Gnas Loss Causes Chondrocyte Fate Conversion in Cranial Suture Formation

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 Gαs. 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 Gαs 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 Gαs 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.

GNAS locus: bone related diseases and mouse models

“…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 ( 142 ). This implies that reducing Hh signaling could be an intervention to suppress the cranial deformity caused by Gnas deficiency ( 140 ).…”

“…Xu et al removed Gsa from osteochondral progenitor cells by generating Prx1-Cre; Gsa flox/flox mice, resulting in accelerated cranial bone formation during craniogenesis and cranial malformation after birth. This is because the loss of Gsa activated the Hh signaling pathway and accelerated osteoblast differentiation and ossification during cranial bone development, but the bone formed was of low quality and low mineral density, which may be due to increased osteoclast differentiation (140).…”

Rapid induction and long-term self-renewal of neural crest-derived ectodermal chondrogenic cells from hPSCs

“…Cluster 7 was monocytes from TMJ-CC. Both ECC-28d and TMJ-CC expressed fibrocartilaginous markers (e.g., COL3A1 and COL1A2), cranial chondrocyte markers (e.g., GNAS, BARX1, MGP) [46][47][48] , and craniofacial markers (e.g., DCN, ITGB1) 49,50 . Cp-28d mainly expressed hypertrophic chondrocyte markers in the growth plate (e.g., CYR61, CXCR4, CTGF) 51,52 , primary chondrocyte markers (e.g., GPX1) 53 and mesoderm markers (e.g., MESDC2) 54 (Supplementary Fig.…”

METTL3 Modulates Ctsk+ Lineage Supporting Cranial Osteogenesis via Hedgehog

“…The cathepsin K (CTSK) lineage of calvarial stem cells (CSCs) forms one of the cell sources for physiologic calvarial mineralization (Debnath et al 2018;Bok et al 2023). Disruption of molecular signaling during calvarial development leads to delayed cranial suture closure (Xu et al 2018) or craniosynostosis (Yu et al 2021;Xu et al 2022), both of which are associated with cranial osseous dysplasia. However, how the cranial suture, a close analogy with a signaling center, orchestrates the intramembranous ossification process of the skull vault by methyltransferase-like 3 (METTL3) remains elusive.…”