09-P004 Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signaling

Hedgehog (Hh) signaling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are critical for proper bone formation. Since many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects of Hh signaling on osteogenesis remain. Using zebrafish larvae mutant in Indian Hedgehog, patched1, patched2 and suppressor of fused (dre), we show that there are two populations of perichondral/endochondral osteoblasts with differential sensitivity to Hh signaling. One, formed outside the cartilage structure, requires low levels of Hh signaling, but still fails to differentiate in Indian Hedgehog mutants. The other derives from transdifferentiating chondrocytes and requires higher levels of Hh signaling to form. This latter population develops significantly earlier in mutants with increased Hh signaling, leading to premature endochondral ossification, and also fails to differentiate in Indian Hedgehog mutants, resulting in severely delayed endochondral ossification. By contrast the number of dermal bone osteoblasts is unaltered in mutants with increased and decreased levels of Hh. Additionally, we demonstrate that the timing of first osteoclast activity positively correlates to Hh levels in both endochondral and dermal bone. Our findings, based on invivo imaging of osteoblasts, offer a possible explanation for some recently reported controversial findings regarding Hh requirements in osteogenesis, and can be integrated in a model that explains how modulated levels of Hh signaling exert a differential effect on specific subpopulations of differentiating osteoblasts.