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1.
Figure 5

Figure 5. Reducing Hh signaling inhibits HO in vivo and in vitro. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a, b) Representative alizarin red and alcian blue staining of the limbs from E18.5 embryos with the indicated genotypes. Less severe HO, particularly in the hindlimb, is indicated (arrow) . In (b), the E18.5 embryos were from the pregnant female mice that had been injected with vehicles or the indicated Hh antagonists three times (E13.5, E15.5 and E17.5). Scale bars, 0.5 mm. (c) Alizarin red staining of the differentiating BMSCs from the Gnasf/f mice with the indicated treatment.

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.
2.
Figure 2

Figure 2. Loss of Gnas in adult subcutaneous tissue leads to HO. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a) Representative alizarin red and alcian blue staining of the Gnasf/f mice injected with either Ad-GFP or Ad-Cre virus in the subcutaneous regions (shown in supplemental Fig. 3). N=18. Extensive ectopic bone formation is indicated (arrow). Scale bar, 1 mm. (b) Histological analyses of ectopic bone formation shown in (a) by Osx and Oc immunohistochemistry (arrows) and Von Kossa staining (dark brown). Scale bar, 0.05 mm. (c, d) Representative Von Kossa staining of the ectopic bone (green arrows) in the subcutaneous (c) and the deep muscular regions (d) in the hindlimbs of Gnasf/f mice 12 weeks after Ad-Cre virus injection. H: hair follicle; M: muscle. Scale bars, 0.2 mm (c), 0.05 mm (d).

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.
3.
Figure 4

Figure 4. Gαs acts through cAMP and PKA to suppress Hh signaling. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a) cAMP levels in SMPs from Gnasf/f mice as measured 3 days after adenovirus infection (mean±SD; n=3; *p=8.5×10−5). (b) PKA activity indicated by Phospho-Creb (P-Creb) protein levels in SMPs from Gnasf/f mice 5 days after adenovirus infection. (c) qRT-PCR assay of Hh target gene expression in SMPs from Gnasf/f mice 5 days after adenovirus infection (mean±SD; n=3; *p=4.4×10−4 for Ptch1; 2.4×10−5 for Gli1; 2.6×10−6 for Hhip). (d) Alizarin red staining of the SMPs from the Gnasf/f mice 14 days after the indicated treatment. (e) PKA activity indicated by P-Creb levels in SMPs from the Gnasf/f mice 5 days after the indicated treatment. (f) q-RT-PCR assay of Hh target expression in SMPs from the Gnasf/f mice that had been infected with adenovirus for 7 days and treated as indicated for 5 days (mean±SD; n=3; *p=2.1×10−4 for Ptch1; 8.7×10−4 for Gli1; 2.6×10−4 for Hhip). (g) PKA activity indicated P-Creb protein levels in WT SMPs infected by dnPKA adenovirus for 5 days. (h) qRT-PCR analysis of Hh target gene expression in WT SMPs infected by dnPKA adenovirus for 5 days (mean±SD; n=3; *p=0.001 for Ptch1; 2.1×10−4 for Gli1; 3.9×10−4 for Hhip). (i) qRT-PCR analysis of osteoblast differentiation marker expression in WT SMPs infected by dnPKA adenovirus for 5 days (mean±SD; n=3; *p=0.003 for Osx; 4.8×10−4 for Alpl). (j) Alizarin red staining of WT SMP cells infected by dnPKA adenovirus for 14 days.

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.
4.
Figure 6

Figure 6. Hh signaling is activated in ectopic bone from POH individuals and activation of Hh signaling is sufficient to cause HO. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a-c) GLI1 immunohistochemical staining of human samples. (a, b) Samples from a normal human subject. Scale Bars: 1 μm. (c) GLI1 expression in ectopic osteoblasts of the POH samples (arrows). Scale Bar: 1 μm. (d) Representative alizarin red and alcian blue staining of the limbs from the R26SmoM2 mice injected with the Ad-GFP (left limb) or the Ad-Cre (right limb). Ectopic bone formation is indicated (arrows). N=8. (e) Alizarin red staining of the cultured SMP cells. (f) Schematic illustration showing the fundamental roles of Gαs in bone formation and the mechanisms of GNAS mutations in bone disease (see text for more details). L: low, H: high, GOF: gain of function, LOF: loss of function.

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.
5.
Figure 3

Figure 3. Removal of Gnas from mesenchymal progenitor cells upregulates Hh signaling in vitro and in vivo. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a, b) Representative Von Kossa (a) or alizarin red staining (b) of Ad-Cre or Ad-GFP infected BMSCs from Gnasf/f mice after 10 days in osteogenic medium. (c) qRT-PCR analysis of osteoblast markers at day 7 following adenovirus infection (mean±SD; n=3; * p=0.017 for Osx; 0.022 for Col1a1; 0.005 for Alpl; 0.003 for BSP; 0.003 for Oc). (d) Representative in situ hybridization performed on E14.5 forelimbs from the WT littermate control (left) and the Prx1-Cre; Gnasf/− embryos (right). Scale bar, 0.5 mm. (e) qRT-PCR analysis of RNA isolated from E14.5 forelimbs. Expression of Gnas and transcription targets of Hh pathway is shown (mean±SD; n=4; * p=2.8×10−5 for Gnas; 0.012 for Ptch1; 0.036 for Gli1; 0.01 for Hhip). (f) qRT-PCR analysis of Hh pathway target genes in the BMSCs 2-3 days following Ad-Cre or Ad-GFP infection (mean±SD; n=3; * p=1.1×10−4 for Ptch1; 0.001 for Gli1; 0.007 for Hhip).

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.
6.
Figure 1

Figure 1. Loss of Gnas in limb mesenchyme leads to HO. From: Activation of Hedgehog signaling by loss of GNAS causes heterotopic ossification.

(a, b) Representative alizarin red and alcian blue staining of forelimbs from wild-type littermate control (WT) and Prx1-Cre;Gnasf/− mutant mice at E17.5 (a) and P4 (b). Regions of initiating HO (a) and overt HO (b) are indicated (arrows). Scale bar, 1 mm. (c) Representative computed tomography (CT) scans of forelimbs from P20 WT littermate and Prx1-Cre;Gnasf/− mutant mice. (d) Representative alizarin red and alcian blue staining of hindlimbs from P20 WT littermate and Prx1-Cre;Gnasf/− mutant mice. A region of unmineralized Achilles tendon (left) and of ossified Achilles tendon (right) are indicated (arrowheads). Regions of HO are also indicated (arrows). Scale bar, 0.5 mm. (e, f) Longitudinal sections of the autopod of a P4 Prx1-Cre;Gnasf/f mouse counterstained with alcian blue and Sirius red and processed by Von Kossa staining (e) or by Osx immunohistochemistry (DAB, brown) (f). Regions of ectopic mineralization (black arrows) and chondrocyte hypertrophy and joint fusion (yellow arrows) are indicated (e), as are the brown nuclear staining of Osx-positive cells (black arrows) in interdigital regions of surrounding light-blued stained ossicles (f). The boxed interdigital regions in each panel are shown in higher magnification on the right. Scale bars, 0.2 mm (left), 0.05 mm (right).

Jean B. Regard, et al. Nat Med. ;19(11):1505-1512.

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