Fig. 1. HO progresses by recruitment of increasing numbers of wild-type cells to the ectopic bone in POH mouse models.

(A) Representative μCT images of the Gnas^f/+; R26^LSL-tdTMT (Ctrl) and Gnas^f/f; R26^LSL-tdTMT (KO) mouse tibia at indicated time points after subcutaneous Ad-Cre injections at 4-weeks-old. Red arrows indicate ectopic bone. White lines indicate section plane. N = 8 biological replicates. Scale bar: 1 mm. (B) Representative von Kossa staining of HO and control tissue sections from the Ad-Cre injected hindlimbs. Dashed lines indicate the interface between epidermis and dermis. Scale bar: 100 μm. (c) Upper panel: Analyses of gene expression in the HO and control tissue by qRT-PCR. (mean±SD; N = 3 biological replicates); Lower panel: quantification of ectopic bone volume from (A) **P < 0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests. (D) Representative immunofluorescent images of Col1a1-GFP and tdTMT in the ectopic bone sections of the indicated mice. Top panel: merged images with lower magnification. Dashed lines indicate the interface between epidermis and dermis. Lower panel: higher magnification images of the boxed regions. Arrows: GFP⁺; tdTMT⁻ cells. DAPI stained the nucleus. N = 5 biological replicates. Scale bar: 100 μm. (E) Quantification of the GFP⁺ cell in (D). Total GFP⁺ cells/field of view (FOV) (left) and GFP⁺; tdTMT⁻ cells vs. total GFP⁺/FOV (right). FOV: 800 × 600 μm **P < 0.01 one-way ANOVA followed by Tukey’s multiple comparisons tests.