Fig. 4. Engrailed misexpression induces changes in the expression of potential Engrailed target genes. In situ hybridization using anti-sense RNA probes were carried out on late third instar wing imaginal discs in different genetic backgrounds: wild-type (WT) (D,G,J,M,P); MS1096 X UAS-VP16-En (E,H,K,N,Q); MS1096X UAS-En (F,I,L,O,R). Overexpression of (VP16-En), driven by MS1096-Gal4, was detected in the wing pouch, using anti-HA antibody (B), when compared with wild type (A). Overexpression of the normal form of Engrailed was detected with polyclonal anti-En antibody (C). Note that (VP16-En) overexpression leads to a posterior distortion of the disc, whereas Engrailed (En) overexpression leads to an anterior distortion. (D-F) In situ hybridization with ß3-tubulin probe. (G-I) In situ hybridization with frizzled 2 probe. (J-L) In situ hybridization with hibris probe. (M-O) In situ hybridization with branchless probe. Normal expression of bnl is indicated by an arrowhead. Arrows indicate the ectopic expression of bnl in the wing pouch. (P-R) In situ hybridization with frazzled probe. Discs are oriented anterior leftwards, ventral upwards.