Figure 5.

Hoxa11-lineage cells differentiate into ectopic chondrocyte, osteoblasts, and adipocytes

(A) Confocal microscopy images of the hindlimb 3 weeks following BT injury, including tile scan image with dashed lines marking native bone and dotted line marking HO (left) and 63× insets of region of interest (n = 2 mice/antigen of interest, n = 3 images/mouse). Sections were immunolabeled with indicated antibodies (bottom). Blue chevrons mark Hoxa11iTom cells co-labeled with specified antibodies. Yellow scale bar, 500 μm; white scale bars, 50 μm. Right: bar chart showing mean with SEM of cell-count quantification of antigen-expressing cells co-labeled with TDTOMATO fluorophore.

(B) snATAC-seq of TdTomato-expressing cluster MSC:0 6 weeks post injury. Genes shown are important in chondrogenesis and osteogenesis. Bars represent mean value of reads.

(C) UMAP plot of cells 6 weeks following BT injury.

(D) UMAP feature plot depicting TdTomato expression with clusters expressing TdTomato circled in red (top left), violin plot showing TdTomato expression 6 weeks following BT injury for reference (top right, from Figure 1C), and cell counts across all clusters (bottom).

(E) Violin plots of genes necessary in differentiation of tenocytes (left), chondrocytes (middle), and osteoblasts (right).

(F) Confocal microscopy images of the hindlimb 9 weeks (bottom) following BT injury with tile scan for reference (n = 2 mice/antigen of interest, n = 3 images/mouse) (left). Dotted line shows the HO site while dashed line shows labeled tibia bone. Calcaneus not labeled at 9-week time point as it is surrounded by heterotopic bone. The colored boxes highlight regions of 63× total zoom inset images (right). Sections were immunolabeled with indicated antibodies. Blue chevrons mark Hoxa11iTom cells co-labeled with specified antibodies. Yellow scale bar, 500 μm; white scale bars, 50 μm.

(G) Bar chart showing mean with SEM of cell-count quantification of antigen-expressing cells co-labeled with TDTOMATO fluorophore.