(A–D) Yki transcriptional activity is reduced by loss of ckIα, but unaffected by loss of gish. XY and XZ confocal images of third instar wing imaginal discs bearing clones mutant for ckIα8B12 (A, C, D), or gish17 (B), co-expressing the Yki transcriptional reporter genes ex-lacZ (A, B), diap1-GFP3.5 (C), or a Yki::GFP fusion protein (a knock-in at the endogenous locus, (D). ex-lacZ is visualised by immunostaining for β-galactosidase (green in A, A’’, B and B’’, grey in A’, (A’’’, B’ and B’’’); diap1-GFP and Yki::GFP are visualised by direct GFP fluorescence (green in C, C’’ and D, grey in C’, (C’’’ and D’). Clones are marked by absence of RFP (red) and highlighted by white dashed lines; DAPI (blue) stains nuclei. Reporter gene expression is drastically reduced in ckIα8B12 (A, C), but not gish17 (B), mutant clones. Yki::GFP appears excluded from the nucleus of ckIα8B12 mutant cells (D). Scale bars 20 μm. (E–H) Overexpression of CkIα, but not GishisoI, results in upregulation of ex-lacZ. Maximum intensity projections of z-stacks of the pouch region of wing imaginal discs from third instar larvae overexpressing no transgene (E), UAS-CkIα (F), or UAS-GishisoI (G) under the control of hh-Gal4. Crosses were raised at 25 °C and larvae were dissected at wandering L3 stage. ex-lacZ expression was detected by immunostaining for β-galactosidase (red in E-G, grey in E’-G’); the posterior compartment is marked by expression of GFP (green); DAPI (blue) stains nuclei. (H) Quantification of the posterior to anterior ratio of ex-lacZ signal intensity in the pouch region; CkIα expression significantly upregulates this Yki target gene (p=0.0001, one-way ANOVA comparing all means to hh >control, with correction for multiple comparisons; n ≥ 8 for all genotypes), while GishisoI does not (p=0.4808). Scale bars 20 μm.