Fig. 6. Mutation of a conserved putative Pitx1 binding site in HLEA results in reduced hindlimb enhancer activity. (A) VISTA plot comparing 768 bp subregion of mouse HLEA against human, cat, dog, cow, armadillo, elephant, opossum and platypus sequences. Horizontal panels show sequence identity across the region, ranging from 50-100%. Pink shading indicates regions of 20 bp or more that are 65% identical. Arrowheads indicate positions of putative Pitx1 binding sites, including one that is perfectly conserved (red arrowhead). The accompanying sequence alignment (beneath) shows perfectly conserved bases (asterisks), the sequence of the wild-type Pitx1 site (red), and the sequence of the mutated site in Pitx1Mut. (B) Plot of the area of lacZ staining in forelimbs and hindlimbs of transgenic mouse embryos carrying wild-type (HLEA-768) or mutant (Pitx1Mut) enhancers. Each data point represents an individual transgenic embryo. (C,D) Whole-mount lacZ staining of E12.5 transgenic mouse embryos carrying wild-type (C) and mutant (D) enhancers.