Methods: To analyze the function of Hand1 we generated mice that conditionally overexpress Hand1 in the osteochondro-progenitor cells via Cre recombinase driven by the Twist2 promoter. Gene expression and tissue morphology were examined histochemically and quantitatively with real-time PCR in Hand1 overexpressing mice. Results were confirmed in vitro using a Hand1 overexpressing chondroprogenitor ATDC5 cell model, real-time PCR and luciferase assays.
Results: Genetic overexpression of Hand1 in the developing limbs (Hand1Tg/+; Twist2-Cre) resulted in prenatal hypoplastic ossification in the long bones identified through decreased detection of bone markers in both primary and secondary ossification centers postnatally mimicking an Indian hedgehog (Ihh) loss-of-function phenotype. Ihh expression was downregulated in femur epiphyses of Hand1 overexpressing mice throughout all stages analyzed from E16.5 to P21. We also confirmed that Hand1 downregulated Ihh gene expression in vitro by inhibiting Runt-related transcription factor 2 (Runx2) transactivation of the Ihh proximal promoter.
Conclusion: Here, we report that Hand1, which is expressed in the cartilage primordia, is required for proper osteogenesis of the bone collar, via its control of Ihh production. These results demonstrate that Hand1 in chondrocytes regulates endochondral ossification, at least in part through the Runx2-Ihh axis.