Fig. 7.

Reduction of NOC1 in cells of the wing imaginal disc induces Xrp1 and Eiger resulting in apoptosis and DILP8-induced developmental delay. (A–C) Confocal images of wing imaginal discs expressing the indicated transgenes using the MS1096-GFP wing-driver. Wingless (WG) expression is visualized using anti-WG antibodies (in red), nuclei are stained with Hoechst (in blue). (D) Larval volume of animals expressing the indicated transgenes using the MS1096-driver was measured at the indicated time AEL until pupariation. The graph is the mean±s.d. for one of three experiments using at least ten animals for each point and genotype. (E) Curves representing the mean±s.d. percentage of larvae that underwent pupariation for the indicated genotypes. A significant delay in pupariation (one-way ANOVA with Tukey's test, P<0.0001) is visible in animals in which NOC1 is reduced using MS1096-Gal4 with both the RNAi lines. Data are expressed as percentage of pupariation over the total number of pupae of the same genotype, and are from five independent experiments. (F–H) Photographs of wings from 3-day-old adults of the indicated genotype. (I–W) Confocal images of wing imaginal discs from third-instar larvae where NOC1-RNAi was expressed using rotund-Gal4 driver, co-expressing DILP8-GFP (I,J), eiger-GFP (N,O) or TRE-dsRED (R,S) reporters, or were stained for apoptotic cells using the anti-Caspase-3 antibody (V,W). (K–Q) Confocal images of wing imaginal discs expressing NOC1-RNAi using the nubbin-Gal4 driver (K,L) or together with eiger-RNAi (P,Q), along with the DILP8–GFP reporter. (M) Quantification of the mean±s.d. GFP intensity in the wing pouch from K,L,O,P. (T,U) qRT-PCRs showing the level of Dilp8 (T) and Xrp1 (U) mRNAs in wing imaginal discs in which NOC1 and Eiger levels were reduced using the rotund-Gal4 promoter; actin5C mRNA was used as control. Data are mean±s.d. for three experiments. (X) Model: NOC1 is necessary for proper rRNA processing. Its reduction decreases protein synthesis and induces a nucleolar stress resulting in apoptosis. This event is accompanied by the upregulation of the pro-apoptotic genes eiger and Xrp1, resulting in DILP8 upregulation that in turn reduces ecdysone delaying animal development. *P<0.05; **P<0.01; ***P<0.001; ****P<0.0001; ns, not significant (one-way ANOVA with Tukey multiple comparisons). Scale bars: 40 µm (A–C); 1 mm (F–H); 100 µm (I-L; N–Q;R,S,V,W).