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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. 6. YAP and SHH are upregulated and required for HO, in an FOP mouse model.

Fig. 6.

(A) Representative μCT images of tibia bones from 4-week-old ACVR1Q207D/+ and ACVR1Q207D/+; ScxCre+ mice. Arrows indicate ectopic bone around tendon region. White lines indicate section position. Right panel: quantification of ectopic bone volume. N=5 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. Scale bar: 100 μm. (B) Gene expression analysis by qRT-PCR of ectopic bone regions in 4-week-old mice. Mean±SD; N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (C) Representative immunofluorescent images of ectopic bone sections from 4-week-old ACVR1Q207D/+ and ACVR1Q207D/+; ScxCre+ mice. Lower panel: higher magnification of the boxed regions. Dashed lines indicate the tendon boundary. Arrows: YAP+; OPN− cells (left panel); SHH+; OSX− cells (right panel). Scale bar: 50 μm. T: Tendon. (D) ALP staining of ACVR1Q207D/+SMPs with Ad-GFP or Ad-Cre infection and VP (200 ng/ml) treatment for 7 days. Right panel: quantification of ALP activity. N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (E) qRT-PCR analysis of gene expression in SMPs in (D). Mean±SD; N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (F) Representative immunofluorescent images of YAP (left panel) or SHH (right panel) in ACVR1Q207D/+ SMPs, with Ad-GFP or Ad-Cre infection. N=3 biological replicates. Scale bar: 20 μm. (G, H) Representative μCT images of tibia bones from the indicated mice 6 weeks after intramuscular injection of indicated virus with cobra venom at 4 weeks of age. Red arrows indicate ectopic bone. Right panel: quantification of ectopic bone volume. N=6 biological replicates. Scale bar: 100 μm. Lower panel: qRT-PCR analysis of the osteoblast markers Col1a1, Osx, Shh/Ihh, YAP target genes (Ctgf and Cyr61); and the HH signaling targets, Ptch1, Gli1 and Hip. Mean±SD; N=3 biological replicates. **p<0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests.