Fig. 1. Hedgehog signaling promotes chondrocyte hypertrophy in the absence of PTHrP signaling. (A,B) Forelimbs of PTHrP^-/- or wild-type E14.5 embryos cultured for 36 hours in the presence or absence of recombinant Shh protein or cyclopamine. Treated limbs were compared with untreated contralateral control ones. Serial sections of the proximal humerus of the cultured limbs were hybridized with ³⁵S riboprobes of Ihh (A) or Col10a1 (B). The distances from the articular end to the Ihh/Col10a1 domain in the untreated wild-type and PTHrP^-/- embryos are indicated by orange lines. This distance was increased in the Shh-treated wild-type humerus but reduced in the Shh-treated PTHrP^-/- humerus (white lines) compared with the untreated contralateral controls. This distance was reduced in the cyclopamine-treated wild type, but increased in the PTHrP^-/- humerus (green lines) compared with the untreated controlateral controls. (C) Statistical analysis (paired Student's t-test) of the distance from articular chondrocytes to the hypertrophic zone between treated and untreated contralateral limbs. Numbers (n) of analyzed limbs are indicated. (D) Serial sections of E14.5 distal humeri of the indicated genotypes stained with Safranin O (upper panel) and hybridized with a ³⁵S Col10a1 riboprobe (lower panel) to detect chondrocyte hypertrophy. The distance from the articular end to the Col10a1 expression domain (orange lines) in the PTHrP^-/- embryos was reduced compared with that in the wild-type embryos. This distance in the PTHrP^-/-;UAS-cIhh;Col2a1-Gal4 double mutant was further reduced compared with that of the PTHrP^-/- embryos.