Generation of Mmp13^–/– mice. (A) Schematic representation of the WT Mmp13 locus, the targeting vector, the targeted allele, the position of EcoRV sites, and the probe used for Southern blot analysis. (B) Southern blot analysis of genomic DNA from littermate progeny of Mmp13^+/– breeding. Probes of EcoRV-digested DNA identified the fragments of 8.5 and 7.3 kb for WT and disrupted alleles, respectively. (C) Northern blot analysis of calvarial RNA after calvarial organ cultures for 5 days with or without IL-1α or PTH, and Western blot analysis of conditioned medium from these organ cultures. Levels of Mmp13 mRNA increased in WT calvariae exposed to IL-1α or PTH in contrast with Mmp13^–/– calvariae. A marked increase in Mmp13 (Ab:Mmp13) is seen in Western blots from WT calvarial conditioned medium, but no increase in Mmp13 is seen in Mmp13^–/– calvarial conditioned medium. (D) Skeletal development in WT and Mmp13^–/– 15.5- to 18.5-dpc embryos. Sections of proximal tibias were stained with Alcian blue. Increases in length of the hypertrophic zones of the growth plates, indicated by the brackets, are seen in the Mmp13^–/– tibias. (Scale bar: 400 μm.)

Analysis of long bones from WT and Mmp13^–/– mice. (A) Sections of femurs from three different WT and Mmp13^–/– 15.5-dpc embryos stained with hematoxylin/eosin. The top panels are low-power views, and the other panels are higher-power views. Primary ossification centers are fully formed in WT samples, but only periosteal cuffs (arrows) are fully formed in Mmp13^–/– samples. (Scale bar: 300 μm.) (B) Adjacent sections of femurs and tibias from 15.5-dpc embryos. In situ hybridization was used for analysis of Mmp13 and -9 mRNAs, and immunohistochemistry was used for analysis of CD31 (seen as red on blue-black background). Note CD31 (arrows) in primary centers of WT bones but restricted to periosteal cuffs in Mmp13^–/– bones. (Scale bar: 300 μm.) (C) Higher-power images of mid-femurs shown in B. (Scale bar: 300 μm.) (D) Mid-femurs as in C. In situ hybridization was used for analysis of Vegf and Col10a1 mRNAs. (Scale bar: 300 μm.) (E) Mid-femurs as in A stained for TRAP. Note the TRAP⁺ cells (arrows). (Scale bar: 300 μm.)

Proximal tibias from 17.5-dpc embryos showing TRAP⁺ cells (arrows) and analysis by in situ hybridization for Mmp13,-9, and -14 mRNAs. (Scale bar: 300 μm.)

Analysis of tibias and femurs. (A) In situ hybridization for Osteopontin and Col10a1 mRNAs in proximal tibias from 18.5-dpc embryos. Brackets indicate the lengths of hypertrophic zones. (Scale bar: 300 μm.) (B) Proximal femurs from newborn (P0) mice. Sections were stained for type X collagen and for QRGIV epitope in collagenolytic TC^B fragments. Brackets indicate the lengths of hypertrophic zones. Note the absence of anti-QRGIV staining [shown as red on blue-black background] in distal growth plates and primary centers of Mmp13^–/– bones. (Scale bar: 300 μm.) (C) SDS/PAGE stained with Coomassie blue (left) and Western blots (right) of collagenase (MMP13) digests of types I and II collagens. The TC^{Bα 1} fragments are indicated by arrows. The faster-moving TC^Bα2(I) fragment of type I collagen moved off the gel. The QRGIV antibody, which detects the neoepitope in the TC^{Bα 1(I)} and TC^{Bα 1(II)} collagenase-cleavage fragments of types I and II collagens, respectively, was used in the immunoblots (IB:QRGIV).

Sections of distal femoral growth plates from 12-wk-old WT and Mmp13^–/– mice. Brackets indicate the approximate lengths of the growth plates. Higher-magnification views of the boxed areas in Left are shown in Right. Note the increased lengths of the growth plates and the irregular arrangement of the chondrocyte columns in Mmp13^–/– samples vs. WT samples. (Scale bar: 300 μm.)