Figure 1. Apoptosis in Uba1 mutant clones is dependent on DRONC and cannot be inhibited by expression of DIAP1.

Shown are eye-antennal imaginal discs from third instar larvae. Posterior is to the right. In each panel, arrows highlight two representative clones. (A) Uba1 mosaic eye-antennal discs labeled for cleaved CASPASE-3 (α-CAS3*) antibody (red). These discs were incubated at 30°C 12 hours before dissection (see Material and Methods). Presence of GFP marks the location of Uba1 clones (see arrow). (B) TUNEL labeling of Uba1 mosaic eye-antennal imaginal discs expressing an RNAi transgene targeting dronc (UAS-droncIR (inverted repeat)) using the MARCM technique (see Material and Methods). Clones are positively marked by GFP. TUNEL-positive cell death is largely blocked by dronc knockdown (B′ and B″). (C) Strong overexpression of diap1 in Uba1 clones (magenta in C′″) fails to rescue the apoptotic phenotype, as visualized by CAS3* labeling (red in C′). Uba1 clones are marked by GFP due to the MARCM technique. Please note that diap1 is so strongly overexpressed in the clones that we had to adjust the settings in such a way that endogenous DIAP1 in wild-type tissue is below the detection limit (C′″). Genotypes: (A) hs-FLP UAS-GFP; FRT42D Uba1^D6/FRT42D tub-Gal80; tub-GAL4. (B) hs-FLP UAS-GFP; FRT42D Uba1^D6/FRT42D tub-Gal80; tub-GAL4/UAS-droncIR. (C) hs-FLP UAS-GFP/UAS-diap1; FRT42D Uba1^D6/FRT42D tub-Gal80; tub-GAL4.