Fig. 3. Identification of a Dlx-regulated enhancer upstream of Gbx2. (A) Evolutionary conservation of the genomic sequence upstream of Gbx2 analyzed using 30-way multiz alignment (Blanchette et al., 2004). Image generated using University of California Santa Cruz Genome Browser. Blue, degree of conservation among mammals; black vertical bars, conservation in each species as indicated; red bar, the Gbx2 enhancer used for the reporter assay in C; black box, coding region of an exon; white box, untranslated region. (B) ClustalW alignment (Larkin et al., 2007) of the 0.6 kb region of the Gbx2 enhancer conserved down to chicken. ^*, conserved nucleotides; putative Dlx-binding sites are highlighted in orange. (C) Results of the luciferase reporter activation assay. pGL, minimal promoter-reporter plasmid without an enhancer; Gbx2en, pGL plasmid with mouse Gbx2 enhancer; - Dlx5, co-transfected with empty expression vector; + Dlx5, co-transfected with Dlx5 expression vector.