Fig. 2
Histological and lineage analysis of FAP-driven HO. a–i IHC (brown staining) for ACVR1 and SOX9 in paraffin sections of pinch-injured gastrocnemius muscle in wild-type (n = 5 mice per timepoint) and FOP (Acvr1tnR206H/+;R26NG/+;Tie2-Cre; n = 5 mice per timepoint) mice. a–c At Day 3 post-injury, areas of weak ACVR1 staining (arrows) are evident in both wild-type (a) and FOP (b) muscle. Occasional SOX9+ cells were observed in FOP (c, arrowheads) and wild-type (see Supplementary Fig. 3) muscle. A few nascent muscle fibers (identified by their central nucleation; arrowheads) are present in wild-type muscle (a). d–f At Day 6, regenerating muscle fibers in wild-type muscle are (d, examples at arrowheads) surrounded by ACVR1+ interstitium (arrows). In FOP muscle, early cartilage and associated mesenchyme (asterisks) is intensely stained for ACVR1 (e) and SOX9 (f). Uninjured muscle adjacent to lesions exhibit thickened bands of interstitial cells that stain strongly for ACVR1 and SOX9 (arrowheads in e, f). g–i At Day 14, regenerated muscle fibers in wild-type muscle are surrounded by an ACVR1+ endomysial layer (g, arrows). In FOP muscle, cartilage (C) continued to strongly express ACVR1 (h, asterisk) and SOX9 (i, arrowheads). ACVR1 staining in some heterotopic bone cells (arrowheads) and encapsulating fibroblastic cells (arrow) is evident in h. M, uninjured muscle. Sections were counterstained with hematoxylin. j–m Fluorescence images of a cryosection through a heterotopic lesion (asterisks) 12 days after cardiotoxin injection (n = 7 mice). j Unrecombined, tdTomato+ cells were absent from skeletal lesional tissue, whereas muscle fibers (M) exhibited intense red fluorescence. k At this stage, most lesional tissue stained positively for the bone marker, osterix (purple). l Virtually all lesional cells are GFP+. m Merge of panels (j–l). Scale bars in a–i and j–m = 100 µm
