USP8 Promotes Smoothened Signaling by Preventing Its Ubiquitination and Changing Its Subcellular Localization (original) (raw)
Figure 6
The function of USP8 in regulating Smo accumulation in the Drosophila wing.
(A–A″) A wing disc expressing UAS-GFP by the dorsal compartment-specific _ap_-Gal4 was stained for Smo and ptc-lacZ to show the wild-type staining. (B–B″) A wing disc from flies co-expressing UAS-USP8RNAi and UAS-GFP by _ap_-Gal4 was stained for Smo and ptc-lacZ. The arrow in (B) indicates the attenuated accumulation of Smo and the arrowheads indicate the down-regulation of ptc-lacZ. GFP indicates that _ap_-Gal4 is expressed in the dorsal compartment cells of the wing disc. (C–D) Wing discs expressing UAS-Flag-USP8C>S by MS1096 Gal4 were stained for Smo (grey in C), Flag (green in C′), and ptc-lacZ (red in D). The arrow in (C) indicates inhibition of Smo accumulation. The arrow in (D) indicates attenuated ptc-lacZ expression. Note that the level of Gal4 driven by MS1096 is higher in the dorsal compartment than in the ventral compartment. (E–E″) A wing disc bearing usp8KO homozygous clones, which were marked by the lack of β-gal staining, was immunostained for Smo. The arrow in (E) shows the reduced Smo accumulation in P-compartment cells. (F–G) Wing discs expressing UAS-Flag-USP8 by MS1096 Gal4 were stained for Smo (grey in F), Flag (green in F′), and ptc-lacZ (red in G). Arrow and arrowhead in (F) indicate the elevated Smo and arrow in (G) indicates the expansion of ptc-lacZ. (H) A wild-type embryo was stained for En. (I–J) Embryos expressing USP8RNAi or USP8C>S by _act5C_-Gal4 were immunostained for En. The arrowheads indicate the reduction and contraction of En expression. (K–K″) USP8 expression patterns in wing and leg discs (arrow and arrowhead, respectively) were determined by FISH with a DIG-labeled mRNA probe against USP8. Rhodamin (red) was used to detect the DIG-labeled mRNA, and DAPI (green, was false colored in green to provide better contrast) was used to label the nuclei. All wing imaginal discs shown in this study were oriented with anterior on the left and ventral on the top. (L) A wild-type adult wing showing interveins 1–5. (M–N) Wings from either male or female flies expressing Flag-Smo by MS1096 Gal4. The arrow in (M) indicates the overgrowth structures between Vein 2 and 3, and the arrow in (N) indicates the thickened Vein 3 caused by the overexpression of Smo. (O) A wing from flies expressing USP8RNAi by MS1096 Gal4. (P–Q) Wings from male or female flies expressing Flag-USP8 by MS1096 Gal4. Arrows indicate the wing overgrowth structure or the thickness of Vein 3. Of note, males of the same genotype have more severe phenotypes than females, presumably due to dosage compensation of the X-chromosome carrying the _MS1096_-Gal4. (R) A wing from ptc mutant flies indicates the overgrowth between Vein 2 and 3. (S–T) Adult wings from flies expressing Flag-USP8 by MS1096 Gal4 in the background of ptc mutants. In the ptc heterozygous or homozygous background, USP8 induced more severe wing overgrowth, which is indicated by wing blisters (S) or sick wings (T).