Fig. 2. Significantly reduced Ihh expression, delayed chondrocyte and osteoblast differentiation in the Wnt5a mutant. (A) Early chondrogenic defect in the limb was examined using whole-mount in situ hybridization. In the Wnt5a mutant (below), Sox9 expression was not detected from the earliest stages of chondrogenic condensation in the distalmost limb mesenchyme (arrowheads). Ihh expression was also significantly reduced. The arrowhead indicates the future humerus. (B) Sections of forelimbs from 12.5 dpc embryos, except those hybridized with the Pthlh probe were from 13.5 dpc forelimbs. The expression of Wnt5a, Ihh and Col10a1 was reduced to below detectable levels in the cartilage of the mutant. Wnt5a expression in the distal limb (arrows) of the mutant was normal. Pthrpr and Cbfa1 expression was significantly reduced, but Pthlh (PTHrP in figure) expression (arrowheads) in the mutant was comparable with that in the wild-type littermate. (C) Strong Ihh expression was detected in the humerus and a few cells in the ulna in the mutant at 15.5 dpc (arrowheads); Ptch was also expressed in the ulna (arrowhead); Pthrpr expression (arrowhead) was weaker and no Col10a1 expression was detected; Cbfa1 expression was reduced in both chondrocytes and perichondrium (arrowhead); osteocalcin was not detected in the mutant. H, humerus; R, radius; U, ulna.