Figure 1.
ebi is required for cellular survival of ommatidia.
(A, B) ey-FLP; ebiP, FRT40A/+, FRT40A (ebi−/− mosaic) ommatidia 1 week (A) and 5 weeks (B) after eclosion. Note ommatidia in ebi mutant clones (−/−). (C–E) Ommatidia from ey-FLP; ebiP, FRT40A/CycEAR95, FRT40A (ebi−/− large clone) flies, in which >95% of compound eyes consist of ebiP homozygous cells, at 2 (C), 3 (D), and 4 (E) weeks after eclosion (Lee et al. 2001). (F) Quantification of abnormal ommatidia with reduced numbers of photoreceptor cells in ebi−/− clones over a 5-week period after eclosion. For comparison, wild-type (WT) ommatidia were analysed. Arrows; scar-like defects. Arrowheads; abnormal ommatidia.
Figure 2.
C-terminal truncation of Ebi causes age-related retinal degeneration.
(A, B) GMR-GFP/+ (GFP) ommatidia 1 week (A) and 5 weeks (B) after eclosion. (C, D) GMR-ebiΔC/+ (ebiΔC) ommatidia 1 day (C) and 5 weeks (D) after eclosion. Arrows; scar-like defects. Arrowheads; abnormal ommatidia. (E) GMR-Gal4/+; GMR-ebiΔC/+; UAS-ebi/+ (ebiΔC+ebi) ommatidia, which express ebi cDNA via GMR-Gal4, 5 weeks after eclosion. (F) GMR-Gal4/+; GMR-ebiΔC/+; UAS-p35/+(ebiΔC+p35) ommatidia. p35 inhibited ebiΔC-induced retinal degeneration.
Figure 3.
ebi interacts with Jra and antagonizes JNK signaling.
Compound eyes from wild type (A), GMR-ebiΔC, GMR-ebiΔC/+ (2×ebiΔC) (B), GMR-ebiΔC, GMR-ebiΔC/Jra1 (2×ebiΔC; Jra1/+) (C), ey-FLP; ebiP, FRT40A/CycEAR95, FRT40A (ebi−/−) (the same genotype as in Figure 1C–E) (D), hepr75/ey-FLP; ebiP, FRT40A/CycEAR95, FRT40A (hepr75/+; ebi−/−) (E), and ey-FLP; ebiP, FRT40A/CycEAR95, FRT40A; pucE68/+(ebi−/−; pucE68/+) (F). (G, H) ebi showed a strong genetic interaction with TNF-α. (G) GMR-Gal4/+; egrGS11687/+, TNF-α overexpression induced a small-eye phenotype (Igaki et al. 2002). (H) GMR-Gal4/+; egrGS11687/ebi4, in which a copy of ebi was removed, enhanced the eye phenotype. (I) Lifespan analysis for wild type and for strains containing ebi mutants (ebi7/ebi90) and ebi mutants with a copy of Jun removed (ebi7, Jra1/ebi90, +) cultured with or without 7.5 mM paraquat. Wild-type flies died within 12 days (open circle and gray line). Strong enhancement occurred in ebi mutant escapers (open square and black line). Removing a copy of Jun suppressed the sensitivity to paraquat (open circle and black line). The median life span was 5.9 d, 4.2 d, and 5.1 d for wild-type, ebi mutant, and ebi mutant with Jun males, respectively. Statistical analysis was performed using the log rank test. For wild type versus ebi mutant, p<0.001. For ebi mutant versus ebi mutant with a copy of Jun removed, p<0.001. n = 100. (J) Immunoprecipitation assay. S2 cells were transfected with a HA-ebi expression vector, and proteins were immunoprecipitated with hemagglutinin antibody and immunoblotted as indicated.
Figure 4.
ebi antagonizes hid expression.
(A, B) Quantitative real-time PCR (qRT-PCR) analysis were performed using specific primers. Each mRNA from S2 cells treated with dsRNA against Jra (A) or ebi (B) was used for the analysis. Data are shown as the mean ± SEM. * = p<0.05; ** = p<0.01; # = p>0.05. (C) Mosaic clones of ebiP. Eye discs from ey-FLP; ebiP, FRT40A/ubi-GFP, FRT40A; hid05014/+ were stained with anti-lacZ and visualized for GFP expression. ebi mutant clones showed slightly increased hid expression. MF: morphogenetic furrow. (D) Mosaic clones of ebiE4. Eye discs from heat-induced hs-FLP; ebiE4, FRT40A/ubi-GFP, FRT40A were stained with anti-Diap1 and visualized for GFP expression. Diap1 was increased in ebi mutant clones.
Figure 5.
ebi downregulates hid expression via the AP-1 target site in the promoter region of hid.
(A) Schematic of the promoter region of hid. The AP-1 half site and its mutant forms are indicated. (B) Luciferase constructs with different lengths of the promoter region of hid. Effects of ebi dsRNA on expression of the wild-type or mutant forms of hid-luc are shown in the graph. ** = p<0.01; # = p>0.05. Data are shown as the mean ± S.E.M.
Figure 6.
Jra is required for ebi-dependent retinal degeneration.
Eye sections of aged flies. (A) GMR-ebiΔC/+; hid05014/+, (ebiΔC; hid/+) ommatidia. 5 weeks after eclosion. (B) GMR-ebiΔC/+; Df(3L)H99/+, (ebiΔC; H99/+) ommatidia. 5 weeks after eclosion. (C, D) GMR-ebiΔC/+; th4/+, (ebiΔC; th/+) ommatidia. Eye sections of 2 days aged flies showed slight degeneration phenotype (C). One week aged flies showed severe degeneration phenotype (D). (E, F) Transmission electron microscope images of ommatidia. 5 weeks after eclosion. Wild-type flies had normal ommatidia, and each mitochondrion was arranged along the cell-cell junction with compact structures (E, inset). ebiΔC flies, in contrast, had electron-dense vesicles (arrows) and showed mitochondrial swelling and changes in mitochondrial cristae (F, inset). mt, mitochondrion; Ra, rhabdomere. (G) GMR-ebiΔC/Jra1, (ebiΔC; Jra/+) ommatidia. 5 weeks after eclosion. (H) hepr75/+; GMR-ebiΔC/+, (hep/+; ebiΔC) ommatidia. 5 weeks after eclosion.
Figure 7.
Light-dependent retinal degeneration in an ebi mutant.
(A–D), Flies were raised under constant illumination (A–E) or darkness (F) at the times shown. Wild-type (A) and ey-FLP; ebiP, FRT40A/CycEAR95, FRT40A (ebiP large clone) (B–F) ommatidia are shown. (G) Models of ebi activity in photoreceptor cells survival.