Ectopic expression of dco suppresses the small eye phenotype induced by overexpression of the proapoptotic genes hid, rpr and grim. Micrographs are of adult female eyes. All flies shown contain GMR-Gal4 and either UAS-lacZ (A, C, E, J) or UAS-dco (B, D, F, H). They contain two copies of GMR-hid^1M (A, B), one copy of GMR-rpr² (C, D), GMR-rpr⁵ (E, F) or a copy each of GMR-grim² and GMR-grim³ (G, H). Overexpression of dco strongly suppresses GMR-hid^IM and other GMR-hid strains (data not shown), while suppression of GMR-rpr and GMR-grim is weaker and variable.

RNAi inhibition of dco enhances Hid-mediated apoptosis in the eye. Adult eyes from GMR-Gal4/UAS-lacZ (A, C), GMR-Gal4/UAS-dco^RNAi (B, D) without (A, B) or with one copy of GMR-hid^1M (C, D). Depletion of dco roughens the eye but does not cause a reduction in size in an otherwise wild-type background (B), but does significantly enhance the GMR-hid^IM phenotype (compare C and D).

dco mutant wing discs have elevated cell death and growth delay that is largely suppressed by removing one copy of hid, rpr and grim. Confocal images of wing discs stained for TUNEL (green) to label apoptotic cells and Wg (red, apical optical section) to identify the developmental age of each disc. The discs in panels A–C are early third instar and those in panels D-F are late third instar. (A, D) Wild type discs 3 and 5 days after egg laying. (B, E) dco mutant discs (dco^P915/dco^P1396), 6 and 8 days after egg laying. (C, F) Df(3L)H99/+, dco (dco^P915/dco^P1396) discs 4 and 6 days after egg laying. H99 is a deficiency that removes a chromosomal region including hid, rpr and grim. The green signals at the edge of the wing discs in D and F are non-specific staining of nuclei from the peripodal membrane. (G, H) Wild type (G) and Df(3L)H99/+, dco (H) pupal wings stained with phalloidin. The wings are 33 hr and 38 hr after the white prepupal stage, respectively. The white arrows denote the position of the longitudinal veins. The overall pattern of the H99/+; dco wings is identical to wild-type. Scale bar: 100μm.

Inhibition of dco has little effect on hid, grim or rpr transcript levels. ptc-Gal4/UAS-dco^RNAidiscs stained for hid (A), grim (B) and rpr (C) transcripts. The location of the ptc expression domain is indicated by the arrows. There is no consistent change in hid expression by dco^RNAi, but a small decrease in grim was sometimes observed, as was a small increase in rpr expression.

Hid, Grim and Rpr do not regulate DIAP1 expression in the wing disc and DIAP1 E3 ligase activity is not required for Dco regulation of DIAP1. (A–C) Confocal images of late third wing imaginal discs containing clones of Df(3L)H99 (A–C), stained for DIAP1 (red). Clones were marked by GFP (green) and the location of the D/V boundary is indicated by the white arrow. The expression pattern of DIAP1 is unaffected in the clones. (D–F) Confocal images of wing imaginal discs stained for DIAP1 (red) and activated Drice (green) from ptc-Gal4/UAS-dco^RNAi; th^6B/+ animals. As in ptc-Gal4/UAS-dco^RNAi controls (see Fig. 4), a strong decrease in DIAP1 levels and increase in activated Drice is observed in the ptc stripe. The scale bars are 20 μM in C and 100μM in F.

Ectopic expression of dco suppresses the small eye phenotype induced by a Ras/MAPK resistant form of Hid. Micrographs are of adult female eyes. Flies contain GMR-Gal4, GMR-hid^ala5 and either UAS-lacZ (A) or UAS-dco (B). Overexpression of dco strongly suppresses the GMR-hid^ala5 phenotype.

Kinase activity is required for dco to suppress Hid-mediated apoptosis in the eye. Adult eyes from GMR-Gal4, GMR-hid^ala5 and either UAS-GFP (A), UAS-dco (B), UAS-dco^D132N (C) or UAS-dco^K38R (D). While the ATP-binding mutant dco^D132N suppresses Hid activity as well as wild-type dco, the kinase dead mutant K38R enhances the small eye phenotype. (E, F) Enhancement was also observed with a weaker GMR-hid transgene. GMR-Gal4, GMR-hid^1M and either UAS-GFP (E) or UAS-dco^K38R (F). Wild type dco and dco^D132N strongly suppressed GMR-hid^1M (data not shown).

Dco regulates DIAP1 protein levels in the wing imaginal disc. Confocal images of late third instar larval wing discs stained for DIAP1 in red (A–D,H,I) and activated Drice in green (E–G,I). ptcGal4 is expressed at an anterior strip next to the anterior-posterior boundary (indicated by arrows in B–D). All discs are oriented anterior to the left. (A) Wild type. (B) ptc-Gal4/UAS-dco. (C, E) ptc-Gal4/UAS-dco^RNAi. (D, F) ptc-Gal4/UAS-dco^RNAi; hid⁰⁵⁰¹⁴/hid^X14. (G–I) Z axis projection of ptc-Gal4/UAS-dco^RNAi disc showing basal localization of Drice positive cells. Arrows indicate the reduction in DIAP1 staining in the ptc expression domain.

dco mutant clones are rescued by inhibition of caspases and exhibit increased Drice activation and decreased DIAP1 protein, but not DIAP-lacZ. dco null (dco^le88) mutant clones were generated in an en-Gal4/UAS-p35 background and are marked by the absence of lacZ staining (green; A,C–H) or GFP (green; I–K) as indicated. (A) dco clones are found in the posterior (P) compartment where p35 is expressed by en-Gal4, but the anterior (A) clones are not observed. The white line marks the AP boundary. The presence of the twins at the anterior indicates that mitotic recombination had been induced. (B) Quantification of the dco clone/twin area. The pixel value of each bar represents the total area of clones or twins in A or P in one disc, and 12 discs containing a total of more than 30 individual clones were analyzed. The difference between the areas of A twins (average: 10451 pixels/disc) and P twins (average: 11138 pixels/disc) is not statistically significant (p>0.20 by the Mann-Whitney test). The average size of the P clones (2855 pixels/disc) is 25.6% of the size of the P twins. (C–E) Active Drice (red) is elevated in dco clones. Occasionally active Drice is observed adjacent to the clones (arrows). (F–H) DIAP1 protein (red) is decreased in dco clones. (I–K) dco clones marked by the absence of GFP were generated in a DIAP-lacZ background (th^i5c8). DIAP-LacZ (red) is not altered in the clones. Scale bar: 20μm.

The dco mutant cells have unaffected Wg and Dpp signaling. Confocal images of late third instar wing imaginal discs containing dco^le88 null clones, generated as described in Fig. 5. Clones are marked by the lack of lacZ staining (green) and the Wg targets Sens and Dll and Dpp target Spalt are stained in red. Sens (A–C) and Dll (D–F) and Spalt (G–I) expression are normal in dco clones.