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 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.

Keywords: cell differentiation; craniofacial anomalies; craniofacial biology/genetics; developmental biology; signal transduction; translational medicine.

Figure 1.

The distinctiveness of cranial cartilage is associated with Gα_s signaling. (A, B) i: Cell populations of cranial sutures and limbs at E18.5 identified by online single-cell RNA sequencing data sets are visualized with UMAP. Clusters of mesenchymal progenitors, chondrocytes, osteoblasts, and others are defined in a descending order by color (pink, green, blue to purple). ii: Violin plot for the expression levels of Gli1 and Ptch1. iii: X-gal⁺ osteoblasts (purple arrow) or chondrocytes (green arrow) are indicated in a stained section from Ptch1^LacZ mice. Scale bar: 100 µm. (C, D) Immunostaining using Sox9, collagen type X (ColX), and phosphorylated-Creb (p-Creb) antibodies on a sagittal section of mastoid fontanel and femur, respectively, from mice at P0. (i–iv) Immunohistochemistry staining with the indicated antibodies counterstained with Alcian blue on cranial suture cartilage or limb. ColX⁺ hypertrophic limb chondrocytes (yellow arrow), p-Creb⁺ cranial suture cartilage, and limb chondrocytes except in the hypertrophic zone (red arrow); mineralization (gray arrow) is indicated in Sox9⁺ chondrocytes (pink arrow). Scale bar: 50 µm.

Figure 2.

Loss of Gnas in osteochondral progenitor cells caused cartilage mineralization in the heterotopic ossification (HO) of cranial sutures and fontanels. (A) Micro–computed tomography 3-dimensional reconstruction image of control calvarias and Prrx1-Cre; Gnas^fl/fl calvarias at P14. N = 6. Blue arrows indicate HO. Scale bar: 1 mm. (B) Skeletal preparation of calvarias of controls and Prrx1-Cre; Gnas^fl/fl at P0. Dotted lines (white) outline the cranial cartilage. The arrow (yellow) indicates mineralization within the cartilage. (C) Quantification based on skeletal preparation. Percentage of the Alizarin red area among total Alcian blue area of mastoid fontanels at P0. N = 6 mastoid fontanels from 6 mice. A.R. area/A.B. area: percentage of area of Alizarin red–positive tissue within cartilage out of area of Alcian blue–positive tissue. Mean ± SD. Two-tailed t test. (D) Costaining with Safranin O (orange to red) and von Kossa (brown to black) on coronal sections from control and Prrx1-Cre; Gnas^fl/fl mutant mice at P0. Dotted lines (gray) outline cranial cartilage. The arrow (blue) indicates ectopic mineralization within the cartilage. Scale bar: 100 μm. (E) Schematics of analysis, as well as immunofluorescence staining of Prrx1-Cre; Gnas^fl/fl; R26R^Tomato and controls using antibodies for collagen type X (ColX), Osterix (Osx), and osteopontin (OPN) at P0. Tomato labels mutant cells (red). Osteogenic differentiated status (arrows indicate ColX [green], Osx [green], OPN [white]) is indicated in the outlined mutant cranial cartilage. Scale bar: 50 μm.

Figure 3.

Sox9⁺ chondrocyte-specific loss of Gnas replayed cranial cartilage heterotopic ossification (HO) and chondrocyte conversion. (A) Immunofluorescence staining of Prrx1-Cre; Gnas^fl/fl; R26R^Tomato and controls using Sox9 antibody at P0. (B) Schematics of experiment design. (C) Skeletal preparation of mouse calvarias from Sox9-CreER; Gnas^fl/fl; R26R^Tomato and their littermate controls (Sox9-CreER; Gnas^fl/+; R26R^Tomato) at P6. Mastoid fontanel is outlined in white. The yellow arrow indicates HO cartilage. Scale bar: 1 mm. Immunostaining using antibody of Osterix (Osx) and osteopontin (OPN) on serial sections. Outlined mutant cranial cartilage (tdTom) in an osteogenic differentiated status (Osx or OPN, green) indicated by arrows. Scale bar: 50 μm. (D) Quantification of suture cartilage in Sox9-CreER; Gnas^fl/fl; R26R^Tomato. Percentage of Osx+tdTom+ double positive cells and percentage of OPN+tdTom+ double positive cells, respectively, among total tdTom⁺ chondrocytes at P6. N = 6, fields from 4 mice. Mean ± SD. Two-tailed t test. (E) Micro–computed tomography 3-dimensional reconstruction of Sox9-CreER; Gnas^fl/fl and their control littermates at P14. Dotted lines (yellow) outline patent sutures and fontanels; the arrow (red) indicates HO. Scale bar: 1 mm.

Figure 4.

Loss of Gnas transformed chondrocytes by activating Hedgehog (Hh). (A) Schematics of mice mating. (B) Whole-mount X-gal staining of Prrx1-Cre; Gnas^fl/fl; Ptch1^LacZ mouse and control littermate calvarias. The arrow (red) indicates ectopic X-gal (green) in outlined sutures and fontanels. Scale bar: 0.5 mm. (C) X-gal staining on coronal cryostat sections of Prrx1-Cre; Gnas^fl/fl; Ptch1^LacZ mouse calvaria. The arrow (red) indicates X-gal⁺ chondrocytes (green) in the outlined cartilage. Scale bar: 100 μm. (D) Quantification of X-gal staining. Percentage of X-gal⁺ chondrocytes among total chondrocytes. N = 6 mice. Mean ± SD. Two-tailed t test. (E) Costaining with Safranin O and von Kossa on X-gal staining of Prrx1-Cre; Gnas^fl/fl; Ptch1^LacZ mouse and control littermate calvarias. The arrow (green) indicates X-gal⁺ chondrocytes contributing to the mineralization of mutant cartilage (outlined). Scale bar: 100 μm.

Figure 5.

Reduction of Gli transcription activity largely rescued cartilage ossification. (A) Upper: micro–computed tomography 3-dimensional image of Prrx1-Cre; Gnas^fl/fl; Gli2^fl/+ mouse and the control littermate calvarias. Dotted lines outline enlarged view of mastoid fontanel. The arrows (red) indicate reduced heterotopic ossification (HO). Scale bars: 1 mm. Lower: skeletal preparation of mouse calvarias and their control littermates. The arrows (yellow) indicate reduced HO in rescue group. Scale bar: 1 mm. (B) Schematics of experiment design. (C) Skeletal preparation of mouse calvarias receiving GANT61, ATO, or DMSO (vehicle). The arrows (red) indicate reduced HO. Scale bar: 1 mm. (D) Schematics of this work: a Gα_s-regulated conversion of cranial chondrocyte identity through Hedgehog signaling strengthens HO during calvaria development.