Ihh determines the location of bone collar formation. (a–d) Sections of the tibiae from d17.5 embryos were stained by von Kossa method and counterstained with Nuclear Fast Red. Mineral is stained black; nuclei are stained red. In the wild-type growth plate (a), bone collars are formed in the perichondrium adjacent to the layers of prehypertrophic and hypertrophic chondrocytes, whereas in the PPR^–/–/wild-type chimeric growth plate (b), ectopic bone collars are induced in the perichondrium adjacent to a cluster of ectopic hypertrophic chondrocytes (brackets). Despite the presence of clusters of ectopic hypertrophic chondrocytes, no ectopic bone collar is induced in the Ihh^–/–;PPR^–/–/wild-type chimeric growth plate (c and d). Arrowheads denote where the eutopic prehypertrophic layer starts. Calcification inside the growth plate (d, asterisk) is ectopic cartilaginous mineralization caused by mutant hypertrophic chondrocytes. (e–g) In situ hybridization of sections of the anterior portion of the ribs from newborn mice with a mouse type X collagen antisense probe. This portion of the wild-type ribs consists of proliferating chondrocytes (e), whereas chondrocytes in the PTHrP^–/– ribs ectopically hypertrophy and express type X collagen mRNA (f). Introduction of a constitutively active PPR transgene driven by the type II collagen promoter (caPPR) reverses this ectopic hypertrophy (g). (h–j) Sections of the anterior portion of the ribs from newborn mice were stained by von Kossa method and counterstained with Nuclear Fast Red. Mineral is stained black; nuclei are stained red. The wild-type ribs have no bone collars (h), whereas the PTHrP^–/– ribs have bone collars in association with the presence of ectopic hypertrophic chondrocytes (i). The PTHrP^–/–;caPPR ribs, however, do not have bone collars, in association with the disappearance of ectopic hypertrophic chondrocytes (j). (k and l) Sections of the humeri from d17.5 Ihh^–/–/wild-type chimera embryos were stained for β-galactosidase activity as well as stained by von Kossa method and counterstained with Nuclear Fast Red. Wild-type cells are stained blue; mineral is stained black; nuclei are stained red. In these mice, bone collars do not form in the perichondrium adjacent to clusters of Ihh^–/– prehypertrophic/hypertrophic chondrocytes (arrowhead denotes where the prehypertrophic layer starts), whereas bone collars do form in the perichondrium adjacent to clusters of wild-type prehypertrophic/hypertrophic chondrocytes (brackets). Horizontal bar = 100 μm.