Endochondral bone formation is largely dependent on cartilage lineage cells. The chondrocytes in growth plates continuously undergo a sequential process from proliferation to terminal hypertrophic differentiation [1]. Once differentiated, hypertrophic chondrocytes elicit multiple functions such as determining bone length, inducing osteogenesis as well as directing bone mineralization, and eventually disappear at the chondro-osseous junction. The fate of the terminally differentiated hypertrophic chondrocytes is conceptually important for understanding its role in endochondral bone formation. It has been debated for decades whether the terminally differentiated hypertrophic chondrocytes die by apoptosis or undergo osteogenic transdifferentiation, however, clear in vivo evidence is lacking [2]. Here, through lineage tracing, we provide the first in vivo evidence that the terminally differentiated hypertrophic chondrocytes are a potent source of osteoblasts, and retain multi-lineage differentiation potential.

First, we generated a novel Col10a1int2-Cre transgenic mouse strain in which Cre expression was under the control of an 8.2 kb promoter and a 3.2 kb 2nd intron of the mouse type X collagen gene (Col10a1), a specific marker for hypertrophic chondrocytes (Figure 1A). In situ hybridization analysis of Col10a1int2-Cre transgenic mice showed that Cre+ cells were exclusively restricted to the hypertrophic zone, but not detectable in the metaphysis and bone marrow cavity from embryonic day 14.5 (E14. 5) to postnatal day 10 (P10)(Figure 1A and Supplementary information, Figure S1A, S1B). Furthermore, double staining showed that all Cre+ cells co-expressed Col10a1, but not Col1a1 (a marker for osteoblasts), endomucin (a marker for endothelial cells) or perilipin (a marker for adipocytes)(Supplementary information, Figure S1A, S1B). In addition, RT-PCR detected Cre-hGH mRNA in growth plate cartilage, but not in bone marrow or cortical bone of Col10a1int2-Cre transgenic mice (Supplementary information, Figure S1C). These data demonstrate that this Col10a1int2-Cre strain is specific for hypertrophic chondrocytes.