Sox9 is a direct transcriptional activator of cartilage-specific extracellular matrix genes and has essential roles in chondrogenesis. Mutations in or around the SOX9 gene cause campomelic dysplasia or Pierre Robin Sequence. However, Sox9-dependent transcriptional control in chondrogenesis remains largely unknown. Here we identify Wwp2 as a direct target of Sox9. Wwp2 interacts physically with Sox9 and is associated with Sox9 transcriptional activity via its nuclear translocation. A yeast two-hybrid screen using a cDNA library reveals that Wwp2 interacts with Med25, a component of the Mediator complex. The positive regulation of Sox9 transcriptional activity by Wwp2 is mediated by the binding between Sox9 and Med25. In zebrafish, morpholino-mediated knockdown of either wwp2 or med25 induces palatal malformation, which is comparable to that in sox9 mutants. These results provide evidence that the regulatory interaction between Sox9, Wwp2 and Med25 defines the Sox9 transcriptional mechanisms of chondrogenesis in the forming palate.

During the healing process after bone fracture, soft callus forms adjacent to the fracture site, is replaced by hard callus, and is finally remodeled to the original bone configuration. Although the cambium layer of the periosteum is reported to play an essential role in callus formation, we still lack direct in vivo evidence of this. To investigate the cell lineage of the soft callus, we analyzed the process of fracture healing in Prx1-Cre;ROSA26 reporter (R26R), Col1a1(3.6 kb)-Cre;R26R, Col1a1(2.3 kb)-Cre;R26R, Sox9-CreERT2;R26R, and Sox9-LacZ mice with X-gal staining. In the Prx1-Cre;R26R, in which the cells of the periosteum stained for X-gal before fracture, all cells in the soft callus were X-gal positive, whereas in the Col1a1(3.6 kb)-Cre;R26R mice, the cells in the periosteum before fracture stained for X-gal and the soft callus was partly composed of X-gal-positive cells. In contrast, in the Col1a1(2.3 kb)-Cre;R26R mice, in which the mature osteoblasts in the cambium layer of the periosteum were marked before fracture, no cells in the soft callus at the fracture site were X-gal positive. These results suggest that most of the cells in the soft callus are derived from the mesenchymal progenitors in the periosteum, and not from mature osteoblastic cells. Interestingly, in the Sox9-LacZ mice, Sox9-expressing X-gal-positive cells emerged in the periosteum adjacent to the fracture site 3 days after fracture. We demonstrated this by injecting tamoxifen into the Sox9-CreERT2;R26R mice for 3 days after fracture, so that these Sox9-expressing periosteal cells gave rise to cells in the soft and hard calli. Our findings show that the periosteal cells in which Sox9 expression is induced just after fracture are the major source of the chondrocytes and osteoblasts in the fracture callus.

Sox9 belongs to the family of Sry-related high-mobility group box transcription factors controlling cell fate, cell proliferation and differentiation in various tissues, including cartilage, testis, the central nervous system, kidney, and gastrointestine. Mice conditionally lacking Sox9 have revealed essential roles for Sox9 in these tissues. To gain further insight into the role of Sox9 in different tissues and at different stages of development, we have generated a transgenic mouse line to express Sox9 in a Cre recombinase-dependent manner. The construct contained the human cytomegalovirus enhancer and chicken β-actin promoter, and a floxed monomeric red fluorescence protein 1 (mRFP1) cassette to direct ubiquitous expression of mRFP1. Cre expression removed the mRFP1 gene, allowing expression of Sox9 and enhanced green fluorescent protein. Expectedly, overexpression of Sox9 in chondrocytes using Col2a1-Cre mice suppressed chondrocyte hypertrophy, and delayed terminal differentiation and subsequent ossification. Misexpression of Sox9 in hypertrophic chondrocytes using Col10a1-Cre knock-in mice also delayed terminal differentiation. This Sox9 conditional transgenic mouse line will be a valuable tool to uncover tissue-specific and developmental stage-specific functions of Sox9.

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