Engineered BAC deletions and comparative sequence analysis pinpoint critical Gdf6 joint regulatory sequences. (a) Recombination strategy used to engineer precise deletions between two 50-bp sequences (A and B boxes) in BAC C-βgeo. (b) Map of three deletions covering the previously defined elbow/knee regulatory region of the Gdf6 locus, and the location of the p(– 65/ – 62)HspLacZ plasmid construct. The 26-kb joint regulatory region initially defined by BACs C-βgeo and D-βgeo is indicated by the red bar. (Right) The number of joint-expressing embryos out of the total transgenic embryos. (c) Close-up of VISTA plot showing highly conserved DNA sequences within the subdomain that is essential for joint expression. The region corresponding to the 2.9-kb PCR fragment in p(– 65/ – 62)HspLacZ is shown. (d,e) Two independently generated embryos transgenic for p(– 65/ – 62)HspLacZ and stained with X-Gal. Arrowheads indicate expression in shoulder, elbow, and knee joints. (f,g) Cryosections through opposite side forelimb elbow (f) and hindlimb knee (g) of embryo shown in d. hu, humerus; ra, radius; ul, ulna; fe, femur; ti, tibia; fi, fibula.
