Fig. 3.
Cg antagonizes transcriptional output from Eya-So. All stained tissues are late third instar wing imaginal discs (n>10 discs examined for all genotypes). (A) Cg limits the ability of Eya-So to activate LMEE-luciferase in S2 cell transcription assays (seven independent replicates). (B) dpp-GAL4-driven eya misexpression always induces ectopic Dac expression in the hinge (yellow arrows, compare with E″), but only occasionally in the pouch (not shown). (C) cg heterozygosity enhances the ability of eya to activate ectopic Dac in the pouch (cyan arrows). (D) Quantification of the number of ectopic Dac patches in the pouch for genotypes in B,C (n≥8). (E-H) A stronger dpp-GAL4 driver. (E″″-H″″) Magnified views of the dorsal patch of ectopic Dac, as boxed in E‴. (E) Driver alone control showing the wild-type pattern of Dac expression. (F) eya misexpression induces ectopic Dac in the hinge (yellow arrow), pouch (blue arrow) and faintly throughout the dpp stripe in the pouch (orange arrow). (G) cg overexpression produces smaller wing discs with aberrant folds in the pouch and loss of the normal dorsal-ventral asymmetric shape and induces weak Dac expression in the pouch, both cell-autonomously and non-autonomously (orange arrows; note lack of overlap with Cg-expressing cells in the dpp stripe). (H) Wing discs co-overexpressing cg and eya no longer ectopically induce Dac in the hinge and pouch and display milder morphological disruptions than with cg overexpression alone. (I) In vitro GST pulldown assays show that Cg can directly bind Eya and can participate in a ternary complex with Eya-So (six independent replicates). Lanes 1-2, GST alone does not pull down So or FLAG-Eya486-760. Full-length So runs as a triplet and the asterisk marks co-purifying degradation products. Lanes 3-6, GST-So pulls down FLAG-Eya486-760. Lanes 7-10, GST-Cg225-404 pulls down FLAG-Eya486-760. Lanes 11-14, GST-Cg225-404 does not effectively pull down So. Lanes 15-18, FLAG-Eya486-760 increases the ability of GST-Cg225-404 to pull down So.