Predicted homeodomain binding sites are required for R2 expression domains in specific joints.
(A) Eight predicted homeodomain binding sites (S1-S8, blue boxes) are found across R2 subregions. Four of these UNIPROBE predicted sites are found in both mice and humans, including S2 in sub-region R2a-c, S5 and S6 in sub-region R2d, and S8 in sub-region R2e. (B) Targeted mutagenesis of conserved S6 within the hindlimb regulatory zone of R2 eliminates normal lacZ signal in the knee (red arrowhead) but not elbow or shoulder, tissues where this enhancer region is predicted to have little to no influence on expression. Mutagenesis of sites S(7+8) within the forelimb regulatory zone of R2 eliminates expression in shoulder (red arrowhead) but not elbow or knee, tissues where this sequence is predicted to have little to no influence on expression. (C) Histology at E14.5 of wild-type R2 (left) versus mutant R2 (right) constructs reveals specific reductions (red arrowheads) in joint domains for each construct. Abbreviations: s, shoulder; e, elbow; k, knee; sc, scapula; h, humerus; f, femur; t, tibia.