Expression of GFP protein traps in known sarcomere proteins. (A-D) Peritoneal sheath labeled with antibodies to α-Actinin (blue) to view Z-discs and phalloidin (red) to reveal F-Actin. The peritoneal sheath is from flies expressing protein traps (green) in Tm1 (A), Mhc (B), Sls (C) and Ilk (D). (E-H) Epithelial sheath labeled as in A-D. (I-L) Oviduct labeled as in A-D. For all panels, the three individual color channels in the boxed regions are shown to the right. Scale bars: 10 μ.

A GFP protein trap in Fasciclin 3 reveals novel cell-cell junctions. (A, A’) An ovariole expressing Fas3::GFP with (A) and without (A’) phalloidin in red. Fas3::GFP localizes to follicle cells in the germaria and then is restricted to polar follicle cells. Additionally a “garland” pattern of Fas3::GFP puncta can be seen in the overlying epithelial sheath. (B, B’) The surface view of follicle cells expressing Fas3::GFP with (B) and without (B’) phalloidin in white. Fas3::GFP puncta can be seen on the cell membrane. (C, C’) A lateral view of follicle cells expressing Fas3::GFP with (C) and without (C’) phalloidin in white and Dapi in blue. Fas3::GFP puncta localize to the apical lateral region of the membrane. Scale bars: A = 50 μ, B = 10 μ. Scale bar in B applies to C.

Epithelial sheath cell end junctions contain Integrin. (A-A”) Immunofluorescence of epithelial sheath expressing Zasp52::GFP (green) stained with antibodies to FLN12−20 (blue) and ß-Integrin (red). In A’, ß-Integrin expression is higher at end junctions between cells (yellow arrowheads) than at Z-discs, while Zasp52 expression is higher in Z-discs than at end junctions (A”). (B-B”). Higher magnification and channel separation of boxed area shown in (A). (C-C”) Immunofluorescence of epithelial sheath expressing Fas3::GFP (green) stained with antibodies to FLN12−20 (blue) and ß-Integrin (red). ß-Integrin is present in a continuous zone at end junctions (C’) while Fas3::GFP is in distinct dots (C”). Scale bars: 10 μ.

Model of epithelial sheath cell structure. (A) Schematic of a section of epithelial sheath muscle showing one surface of an ovariole. Each muscle cell contains a single nucleus (yellow) and has a mesh like structure (blue). Thin cytoplasmic projections extend between cells in the anterior/posterior direction. Sarcomeric actin (red) forms branching structures within cells. Fas3 cell-cell contacts (green) are located at the both the end-to-end junctions between cells and at the intersection of lateral cytoplasmic extensions between adjacent cells. (B) Schematic of two epithelial sheath muscle cells filleted flat to emphasize that where two cells connect there is a continuation of the sarcomeric structure across cell membranes.

Drosophila ovarian muscle types. (A) Drosophila ovaries have three types of muscle: the Peritoneal sheath, which surrounds each intact ovary, the Epithelial sheath, which surrounds each ovariole, and the muscle of the oviducts. (B) Sarcomeres contain Z-discs that anchor the F-actin thin filaments, and myosin thick filaments bound to F-actin. The registration and position of thick filaments gives a characteristic banding pattern with the I band containing thin filaments, A band containing thick and thin filaments and the M band containing only thick filaments. The model also shows approximate positions of Sallimus, Projectin and Tropomyosin. The shading represents the locations of the epitopes recognized by Sallimus and Projectin antibodies. (C) Peritoneal sheath muscle labeled with phalloidin (red) to reveal F-Actin distribution (C’), and antibodies to Projectin (green) (C”) and Sallimus (blue) (C’”). (D) Epithelial sheath muscle labeled as in C. (E) Oviduct muscle labeled as in C. Scale bars: 10 μ.

Ovarian muscle expression of GFP protein traps in uncharacterized proteins. Top: Schematic representations of isoforms predicted through differential splicing of Zasp52, Zasp66 and CG15926 from Flybase. The location of the GFP gene insertion with respect to each isoform is indicated with a green triangle and the locations of exon boundaries are indicated by black lines. Bottom: Expression patterns of GFP-tagged proteins (green) in peritoneal sheath (A-C), epithelial sheath (D-F), oviduct (G-I), larval midgut (J-L), larval body wall (M-O), and adult indirect flight muscle (P-R) stained with phalloidin (red) and α-Actinin antibodies (blue, A, B, D, E, G and H) or Dapi (blue, R). Zasp52::GFP (first column) and Zasp66::GFP (second column) colocalize with α-Actinin in Z-discs in peritoneal sheath (A,B) epithelial sheath (D,E) and oviduct (G,H). Z-discs in both the longitudinal (arrows) and circular (arrowheads) muscles of the larval midgut contain Zasp proteins (J, K), as do Z-discs in larval body wall (M, N) and adult flight muscles (P, Q). CG15926::GFP localizes to cell membranes in all muscle types. Additional localization to membranes on the interior of muscle cells can be seen in oviduct (I, arrow), larval midgut (L), larval bodywall (O) and flight muscle (R). This may be sarcoplasmic reticulum. Scale bars: 10 μ. Scale bar in D applies to D-R.

FLN12−20 and Fas3::GFP expression reveal epithelial sheath cell shape. (A) Immunofluorescence of epithelial sheath expressing Fas3::GFP (green) with FLN12−20 labeled white and phalloidin red. (B-B”) Higher magnification and channel separation of boxed area shown in (A). (C) Immunofluorescence of epithelial sheath showing mosaic expression levels of FLN12−20 (green). Phalloidin-labeled F-Actin is red. FLN12−20 is present in cell bodies around nuclei (arrows). (D-D”) Higher magnification and channel separation of boxed area in panel C. Sarcomeric F-Actin exhibits a more pronounced periodicity in cell expressing a lower level of FLN12−20 (compare sarcomeric bundles in the upper part of D’ with those in the lower half). (E-E”) Higher magnification and channel separation of boxed area in panel C. F-Actin “joints” coincide with borders between regions of high and low FLN12−20 expression. (F-F”) Epithelial sheath muscle expressing Fas3::GFP (green) and also displaying FLN12−20 (white) mosaicism. F-Actin is red. Region expressing low levels of FLN12−20 (arrow) is outlined by Fas3::GFP dots. (G-G’) Higher magnification and channel separation of boxed area shown in (F). Two types of fine projections are evident extending from muscle cell bodies: those associated with Fas3 dots (G, white arrowheads), and those that extend from one portion of a cell body to another (G, yellow arrowheads). Scale bars: A, C and F = 20 μ, B, E and G = 10 μ. Scale bar in C applies to D.

Epithelial sheath cells are mononucleate. (A-A’) Confocal projection of top half of an ovariole expressing both Fas3::GFP (green dots) and Meso18::GFP (green nuclei) in epithelial sheath cells stained with antibodies to FLN12−20 (red). (A’) A single cell is clearly outlined by Fas3::GFP dots and contains only one nucleus, as seen with Meso18::GFP. (B-B’) Rendering of optical sections taken through the entire ovariole shown in A. Only green signal from Fas3::GFP and Meso18::GFP was rendered. The top half of the confocal stack is colored cyan, the bottom half red. B’ shows the top half only, and represents a rendering of the raw data shown in A’. See Supplemental movies for rotation of the raw data and rendered stacks. (C-C’) Confocal image of an ovariole with Flp/FRT-induced mitotic clones. F-Actin is in red and Zasp52::GFP is in green. C’ shows Zasp52::GFP only, with the inferred copy number of GFP genes indicated in yellow. Scale bars: 20 μ.