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. 2021 Jan 28;17(1):e1009300. doi: 10.1371/journal.pgen.1009300

Fig 3. Aberrant Wg signaling gradient is essential for the induction of massive cell-turnover.

Fig 3

(A-B”) The relative position between dying cells (cleaved-PARP, magenta) and Wg expression (anti-Wg, green) in the wild-type (A) or RpS3/+ (B) wing pouch. Scale bar, 100 μm. (C-D”) The relative position between dying cells (cleaved-PARP, magenta) and Wg signaling activity (anti-β-galactosidase, green) in the nmo-LacZ/+ (C) or RpS3/+, nmo-LacZ/+ (D) wing pouch. These images are Z-stacked images from 8 (C) and 8 (D) confocal images (3.02 μm and 2.39 μm, respectively) for whole wing pouch that is acquired every 1.01 μm or 0.8 μm, respectively. Scale bar, 50 μm. (E-F’) RpS3 was overexpressed in the posterior compartment of wing discs of nmo-lacZ/+ (E) or RpS3/+, nmo-lacZ/+ (F) flies using the en-Gal4 driver. nmo expression was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (G-H’) RpS3 was overexpressed in the posterior compartment of wing discs of nkd-lacZ/+ (G) or RpS3/+, nkd-lacZ/+ (H) flies using the en-Gal4 driver. nkd expression was visualized by anti-β-galactosidase staining (white). Scale bar, 100 μm. (I-K’) Dying cells were visualized by anti-cleaved PARP staining (white) in the wing discs of RpS3/+, wg/+ (I), RpS3/+, nub-Gal4, UAS-Wg (J), or RpS3/+, nub-Gal4, UAS-dsh-RNAi (K) flies expressing CD8-PARP-Venus. Wg expression was visualized by anti-Wg staining (white). Scale bar, 100 μm. (L) Boxplot with dots representing cleaved-PARP-positive dying cells per pouch in genotypes shown in (B) (n = 12, number of wing pouches), (I) (n = 9), (J) (n = 10), and (K) (n = 11). Error bars, SEM; ***, p<0.001; non-parametric Mann-Whitney U-test. Genotypes are as follows: nub-Gal4/+; UAS-CD8-PARP-Venus/+ (A), nub-Gal4, UAS-CD8-PARP-Venus, RpS3Plac92/+ (B), nub-Gal4/+; UAS-CD8-PARP-Venus/nmoP1 (C), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/nmoP1 (D), en-Gal4, UAS-GFP/+; UAS-RpS3/nmoP1 (E), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/nmoP1 (F), en-Gal4, UAS-GFP/+; UAS-RpS3/nkd04869a (G), en-Gal4, UAS-GFP/+; RpS3Plac92, UAS-RpS3/nkd04869a (H), nub-Gal4/wgl-8; UAS-CD8-PARP-Venus, RpS3Plac92/+ (I), nub-Gal4/+; UAS-CD8-PARP-Venus, RpS3Plac92/UAS-Wg (J), and nub-Gal4/UAS-dsh-RNAi; UAS-CD8-PARP-Venus, RpS3Plac92/+ (K).