Suppressor of Fused restraint of Hedgehog activity level is critical for osteogenic proliferation and differentiation during calvarial bone development.

Hedgehog signaling plays crucial roles in the development of calvarial bone, relying on the activation of Gli transcription factors. However, the molecular mechanism of the role of regulated Gli protein level in osteogenic specification of mesenchyme still remains elusive. Here, we show by conditionally inactivating Suppressor of Fused (), a critical repressor of Hedgehog signaling, in -mediated cranial neural crest (CNC) or -mediated mesodermal lineages that restraint of Hedgehog activity level is critical for differentiation of preosteogenic mesenchyme. Ablation of results in failure of calvarial bone formation, including CNC-derived bones and mesoderm-derived bones, depending on the Cre line being used. Although mesenchymal cells populate to frontonasal destinations, where they are then condensed, deletion significantly inhibits the proliferation of osteoprogenitor cells, and these cells no longer differentiate into osteoblasts. We show that there is suppression of and , the osteogenic regulators, in calvarial mesenchyme in the mutant. We show that down-regulation of several genes upstream to and is manifested within the calvarial primordia, including and its downstream genes and By contrast, we find that , the Hedgehog activity readout gene, is excessively activated in mesenchyme. Deletion of in CNC leads to a discernible decrease in the repressive Gli3 form and an increase in the full-length Gli2. Finally, we demonstrate that simultaneous deletion of and in CNC completely restores calvarial bone formation, suggesting that a sustained level of Hedgehog activity is critical in specification of the osteogenic mesenchymal cells.