Fig 2: Expression of cyclooxygenases during chondrogenic differentiation from progenitor cells.A: Expression of COX-1 and COX-2 in growth plates was determined by IHC. RZ = resting zone, PZ = proliferative zone, HZ = hypertrophic zone. B: Col2a1, Col10a1, Sox9 and Runx2 mRNA expression during chondrogenic differentiation of ATDC5 cells. C: COX-1 and COX-2 mRNA expression. In graphs, error bars represent mean ± s.e.m.(standard error of the mean; n = 3). * indicates p < 0.05. D: Protein expression of COX-1 and COX-2 in differentiation. Molecular weight markers (kDa) are depicted on the left of immunoblots and relative quantifications are depicted on top of immunoblots. E: Medium concentrations of PGD2, PGE2, PGF2a and TXA2 during chondrogenic differentiation.

Fig 3: Generation and gross phenotype of Col2a1-Cre; Npr2flox/flox mice.(a) Conditional targeting of Npr2. Restriction enzymes: E, EcoRI; S, SacI; V, EcoRV; Arrows: PCR primers. Triangles (black): loxP sites. Triangles (white): FRT sites. (b) RT-PCR analysis of Npr2 gene in cartilage and brain of Col2a1-Cre; Npr2flox/flox and Npr2flox/flox mice. The full-length gel is presented in Supplementary Figure S4. (c) Quantitative real-time RT-PCR analysis for GC-B expression in Npr2flox/flox and Col2a1-Cre; Npr2flox/flox mice. (d) Immunohistochemical analysis of GC-B in growth plates of Npr2flox/flox and Col2a1-Cre; Npr2flox/flox mice. (e) Gross appearance of Npr2flox/flox (upper) and Col2a1-Cre; Npr2flox/flox (lower) mice. (f) Growth curves of naso-tail length of Npr2flox/flox (¦) and Col2a1-Cre; Npr2flox/flox (?) mice. *: P < 0.01.

Fig 4: Impaired skeletal growth in Col2a1-Cre; Npr2flox/flox mice.(a) Soft x-ray picture and (b) graph of lengths of bones (skull length, skull width, humerus, radius, ulna, femur, tibia, and lumber) of Npr2flox/flox and Col2a1-Cre; Npr2flox/flox mice at 10-week-old. *: P < 0.01, n = 5, each. (c)–(f) Histological analyses of tibial growth plates of Npr2flox/flox and Col2a1-Cre; Npr2flox/flox mice at the age of 2 weeks. Scale bar: 100 µm in each panel. (c) Micrographs of growth plates stained by Alcian Blue-hematoxylin and eosin (left pictures), and the thickness of growth plates (right graph). *: P < 0.01, n = 5, each. In micrographs, right panel in each set of panels is exhibited with higher magnification. (d), (e) Pictures of tibial growth plate stained by type II (d) and X (e) collagen antibodies (upper panels), and the thickness of nonhypertrophic (d) and hypertrophic (e) chondrocyte layers of the growth plates (lower graphs). *: P < 0.01, n = 5, each. (f) Pictures of tibial growth plates with BrdU staining (left panels) and the proliferative rate of chondrocytes in growth plate shown as the average percent of BrdU-positive cells relative to the total cell count (right graph). *: P < 0.05, n = 5, each.

Fig 5: Regeneration involves skeletal cells with hybrid osteochondral properties.(A) Schematic of the murine rib resection model. A 3 mm bone segment is resected from one rib (8-11), while the periosteum is carefully released and left in the mouse. Alizarin red and alcian blue whole mount staining indicates that repair occurs through a cartilage intermediate. The images show the outcome immediately after the resection at 0 wpr (weeks post resection, n = 2). At 1 wpr alcian blue positive material is evident between the cut ends (n = 3), by 4 wpr (weeks post resection) the lesion is fully-spanned by a mineralized callus (n = 2), while by 10 wpr remodeling has occurred (n = 2). (B–D) Histological sections stained with hematoxylin and eosin (H and E) (n > 5 for each time point) and near-adjacent double fluorescent RNA in situ hybridization (RNA-ISH) assays confirm the presence of a cartilage intermediate and show expression patterns in the repair callus. (B) At 5 dpr (days post resection) mesenchymal-like progenitor cells have moved into the resected region and are positive for the expression of both Sox9 (green) and Runx2 (red). The enlarged boxes with the separated color channels show co-expression in many of the cells within the resected region (overlap is yellow) (n = 3). (C) By 10 dpr bone and cartilage formation spans the resected region. Many cells that mediate repair express both the chondrocyte-associated gene Col2a1 (green), as well as the osteoblast-associated gene Bglap (red). The enlarged boxes show cells that have chondrocyte morphology expressing both Col2a1 and Bglap (n = 3). (D) At 14 dpr trabecular bone spans almost the entire resected region with only a small amount of cartilage at the cut ends; cells expressing both Col2a1 (green) and Col1a1 (red) are widespread. The enlarged boxes show the surface of newly formed trabecular bone where cells can be found that co-express Col1a1 and Col2a1 (n = 4). (E) At 10 dpr, animals double transgenic for Col1(2.3)-GFP;ColX-mCherry have mCherry (red) positive cells that are also expressing the osteoblast-specific reporter for Col1 (green). (F) Expression of Col2a1 (green) and Col1a1 (red) of the rib growth plate from an uninjured animal does not show a high degree of overlap (n = 5). Col2a1 is highly expressed in chondrocytes of the growth plate but not in osteoblasts forming new bone, while Col1a1 is highly expressed in the osteoblasts/cytes below the growth plate but not in cartilage cells. Scale bar for A = 500 microns, B-F = 200 microns, enlarged box H and E = 100 microns, enlarged box = 50 microns.