Abstract
Calvaria development is distinct from limb formation. Craniosynostosis is a skull deformity characterized by premature cranial suture fusion due to the loss of the GNAS gene and, consequently, its encoded protein Gα(s). This birth defect requires surgery, with potential lethal consequences. So far, hardly any early-stage nonsurgical interventions for GNAS loss-related craniosynostosis are available. Here, we investigated the role of the Gnas gene in mice in guarding the distinctiveness of intramembranous ossification and how loss of Gnas triggered endochondral-like ossification within the cranial sutures. Single-cell RNA sequencing (scRNA-seq) of normal neonatal mice cranial suture chondrocytes showed a Hedgehog (Hh) inactivation pattern, which was associated with Gα(s) signaling activation. Loss of Gnas evoked chondrocyte-to-osteoblast fate conversion and resulted in cartilage heterotopic ossification (HO) within cranial sutures and fontanels of the mouse model, leading to a skull deformity resembling craniosynostosis in patients with loss of GNAS. Activation of ectopic Hh signaling within cranial chondrocytes stimulated the conversion of cell identity through a hypertrophy-like stage, which shared features of endochondral ossification in vivo. Reduction of Gli transcription activity by crossing with a loss-of-function Gli2 allele or injecting GLI1/2 antagonist hindered the progression of cartilage HO in neonatal stage mice. Our study uncovered the role of Gα(s) in maintaining cranial chondrocyte identity during neonatal calvaria development in mice and how reduction of Hh signaling could be a nonsurgical intervention to reduce skull deformity in craniosynostosis due to loss of GNAS.