The parathyroid hormone/parathyroid hormone-related peptide receptor coordinates endochondral bone development by directly controlling chondrocyte differentiation

Abstract

During vertebrate limb development, growth plate chondrocytes undergo temporally and spatially coordinated differentiation that is necessary for proper morphogenesis. Parathyroid hormone-related peptide (PTHrP), its receptor, the PTH/PTHrP receptor, and Indian hedgehog are implicated in the regulation of chondrocyte differentiation, but the specific cellular targets of these molecules and specific cellular interactions involved have not been defined. Here we generated chimeric mice containing both wild-type and PTH/PTHrP receptor (-/-) cells, and analyzed cell-cell interactions in the growth plate in vivo. Abnormal differentiation of mutant cells shows that PTHrP directly signals to the PTH/PTHrP receptor on proliferating chondrocytes to slow their differentiation. The presence of ectopically differentiated mutant chondrocytes activates the Indian hedgehog/PTHrP axis and slows differentiation of wild-type chondrocytes. Moreover, abnormal chondrocyte differentiation affects mineralization of cartilaginous matrix in a non-cell autonomous fashion; matrix mineralization requires a critical mass of adjacent ectopic hypertrophic chondrocytes. Further, ectopic hypertrophic chondrocytes are associated with ectopic bone collars in adjacent perichondrium. Thus, the PTH/PTHrP receptor directly controls the pace and synchrony of chondrocyte differentiation and thereby coordinates development of the growth plate and adjacent bone.

Figure 1

Ectopic differentiation of PTH/PTHrP receptor (−/−) chondrocytes and elongation of chimeric growth plates. (A and B) Sections of the tibiae from d17.5 wild-type embryos (A) and d17.5 wild-type embryos containing cells with one β-galactosidase transgene (stained blue) and cells with no transgene (B) were stained for β-galactosidase activity and counterstained with nuclear fast red. Cells with and without the transgene behave indistinguishably. p, proliferating layer; h, hypertrophic layer including prehypertrophic chondrocytes. (C and D) Sections of the tibiae from d17.5 chimeric embryos containing both wild-type and PTH/PTHrP receptor (−/−) cells were stained for β-galactosidase activity and counterstained with nuclear fast red. PTH/PTHrP receptor (−/−) cells (stained blue) ectopically differentiate into hypertrophic-like cells in the middle of wild-type proliferating chondrocytes. All mutant cells below a line approximately corresponding to the level at which the growth plate narrows (arrowhead) appear hypertrophic. (E–H) Sections of the tibiae from wild-type (E) and chimeric embryos with various contributions of PTH/PTHrP receptor (−/−) cells (F–H) were stained for β-galactosidase activity and counterstained with H&E. Contributions of mutant cells are estimated ≈10% (F), 30% (G), and 60% (H). In proportion to the contribution of mutant cells, columns of wild-type proliferating chondrocytes elongate (two-headed arrows). (Bar = 100 μm.)

Figure 2

Expression of type X collagen and Ihh by ectopically differentiated PTH/PTHrP receptor (−/−) chondrocytes. (A–D) In situ hybridization of sections of the humeri from d17.5 embryos with a mouse type X collagen antisense probe. In the wild-type (A, bright field; B, dark field), type X collagen is expressed in hypertrophic chondrocytes. In the chimera containing both wild-type and PTH/PTHrP receptor (−/−) cells (C, bright field; D, dark field), ectopically differentiated mutant cells also express type X collagen in the proliferating layer. (E–H) In situ hybridization of sections of the humeri from d17.5 embryos with a mouse Ihh antisense probe. In the wild-type (E, bright field; F, dark field), Ihh is expressed in prehypertrophic and hypertrophic chondrocytes. In the chimera (G, bright field; H, dark field), ectopically differentiated mutant cells also express Ihh in the proliferating layer. (Bar = 200 μm.)

Figure 3

Up-regulation of Ptc and PTHrP expression in chimeric growth plates. (A–D) In situ hybridization of sections of the humeri from d17.5 embryos with a mouse Ptc antisense probe. In the wild-type (A, bright field; B, dark field), Ptc is expressed in proliferating chondrocytes, most strongly adjacent to Ihh-producing prehypertrophic chondrocytes, with expression decreasing toward the articular surface. In the chimera containing both wild-type and PTH/PTHrP receptor (−/−) cells (C, bright field; D, dark field), proliferating chondrocytes surrounding ectopic hypertrophic chondrocytes overexpress Ptc mRNA. (E–H) In situ hybridization of sections of the humeri from d17.5 embryos with a rat PTHrP antisense probe. In the wild-type (E, bright field; F, dark field), PTHrP is weakly expressed in the periarticular region. In the chimera (G, bright field; H, dark field), PTHrP expression is strongly up-regulated in the same region. (Bar = 200 μm.)

Figure 4

Ectopic mineralization of cartilaginous matrix and ectopic bone collar formation in chimeric bones. (A–D) Sections of the tibiae from d17.5 embryos were stained for β-galactosidase activity and also with alizarin red S for detection of mineralization. In the wild-type (A), mineralization of cartilaginous matrix occurs adjacent to late hypertrophic chondrocytes (arrows). No mineralization is observed adjacent to ectopic hypertrophic chondrocytes in the tibiae from chimeras with ≈60% contribution of PTH/PTHrP receptor (−/−) cells (B), whereas in chimeras with >90% contribution, matrix adjacent to ectopic hypertrophic chondrocytes in large clusters mineralize (C and D, arrows). (E–H) Sections of the radii from d17.5 chimeric embryos with ≈60% contribution of mutant cells were stained for β-galactosidase activity and also stained with alizarin red S (E) or H&E (F–H). In the wild-type (A), bone collars are formed in the perichondrium abutting prehypertrophic and hypertrophic chondrocytes (arrowheads). No ectopic bone collar formation is observed in the tibiae of chimeras with ≈60% contribution (B). However, in smaller bones such as the radii of the same chimeras, ectopic bone collars are formed (E and F, arrowheads). At higher magnification, ectopic bone collars are seen near clusters of ectopic hypertrophic chondrocytes; ectopic bone collars include both wild-type and PTH/PTHrP receptor (−/−) osteoblasts (stained blue) (G and H, arrowheads). In the tibiae of chimeras with >90% contribution, there is intensive ectopic bone collar formation (C and D). (Bar = 100 μm.)