated within one single cell. Shown are z-stack projections through the whole germarium. Orb is in red and as a single channel shown in white in the middle panels, Shot, marking the fusome, is in blue and as a single channel shown in white in the right panels. Bar, 10 µm. (D) Germaria of homozygous rtnl1^null mutant females still show concentration of Me31b into the oocyte in budding egg chambers, although similar to Orb, the concentration on the fusome is less tight (top panel, arrows point to budding egg chambers). In later egg chambers, Orb and Me31b still colocalize in foci within the oocyte (bottom panels, white arrows) and Me31b still accumulates in foci within the nurse cells (bottom panels, green arrowheads). Top panel is a z-stack projection through a whole germarium, bottom panel is a single confocal section. Me31b is in green and as a single channel shown in white in the middle panels, Orb is in red and as a single channel shown in white in the right panels.