Fig. 3. Mapping of EY and TOY binding sites on the so10 enhancer. (A) Sequence alignment of the so10 wild-type (wt) and mutated (mt) enhancer. The boxes indicate the protected regions revealed by the footprint experiment. The common EY- and TOY-binding sites are shaded grey and the TOY-specific binding sites are white. The bold letters on the wt so10 enhancer indicate the sequence most related to the Pax6 consensus sequence (P6CON), also shown in C. The 128 bp fragment used for the bandshift assay is underlined between the two arrowheads. (B) Footprint experiment on the wild-type (WT) and mutated (MT) so10 enhancer. Sites 1, 2 and 5 are protected by EY- and TOY-PD whereas sites 3 and 4 are TOY-PD specific. No binding was detected after mutagenesis (MT) of the five binding sites. (C) Sequence alignment of the five binding sites identified on so10 with the Pax6 consensus binding site (P6CON) (Epstein et al., 1994). The bases fitting with the consensus are shown in capital letters. (D) Wing discs where either EY or TOY were misexpressed by dpp^blink-Gal4 in flies carrying the lacZ reporter gene under the control of the so10^EY+TOYmt enhancer. In both cases no ß-galactosidase expression was detected indicating that the mutated enhancer was not inducible anymore by TOY or EY.