Overexpression of transcription factor FoxA2 in the developing skeleton causes an enlargement of the cartilage hypertrophic zone, but it does not trigger ectopic differentiation in immature chondrocytes

We previously found that FoxA factors are necessary for chondrocyte differentiation. To investigate whether FoxA factors alone are sufficient to drive chondrocyte hypertrophy, we build a FoxA2 transgenic mouse in which FoxA2 cDNA is driven by a reiterated Tetracycline Response Element (TRE) and a minimal CMV promoter. This transgenic line was crossed with a col2CRE;Rosa26rtTA/+ mouse line to generate col2CRE;Rosa26rtTA/+;TgFoxA2+/− mice for inducible expression of FoxA2 in cartilage using doxycycline treatment. Ectopic expression of FoxA2 in the developing skeleton reveals skeletal defects and shorter skeletal elements in E17.5 mice. The chondro-osseous border was frequently mis-shaped in mutant mice, with small islands of col.10+ hypertrophic cells extending in the metaphyseal bone. Even though overexpression of FoxA2 causes an accumulation of hypertrophic chondrocytes, it did not trigger ectopic hypertrophy in the immature chondrocytes. This suggests that FoxA2 may need transcriptional co-factors (such as Runx2), whose expression is restricted to the hypertrophic zone, and absent in the immature chondrocytes. To investigate a potential FoxA2/Runx2 interaction in immature chondrocytes versus hypertrophic cells, we separated these two subpopulations by FACS to obtain CD24+CD200+ hypertrophic chondrocytes and CD24+CD200− immature chondrocytes and we ectopically expressed FoxA2 alone or in combination with Runx2 via lentiviral gene delivery. In CD24+CD200+ hypertrophic chondrocytes, FoxA2 enhanced the expression of chondrocyte hypertrophic markers collagen 10, MMP13, and alkaline phosphatase. In contrast, in the CD24+CD200− immature chondrocytes, neither FoxA2 nor Runx2 overexpression could induce ectopic expression of hypertrophic markers MMP13, alkaline phosphatase, or PTH/PTHrP receptor. Overall these findings mirror our in vivo data, and suggest that induction of chondrocyte hypertrophy by FoxA2 may require other factors in addition to Runx2 (i.e., Hif2α, MEF2C, or perhaps unknown factors), whose expression/activity is rate-limiting in immature chondrocytes.