Fig. 5. Engrailed is a direct repressor of frizzled 2 expression. (A) fz2 expression was analyzed by in situ hybridization (in blue) in wild-type (WT) and engrailed mutant (en^-) genetic backgrounds. Embryos were double stained with anti-Engrailed antibody (in brown). (B) Schematic representation of the (1A4-GFP) transgene, where a trimer of 170 bp 1A4 fragment has been cloned upstream to hsp70 minimal promoter and GFP reporter gene. (C) GFP expression is shown in different genetic backgrounds: (1A4-GFP) corresponds to the normal expression of the transgene (1, 3, 5, 7, 9); (1A4-GFP/VP16-En) corresponds to GFP expression in the presence of the activator form of Engrailed, driven either by (hs-Gal4) (2, 4, 6, 8) or by (en-Gal4) (10). GFP expression was analyzed in either late L3 larval tissues (1-8) or in embryos (9, 10): (1, 2) hindgut (d, dorsal; v, ventral); (3, 4) midgut; (5, 6) salivary gland; (7, 8) wing imaginal disc; (9, 10) embryo. White arrowheads show the position of stripes. Bracket in 2 indicates GFP expression in both dorsal and ventral compartments. (D) GFP expression of late third instar larvae is shown in the hindgut (GFP) and can be compared with endogenous fz2 expression, detected by an anti-Frizzled 2 antibody in red (anti-Fz2) or with engrailed expression, detected by polyclonal anti-Engrailed antibody in red (anti-En). Merged images are shown.