Gαs signaling controls intramembranous ossification during cranial bone development by regulating both Hedgehog and Wnt/β-catenin signaling

Abstract

How osteoblast cells are induced is a central question for understanding skeletal formation. Abnormal osteoblast differentiation leads to a broad range of devastating craniofacial diseases. Here we have investigated intramembranous ossification during cranial bone development in mouse models of skeletal genetic diseases that exhibit craniofacial bone defects. The GNAS gene encodes Gα_s that transduces GPCR signaling. GNAS activation or loss-of-function mutations in humans cause fibrous dysplasia (FD) or progressive osseous heteroplasia (POH) that shows craniofacial hyperostosis or craniosynostosis, respectively. We find here that, while Hh ligand-dependent Hh signaling is essential for endochondral ossification, it is dispensable for intramembranous ossification, where Gα_s regulates Hh signaling in a ligand-independent manner. We further show that Gα_s controls intramembranous ossification by regulating both Hh and Wnt/β-catenin signaling. In addition, Gα_s activation in the developing cranial bone leads to reduced ossification but increased cartilage presence due to reduced cartilage dissolution, not cell fate switch. Small molecule inhibitors of Hh and Wnt signaling can effectively ameliorate cranial bone phenotypes in mice caused by loss or gain of Gnas function mutations, respectively. Our work shows that studies of genetic diseases provide invaluable insights in both pathological bone defects and normal bone development, understanding both leads to better diagnosis and therapeutic treatment of bone diseases.

Fig. 1

Hh signaling is activated in the forming calvarial bone in which Smo function is dispensable. a Alizarin red and alcian blue staining of controls and Prrx1Cre; Smo^f/f embryos at E18.5. The limbs are shown in high magnification on the side. Loss of long bone ossification in the limb (black arrow) and well-developed parietal bone and inter-parietal bone (white arrows) are indicated. b Alizarin red and alcian blue staining of control mouse heads from E15.5 and E18.5 embryos. E15.5 coronal sectioning positions (dotted lines) are indicated. Left panel: dorsal view; right panel: lateral view. c X-gal staining of mouse heads from the Ptch1^LacZ/+ embryos at E15.5 and E18.5. Left panel: dorsal view; right panel: lateral view. Scale bar in a–c: 0.5 mm. d Whole-mount X-gal staining of mouse embryo head with the indicated genotypes at E18.5. X-gal staining was mildly reduced in mutants. Removal of Smo led to mildly reduced Hh signaling in the developing skull. e Coronal sections of the parietal bone at E15.5 (indicated in b) were stained with von Kossa method and Safranin O. The osteogenic front regions (dotted box) are shown in high magnification after being stained with the indicated antibodies. Leading edge of osteogenic front (white arrow and white dotted line) is indicated. Scale bar: 100 μm

Fig. 2

Gα_s activation delays intramembranous ossification. a Alizarin red and alcian blue staining of the mouse heads from embryos at E18.5. Increased suture space (white arrow) and reduced parietal and inter-parietal bone (red and blue arrow) are indicated. Scale bar 0.5 mm. Maximum length and width of the head (double arrows) are reduced in the mutant. a apex, b base. b von Kossa and Safranin O staining of parietal bone sections from E18.5 embryos; section position is indicated in a by a dotted line. M midline (black arrow). Mineralized parietal bone region is indicated by dotted lines. Boxed region is shown in higher magnification at the upright corner. Scale bar: 500 μm. c μCT image of 2-month-old mice. Upper panel: Dorsal view of a three-dimensional (3D) reconstructed image. Enlarged fontanel (red arrow) is indicated. Lower panel: Coronal two-dimensional (2D) image at a position indicated by the dotted lines in the upper panel. Thickened bone (white arrow) and bone gaps (red arrow) are indicated. Scale bar: 5 mm. Right panel: Cephalic index of the skull from 2-month-old mice. Cephalic index = 100 × width/length. Results are shown as average of measurements of three different mice±SD. *P < 0.05. d von Kossa and Safranin O staining of parietal bone from E15.5 embryos. The boxed osteogenic front region in the WT embryo and a region in the same position in the mutant were processed for immunofluorescent staining with the indicated antibodies. Images are shown in the lower panel. Osteogenic front is indicated (dotted area). Scale bar: 100 μm. e qRT-PCR analysis of osteoblast differentiation genes in the parietal bone tissues from P0 mice. Results are shown as average of three independent experiments±SD. *P < 0.05. The two-tailed Student’s t test was used in the statistic analysis

Fig. 3

Loss of Gα_s accelerates intramembranous ossification. a Alizarin red and alcian blue staining of the mouse heads from E18.5 embryos. Accelerated ossification in the parietal bone (black dotted area), inter-parietal bone, and ectopic bone in between (white arrow) in the posterior fontanel are indicated. Upper: dorsal view. Lower: lateral view, position of coronal sections is indicated by a white dotted line. Maximum width and length of the skull (double arrows) are indicated. Scale bar: 0.5 mm. a apex, b base. b von Kossa and Safranin O staining of the parietal bone section (coronal section) at E18.5. M midline (arrow). Mineralized parietal region is indicated by a dotted line. Ectopic ossification in the suture is indicated (red arrows). Scale bar: 500 μm. c μCT image of the mouse heads from mice at P6. Upper panel: 3D reconstruction, dorsal view. Ectopic ossification (red arrow) and porous bone (blue arrow) are indicated. Lower panel: Coronal 2D view at the position indicated by the dotted line in 3D. Thickened bone (red arrow) and porous bone (blue arrow) are indicated. Scale bar: 1 mm. Quantitative analysis of cephalic index of the skull from P6 mice. Cephalic index = 100 × width/length. Results are shown as average measurements of three different mice±SD. *P < 0.05. d von Kossa and Safranin O staining of parietal bone from E15.5 embryos. The boxed osteogenic front regions were processed for immunofluorescent staining with the indicated antibodies. Images are shown in the lower panel. Osteogenic front is indicated (dotted line). Scale bar: 100 μm. e qRT-PCR analysis of osteoblast differentiation genes in the parietal bone tissues from P0 mice. Results are shown as average of three independent experiments±SD. *P < 0.05. **P < 0.01. The two-tailed Student’s t test was used in the statistic analysis

Fig. 4

Gα_s regulates Hh signaling during cranial bone formation. a, b Whole-mount X-gal staining of the mouse heads from E18.5 embryos. Prrx1Cre expression regions are indicated. Upper: Dorsal view. Lower: Lateral view. Increased space devoid of X-gal staining or ectopic X-gal staining is shown (arrows). Islands of strong X-Gal staining are pointed by red arrows in a. Section position (white dotted line) is indicated. a apex, b base. Scale bar: 0.5 mm. c, d X-gal staining of cryostat sections from E18.5 embryos. M midline (arrow). The region with X-gal staining is shown by dotted line. Boxed regions in c are shown in higher magnification. Reduced (c) or ectopic and enhanced (d) Hh signaling is shown (black arrows). Scale bar: 0.5 mm. e–g qRT-PCR analysis of Hh signaling target gene (e, f) and Hh ligand gene (g) expression in the P0 parietal bone tissue. Results are shown as average of three independent experiments±SD. *P < 0.05, **P < 0.01. The two-tailed Student’s t test was used in the statistic analysis

Fig. 5

Increasing Hh signaling partially rescues the phenotypes caused by Gα_s signaling activation. a Alizarin red and alcian blue staining of the mouse heads from E16.5 embryos. Upper: lateral view. Lower: higher magnification view of the boxed area, parietal bone is circled. Right: The dissected parietal bone was flattened and shown in higher magnification. Orientation of skull (apex, base, anterior, posterior) is indicated in the first set of images. Scale bar; 0.5 mm. a apex, b base, pt posterior, at anterior. b Alizarin red and alcian blue staining of the mouse heads from P0 mice. Images were captured after removing mandibles and partial skull base. Upper: dorsal view. Lower: higher magnification view of the boxed region in the upper panel. Progressive rescue of accelerated ossification in the sagittal suture (yellow arrow) and posterior fontanel (black arrow) are shown. Scale bar; 0.5 mm. c The i.p. injection scheme of DMSO or ATO to pregnant mice is shown. P0 pups were harvested and analyzed by alizarin red and alcian blue staining. Black boxed regions are shown in higher magnification on the right. At posterior fontanels, ectopic ossification is shown by black arrows. Porous bone is shown by blue arrows. Scale bar: 0.5 mm

Fig. 6

Gα_s regulates Wnt/β-catenin signaling during cranial vault formation. a, b Whole-mount X-gal staining of the mouse heads from P0 pups with the indicated genotypes. Boxed regions are shown in higher magnification in the lower panel. Enhanced (a) or reduced (b) X-gal staining at the osteogenic front and parietal bone is indicated (black arrow). Scale bar; 0.5 mm. c, d In situ hybridization using the probes of Wnt/β-catenin target genes Lef1 and Tcf1 on parietal bone sections from P0 pups. Osteogenic front (box 1) and matured bone (box 2) are shown in higher magnifications in the middle and bottom panels, respectively. Scale bar: 0.5 mm. e, f qRT-PCR analysis of Wnt/β-catenin signaling target gene expression in parietal bone tissues from P0 pups. Results are shown as average of three independent experiments±SD. *P < 0.05, **P < 0.01. The two-tailed Student’s t test was used in the statistic analysis

Fig. 7

Decreased Wnt/β-catenin signaling partially rescues the phenotypes caused by Gα_s signaling activation. a Alizarin red and alcian blue staining of the mouse heads from P0 mouse pups with the indicated genotypes. Boxed areas are shown in higher magnifications on the right. Delayed ossification is indicated with wider suture in mutants and partial rescue is indicated by narrower suture (double arrows). Scale bar: 0.5 mm. b, c Alizarin red and alcian blue staining of the head from P0 pups with the indicated genotypes. Schemes of injection to the pregnant females (1 mg/kg LGK974 or DMSO) are shown. Boxed areas are shown in higher magnifications on the right. The parietal bone was dissected out and shown in higher magnification. Delayed ossification is indicated by larger suture space and higher bone porosity. Both were partially rescued by LGK974 treatment. Delayed cartilage dissolution is indicated (yellow arrow). Images were captured after removing mandibles and partial skull base. Scale bar: 0.5 mm

Fig. 8

Activated Gα_s does not cause osteoblast to chondrocyte fate change. a Alizarin red and alcian blue staining of P0 mouse head with the indicated genotypes. Section position is indicated by dotted line on parietal bone. Coronal sections of the parietal bone showed increased cartilage (red dotted line) and decreased ossification (black dotted line) in the mutant after von Kossa and Safranin O staining (lower panel). Scale bar: 0.5 mm. b Schematics of lineage tracing of Osx-expressing cells and their descendants. c Lineage tracing in the Osx1-GFP::Cre; Rosa 26^tdTomato mice with immunofluorescence co-staining using the indicated antibodies on frozen sections from P0 pups with the indicated genotypes. Boxed regions are shown in higher magnification in the lower panel. Scale bar: 0.5 mm. d Schematics for the role of Gα_s signaling that controls skeletal progenitor cells commitment to osteoblast cells and osteoblast maturation during intramembranous ossification in cranial bone development. Gα_s inhibits Hedgehog signaling while promoting Wnt/β-catenin signaling. Hh signaling induces osteoblast commitment (Osx⁺) from progenitor cells (Prrx1⁺), whereas Wnt signaling inhibits osteoblast maturation (Opn^high and MMPs⁺)