Fig. 7. An Nkx2 binding site is required for SBE3 enhancer function. (A) Vista plot of the alignment between human and mouse SBE3 sequences. The target recognition sequence for the Nkx2 homeodomain protein (red) is present in both human and mouse SBE3 sequences. (B) Binding of Nkx2.1 protein to a site in the SBE3 sequence. Cell lysates transfected with pCMV (lane 1) or pCMV-Nkx2.1 (lanes 2-6) were analyzed for binding to a 39 bp probe overlapping the Nkx2 recognition sequence in SBE3. Wild-type cold competitor (lanes 3, 4) interfered with binding of Nkx2.1 protein, while mutant competitor with nucleotide substitutions in the core binding site (AAGTAGGGAGCA) did not alter the shifted complex (lanes 5, 6). (C-F) X-gal staining of transgenic embryos carrying a wild-type (C,E) or mutant SBE3 reporter construct in which the Nkx2 core recognition sequence (AAGTAG) was deleted (D,F). Embryos carrying the wild-type SBE3 reporter construct show consistent X-gal staining in the svz of the mge (C,E). By contrast, embryos carrying an SBE3 reporter construct lacking the Nkx2 site (Nkx2) showed no staining in the ventral forebrain (D,F). Ectopic X-gal staining in the hindbrain (asterisk) of embryos carrying either wild-type or mutant SBE3 transgenes was detected in equal frequency and thus served as an internal staining control. The ratio of embryos exhibiting reproducible Shh-like reporter activity over the total number of transgenic embryos is indicated for each construct.