Chondrocytes Transdifferentiate Into Osteoblasts In Endochondral ...

Abundance of Col10a1-Cre induced reporter+ cells throughout the primary spongiosa of Col10a1-Cre reporter-containing embryos and mice

In Col10a1-Cre transgenic mice, Cre recombinase activity was previously detected specifically in all hypertrophic chondrocytes starting from E13.5 throughout endochondral skeletal development and into the postnatal stage [18]. Here we further confirmed that in the femurs and tibias of E15.5 Col10a1-Cre; ROSA26R mice, only hypertrophic chondrocytes, not cells in the perichondrium and periosteum, were positive for LacZ (S1B-a, b Figure.), indicating that Cre activity driven by the Col10a1 regulatory elements occurred specifically in hypertrophic chondrocytes in these Col10a1-Cre transgenic mice. This was also confirmed by in situ hybridization of Col10a1 and Cre mRNA which was only observed in the hypertrophic zone (S1C Figure). To test the hypothesis that some of the Col10a1-expressing hypertrophic chondrocytes might transdifferentiate into osteoblasts, we crossed Col10a1-Cre with Osx flox/flox mice to generate Col10a1-Cre; Osx flox/+ embryos. In these embryos EGFP expression labels Osx-expressing cells, which either expressed Col10a1 or were derived from Col10a1-expressing cells (Figure S1A).

Immunofluorescence (IF) with anti-EGFP showed that in the femurs of Col10a1-Cre; Osx flox/+ embryos, abundant EGFP-positive (EGFP+) cells were present throughout the primary ossification centers (Fig. 1), where only very few, if any, Col10a1- or Cre-expressing cells were detected (S1C Figure). The appearance of these EGFP+ cells was concurrent with the onset of primary ossification at E15.5 (Fig. 1A). These EGFP+ cells continued to be present in the primary spongiosa at E16.5 (Fig. 1B), E18.5 (Fig. 1C) to after birth (Fig. 1D). The EGFP expression levels of these cells increased from E15.5 to E18.5 in parallel to the increasing levels of Osx expression in the same regions. In the 2-week-old Col10a1-Cre; Osx flox/+ mice, EGFP+ cells were found throughout the trabecular surfaces, also lining the endosteum of the distal half of the femur, and even embedded within the bone matrix of the cortical bone and trabeculae (Figure S1D, Fig. 1D).

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Figure 1. Presence of abundant EGFP+ (Osx−/+) cells throughout the primary spongiosa of Col10a1-Cre; Osxflox/+ embryos and mice.

Anti-EGFP immunofluorescence (IF) showed that EGFP+ (Osx−/+) cells (red) were found in the primary spongiosa of E15.5 (A), E16.5 (B) and E18.5 (C) Col10a1-Cre; Osx flox/+ femurs (right panels), but not in the Osx flox/+ control embryos (left panels). The controls showed auto-fluorescence produced by mineralized tissue and bone marrow cells, but no EGFP+ cells. Upper panels: anti-EGFP (red); Lower panels: anti-EGFP and DAPI (blue). There were virtually no EGFP+ cells in the perichondrium (between white arrows) or periosteum (between white arrowheads) of the Col10a1-Cre; Osx flox/+ embryos. D: In the femurs of 2-week-old Col10a1-Cre; Osxflox/+ mice, EGFP+ (Osx−/+) cells (green arrows) were found throughout the trabeculae (panel a), on the endosteum surface (panel b), and embedded within the cortex (green asterisks). Red arrowhead: periosteum; Green arrowhead: endosteum; gp: growth plate (white brackets); ps: primary spongiosa; bm: bone marrow. In the Osx flox/+ control embryos, weak EGFP+ cells were present in the perichondrium and periosteum area (also in Fig. 3C), indicating that there was a low level read through of EGFP independent of Cre-mediated excision of floxed Osx allele.

https://doi.org/10.1371/journal.pgen.1004820.g001

Such EGFP+ non-chondrocytic cells were completely absent in intramembranous skeletal elements such as the calvariae of E18.5 Col10a1-Cre; Osx flox/+ embryos (Figure S1E). In addition to its expression in hypertrophic chondrocytes and osteoblasts in the primary spongiosa, Osx was also highly expressed in periosteum and perichondrium [16]. However, EGFP+ cells were only detected in primary ossification centers and, as to be expected at lower levels in the hypertrophic zone, not in periosteum and perichondrium. This indicated that these EGFP+ non-chondrocytic cells were cells in which the Osx floxed allele was recombined by Col10a1-Cre and that these cells were unlikely to be derived from periosteum or perichondrium cells (Fig. 1). Staining of EGFP+ hypertrophic chondrocytes was not strong enough to be clearly shown due to strong autofluorescence from mineralized cartilage and bone matrix and from blood cells in the bone marrow.

Similarly, Col10a1-Cre induced Tomato+ cells were found throughout the primary spongiosa of Col10a1-Cre; ROSA-tdTomato embryos (Fig. 2A, B and C) in the same pattern as in the Col10a1-Cre; Osx flox/+ embryos. This result confirmed our observation that cells derived from Col10a1-Cre expressing hypertrophic chondrocytes were indeed present in the primary spongiosa. In the femurs of 6-month-old Col10a1-Cre; ROSA-tdTomato;Osx flox/+ mice, Tomato+ cells continued to be present, however, the number of these cells on the endosteum surface was reduced and these cells were distributed more evenly from endosteum to periosteum within the cortex (S1F Figure). By 8 months, there were almost no Tomato+ cells present in the primary spongiosa. The Tomato+ cells were distributed mostly within the bone matrix or in the growth plate. Besides a few strong Tomato+ chondrocytes in the growth plate, the fluorescence intensity of a majority of Tomato+ cells were much weaker than in the younger mice (Figure S1G).

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Figure 2. Presence of abundant Tomato+ cells throughout the primary spongiosa of Col10a1-Cre; ROSA-tdTomato embryos.

Col10a1-Cre;ROSA-tdTomato embryos were generated to verify the observations made in the Col10a1-Cre;Osx flox/+ embryos. Abundant Tomato+ non-chondrocytic cells (red) were present throughout the primary spongiosa in femurs of E15.5 (A), E16.5 (B) and E18.5 (C) Col10a1-Cre;ROSA-tdTomato embryos (right panels). Tomato+ non-chondrocytic cells were completely absent from the ROSA-tdTomato control embryos in all stages evaluated (left panels in A and B, upper panel in C). Upper panels in A and B and middle panel in C: red channel; Lower panels: red and blue channels. Right panels in C: magnified images. There were virtually no Tomato+ cells within the perichondrium (between white arrows) or periosteum (between white arrowheads) of the Col10a1-Cre; ROSA-tdTomato embryos. gp: growth plate (white brackets); ps: primary spongiosa; bm: bone marrow.

https://doi.org/10.1371/journal.pgen.1004820.g002