Analysis of crh, fbxl7, and ds phenotypes and Ds binding. (A–B′) Loss of Crb has no effect on Dachs and Dlish. XY confocal micrographs of third instar wing imaginal discs containing crb11A22 mutant clones (marked by absence of GFP shown in green) with Dachs staining (A and A′—shown in gray) and Dlish staining (B and B′—shown in gray). (C and C′) RNAi-mediated knockdown of Fbxl7 has no effect on Ex. XY confocal micrographs of third instar wing imaginal discs where en-Gal4 was used to drive expression of UAS-Dicer2,UAS-GFP,UAS-fbxl7^IR with Ex staining (shown in gray). GFP marks the Gal4 positive posterior compartment (shown in green). (D and D′) RNAi-mediated knockdown of Fbxl7 has no effect on ubi-Ex1-468::GFP. XY confocal micrographs of third instar wing imaginal discs where hh-Gal4 was used to drive expression of UAS-fbxl7^IR with direct GFP fluorescence representing ubi-Ex^1-468::GFP (shown in gray). Cubitus Interruptus (Ci) staining marks the Gal4 negative anterior compartment (shown in red). (E and E′) Validation of ds^38k as a protein null. XY confocal micrographs of third instar wing imaginal discs containing ds^38k mutant clones (marked by absence of RFP shown in red) with Ds staining (shown in gray). (F–G′) Loss of ds dramatically increases apical Dachs and Dlish. XY confocal micrographs third instar wing imaginal discs containing ds^38k mutant clones (marked by absence of RFP shown in red) with Dachs staining (F and F′, shown in gray) and Dlish staining (G and G′, shown in gray). (H and H′) Loss of ds has no effect on Crb. XY confocal micrographs third instar wing imaginal discs containing ds^38k mutant clones (marked by absence of RFP shown in red) with Crb staining (shown in gray). (I and I′) Loss of ds has a minimal effect on Ft in the pouch. XY confocal micrographs third instar wing imaginal discs containing ds^38k mutant clones (marked by absence of RFP shown in red) with Ft staining (shown in gray). All XY images are orientated as dorsal up. Clonal boundaries are marked by yellow dotted lines. Scale bars are 10 µm. (J) Ds^ICD does not bind to Ex^NT or Ex^CT. In vitro transcribed and translated Ex^NT or Ex^CT were incubated alone or with in vitro transcribed and translated Ds^ICD and subjected to FLAG-IP. (K) Ds^ICD does not bind to Dlish. In vitro transcribed and translated Dlish was incubated alone or with in vitro transcribed and translated Ds^ICD and subjected to FLAG-IP. The expression and presence of proteins was analyzed by immunoblotting with the indicated antibodies. WB, Western blot. Source data are available for this figure: SourceData FS5.