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- Fig. 1. crb and chp synergistically control the width of the rhabdomere.
(A–F) Electron micrographs of cross-sections of control (w) (A), crb11A22 (B), chp2 (C), crb11A22 +/+ + (D); + +/+ chp2 (E) and crb11A22 +/+ chp2 (F) adult Drosophila ommatidia. Scale bar: 1 µm. (G) Box-plot representing the width of rhabdomeres of indicated genotypes. Whiskers indicate 10–90% confidence interval. The width is indicated in µm2 and estimated by measuring cross-sectional areas of rhabdomeres from outer PRCs (R1–R6) in 1–2-day-old adult female Drosophila eyes. n = number of ommatidia.
- Fig. 2. Mutation in crb suppresses interrhabdomere adhesion in prom and eys mutants.
(A–F) Electron micrographs showing tangential sections of ommatidia of Drosophila with the following genotypes in w background: cn bw prom1 (A), cn bw prom1; crb11A22 (B), eys1 cn bw (C), eys1 cn bw; crb11A22 (D), + cn bw prom1/eys1 cn bw +; +/+ (E) and + cn bw prom1/eys1 cn bw +; crb11A22/+ (F). Numbers in A–F depict the numbers of individual rhabdomeres or rhabdomere clusters, not the identity of PRCs. Asterisk: IRS; white arrow: rhabdomere adhesion. Scale bar: 1 µm. (G,H) Quantification of interrhabdomeral adhesion of PRCs with different genotypes. Column chart (mean ± s.d.) represents the number of single rhabdomeres or rhabdomere clusters per ommatidium (G) and average individual rhabdomeres per ommatidium (H). n = number of ommatidia.
- Fig. 3. Localisation of Crb and Chp in wild-type and mutant PRCs.
(A,B,D) Drosophila adult ommatidia. Genotypes are w (A), crb11A22 mosaic (B,D,E) and chp2 mosaic (F). Confocal images of immunostainings on tangential (A,B,F) and longitudinal (D) sections of PRCs stained for F-actin (blue), Chp (magenta) and Crb (green). White arrows in panels B,B″ and D point to intracellular Chp punctae in crb mutant cells. (C) Box-plot representing the number of cytosolic Chp positive punctae per cell per cross-section of wt, crb/+ and crb/crb mutant PRCs. Whiskers indicate 5–95% confidence interval. Statistical significance is analysed with Kruskal–Wallis test, followed by Dunn's multiple comparisons test. ****p<0.0001. (E–E″) Immunoelectron micrograph showing the localization of Chp (10 nm gold particle) (magenta arrows) in a cross-section of crb11A22 mutant PRC. Chp is localized in the rhabdomere (E,E′, asterisk) and in a multivesicular body (E″, arrow). (F) Confocal images of immunostainings on tangential sections of PRCs stained for F-actin (blue), Chp (magenta) and Crb (green). White arrowhead points to the apical membrane in chp mutant cells, which is no longer subdivided into rhabdomere and stalk. Scale bars: 5 µm (A,B,D,F), 100 nm (E).
- Fig. 4. Development of the apical compartment in chp mutant PRCs.
(A–D) Cartoons of different developmental stages in wild-type ommatidia (not taking into account the correct contacts made by individual rhabdomeres). Magenta: adherens junction. Cyan: common apical surface in panel A, as deduced by co-localisation of Crb and F-actin. Green: Crb, highlighting the stalk membrane. Blue: F-actin, highlighting the microvilli. Grey: interrhabdomerel space. R1–R7 = number of PRCs. pd = pupal development. (E–L′): Electron micrographs of tangential sections of wild-type (E–H′) and chp2 mutant (I–L′) ommatidia at 38% pd (E,E′,I,I′), 54% pd (F,F′,J,J′), 79% pd (G,G′,K,K′) and in the adult (H,H′,L,L′). The first defects in chp mutant ommatidia can already be detected at 38% pd, in that the microvilli are not as closely associated with each other as in wild type (compare panel E′ to panel I′). Scale bars: 1 µm (E–H,H′,I–L,L′) and 100 nm (E′–G′,I′–K′); blue asterisk, IRS; magenta arrows, stalk membrane (labelled in green).
- Fig. 5. Effect of constant light exposure on the survival of crb, prom, eys and chp mutant PRCs.
Electron micrographs of ommatidial cross-sections of Drosophila raised on standard (A–L) or vitamin A-depleted Drosophila medium (M–P) and kept under various light conditions. Genotypes are: crb11A22 (A,E,I,M), cn1 bw1 prom1 (B,F,J,N), eys1 cn1 bw1 (C,G,K,O) and chp2 (D,H,L,P), all in a w genetic background. crb, prom and eys mutant PRCs do not show any signs of retinal degeneration when kept under a 12 hrs light/dark cycle (A–C) or in constant darkness (E–G). When kept in constant light, they display characteristics of degeneration (I–K), such as condensed cytoplasm (white arrows) and missing rhabdomeres. Degeneration under constant illumination is prevented when animals were raised in a vitamin A-depleted medium (M–O). Under all tested conditions, chp mutant PRCs do not show any major signs of retinal degeneration (D,H,L,P). Scale bar: 1 µm; asterisk: IRS.
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