Fig. 6. Mutating the putative So binding sites in 3EE abolishes enhancer activity in vivo. (A) A multiple sequence alignment of the 40 conserved bases in the 3' eye enhancer. Mismatched bases are shown in grey and the two putative So-binding sites are shown in green. Mutated So-binding sites are shown in red. (B-E) Each panel shows a single eye disc co-stained with anti-Dac (blue) and anti-GFP (green). Mutating each putative So-binding site individually (C,D) results in the dramatic reduction of GFP reporter expression from the 3EE enhancer when compared with the wild-type version of same enhancer (B). When both So-binding sites are mutated, enhancer activity and GFP expression is completely abolished (E). Endogenous Dac expression is unaffected in all cases.