Figure 4. Caspase-generated ROS are required of recruitment and activation of hemocytes to scrib^−/− Ras^V12 mosaic eye/antennal imaginal discs.

Hemocytes were labeled with the NimC antibody (Kurucz et al., 2007) (red in top panels; grey in (‘) panels and in (L–R)). Scale bars in (A–J’): 50 μm; in (L–R): 100 μm. (A,A’) Control mosaic discs (FRT +) carry small hemocyte clusters mostly at the antennal portion of the disc. (B,B’) scrib^−/− Ras^V12 mosaic discs are covered by large quantities of hemocytes. + indicates scrib^−/− Ras^V12 in otherwise wt background. (C–I) Hemocyte recruitment to scrib^−/− Ras^V12 eye/antennal imaginal discs is strongly impaired upon loss of ROS (C–F’) and caspase activity (G–I'). (J,J’) Expression of a dominant negative JNK transgene (JNK^DN) in scrib^−/− Ras^V12 mutant clones blocks hemocyte recruitment. (K) Quantification of NimC labelings reveals that the number of hemocytes attached to scrib^−/− Ras^V12 mosaic discs is significantly lower when ROS levels or caspase activity are reduced in scrib^−/− Ras^V12 clones. To facilitate the quantification, the mean intensity of NimC labelings across the entire disc was determined, normalized to GFP (to account for the reduced size of ROS-depleted or caspase-inhibited scrib^−/− Ras^V12 clones) and analyzed by one-way ANOVA with Holm-Sidak test for multiple comparisons. Error bars are SD. P values are referenced to scrib^−/− Ras^V12 and are indicated by asterisks above each column. ****p<0.0001. Ten discs per genotype were analyzed. + indicates scrib^−/− Ras^V12 in otherwise wt background. (L–R) High magnification images of hemocytes attached to the discs of indicated genotype. Note that in (M) hemocytes attached to scrib^−/− Ras^V12 discs extend cellular protrusions (arrows), similar to cytonemes. These protrusions are absent in hemocytes attached to control (L) and caspase-inhibited or ROS-depleted discs (N–R). + in (M) indicates scrib^−/− Ras^V12 in otherwise wt background. Genotypes: (A,L) yw ey-FLP/+; act>y⁺>Gal4, UAS-GFP^56ST/+; FRT82B tub-Gal80/. FRT82B w⁺; (B,M) yw ey-FLP/+; act>y⁺>Gal4, UAS-GFP^56ST/+; FRT82B tub-Gal80/UAS-Ras^V12 FRT82B scrib²; (C–I, N–R) yw ey-FLP/+; act>y⁺>Gal4, UAS-GFP^56ST/UAS-X; FRT82B tub-Gal80/UAS-Ras^V12 FRT82B scrib² with UAS-X being UAS-Duox^RNAi (C), UAS-hCatS (D,N), UAS-SOD1 (E), UAS-Catalase (F,O), UAS-SOD2 (P), UAS-dronc^RNAi (G,Q), UAS-drICE^RNAi (H,R) and UAS-p35 (I). (J) yw ey-FLP/UAS-JNK^DN; act>y⁺>Gal4, UAS-GFP^56ST/+; FRT82B tub-Gal80/UAS-Ras^V12 FRT82B scrib².