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. Author manuscript; available in PMC: 2022 Jun 23.
Published in final edited form as: Sci Transl Med. 2021 Jun 23;13(599):eabb2233. doi: 10.1126/scitranslmed.abb2233

Fig. 7. Yap and SHH are upregulated in and required for HO in an injury-induced HO mouse model.

Fig. 7.

The Achilles tendon puncture (ATP) surgery was performed in 4-week-old WT mice. (A) Representative μCT images and the quantified ectopic bone volume of tibia bone and the Achilles tendon region, 6 or 12 weeks after Achilles tendon puncture (ATP) surgery. Arrows indicate ectopic bone around tendon region. N=5 biological replicates. Sub: Subcutaneous region. Scale bar: 100 μm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. Right panel: Representative images of von Kossa staining of tendon sections, 12 weeks post ATP surgery. The boxed region is shown at higher magnification in the lower panel. Scale bar: 100 μm. Arrows indicate mineralization in the injured subcutaneous region in the sham sample. (B) QRT-PCR analysis of Achilles tendon samples, 6 weeks post ATP surgery. Expression of the YAP target gene Ctgf, Cyr61, osteoblast marker Col1a1, and Runx2, Shh/Ihh, and HH-signaling targets, Ptch1, Gli1 Hip, and Tgfβ1-3 are shown. Mean±SD; N=3 biological replicates.). **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (C) Top panel: X-ray images of the mouse hindlimbs. Low panel: β-Gal staining of tibia-tendon of 6-week-old Ptch1LacZ+ mice, 10 days after ATP surgery. Arrows indicate ectopic bone around the tendon. Scale bars: 100 μm. (D) Representative immunofluorescent images of tendon marker TNMD, GFP, and YAP in tendon sections from the Ctgf-GFP mice 6 weeks post ATP surgery in 4-week-old mice. Lower panel: higher magnification of boxed region. Scale bar: 50 μm. (E) Representative immunofluorescent staining for SHH and OSX in ectopic bone sections from WT mice, 6 weeks after ATP surgery. Arrows: SHH+ OSX− cells. Scale bar: 20 μm. (F, G) μCT images of the tibia/tendon region 6 weeks after ATP surgery with topical treatment of VP (20 μg/ml or 200 μg/ml) and THZ1 (1 mM) (F) or IP injection of VP (2.5 mg/kg) (G) 5 days per week for 6 weeks. Arrows indicate ectopic bone around tendon region. Lower panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar: 100 μm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (H) μCT images of tibia/tendon region from the indicated mice 6 weeks after Ad-Cre tendon injection in 4-week-old mice. ATP was performed 1 day after Ad-Cre injection. Lower panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar: 100 μm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (I) μCT images of the tibia/tendon region of WT mice 6 weeks after ATP surgery followed by IP injection of SHH monoclonal antibody (0.25 mg/kg, 5 days per week). Arrows: ectopic bone around tendon region. Lower panel: quantification of ectopic bone volume. N=5 biological replicates. Scale bar: 100 μm. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests.