Novel Lineage-Tracing System to Identify Site-Specific Ectopic Bone Precursor Cells

Abstract

Heterotopic ossification (HO) is a form of pathological cell-fate change of mesenchymal stem/precursor cells (MSCs) that occurs following traumatic injury, limiting range of motion in extremities and causing pain. MSCs have been shown to differentiate to form bone; however, their lineage and aberrant processes after trauma are not well understood. Utilizing a well-established mouse HO model and inducible lineage-tracing mouse (Hoxa11-CreER^T2;ROSA26-LSL-TdTomato), we found that Hoxa11-lineage cells represent HO progenitors specifically in the zeugopod. Bioinformatic single-cell transcriptomic and epigenomic analyses showed Hoxa11-lineage cells are regionally restricted mesenchymal cells that, after injury, gain the potential to undergo differentiation toward chondrocytes, osteoblasts, and adipocytes. This study identifies Hoxa11-lineage cells as zeugopod-specific ectopic bone progenitors and elucidates the fate specification and multipotency that mesenchymal cells acquire after injury. Furthermore, this highlights homeobox patterning genes as useful tools to trace region-specific progenitors and enable location-specific gene deletion.

Keywords: Hoxa11; aberrant differentiation; heterotopic ossification; mesenchymal progenitors; tendon.

Graphical abstract

Figure 1

Combined time-point analysis of scRNA sequencing shows distinct TdTomato-expressing clusters (A) Experimental schematic showing injury model and cells harvested in the uninjured limb (n = 1), 1 week following BT injury (n = 1), and 6 weeks following BT injury (n = 2) from Hoxa11-CreER^T2:ROSA-TdTomato mice that were processed and sequenced. Mouse schematic adapted from our group's previous work (Agarwal et al., 2016a). (B) UMAP cluster, cluster definitions, and cell counts of combined time-point analysis. (C) Violin plots of combined time-point and individual time-point TdTomato expression. (D) Violin plots of gene markers for mesenchymal and bone progenitors.

Figure 2

Trajectory analysis of TdTomato⁺ cells shows osteo/chondro branch following traumatic injury (A) Monocle 2 pseudo-time analysis separated by TdTomato-expressing cluster. (B) Feature plots of Pdgfra and Clec3b gene expression levels. (C) Feature plots of chondrocyte differentiation genes Acan and Sox9 expression levels. (D) Feature plots of osteoblast differentiation genes Runx2 and Spp1 expression levels. (E) Feature plots of tendon differentiation genes Scx, Tnmd, and Comp expression levels. (F) Pseudo-time plots separated by time point with osteo/chondro branch circled in orange. (G) UMAP steam plot of RNA velocity of TdTomato⁺ cells in clusters MSC:0, MSC:2, MSC:14, and Teno:16 (left) with arrows showing velocity and UMAP latent time plot (right). (H) CytoTrace analysis of TdTomato-expressing cells including a UMAP plot with predicted ordering score (left), a UMAP plot colored by cluster (center), and a box plot showing predicted score of clusters (right).

Figure 3

Hoxa11-lineage cells are found within tendon and enthesis and do not express chondro/osteo differentiation genes in the uninjured limb (A) SnATAC-seq of TdTomato-expressing cluster MSC:0 in the uninjured limb (n = 1). Genes shown are important in chondrogenesis and osteogenesis. Bars represent mean value of reads. (B) UMAP plot of cells in the uninjured limb. (C) UMAP feature plot depicting TdTomato expression in the uninjured limb (top left), violin plot showing TdTomato expression across clusters (top right, from Figure 1C), and cell counts across all clusters (bottom). (D) Violin plots of genes necessary in differentiation of tenocytes (top), chondrocytes (middle), and osteoblasts (bottom). (E) Confocal microscopy images of the uninjured hindlimb with tile scan (left) and 63× total zoom inset images of regions of interest (n = 2 mice/antigen of interest, n = 3 images/mouse). Colored boxes in tile show region of inset. Sections were immunolabeled with PDGFRα and SOX9 (bottom). Blue chevrons mark Hoxa11iTom cells co-labeled with specified antibodies. Yellow scale bar, 500 μm; white scale bars, 50 μm.

Figure 4

Hoxa11 marks a unique osteo/chondro/teno cell type following injury (A) snATAC-seq of TdTomato-expressing cluster MSC:0 1 week post injury. Genes shown are important in chondrogenesis and osteogenesis. Bars represent mean value of reads. (B) UMAP plot of cells 1 week following BT injury. (C) UMAP feature plot depicting TdTomato expression (top left), violin plot showing TdTomato expression at 1 week following BT injury for reference (top right, from Figure 1C), and cell counts of all clusters (bottom). (D) Violin plots of genes necessary in differentiation of tenocytes (left), chondrocytes (middle), and osteoblasts (right). (E) Bar chart visualizing the number of TdTomato⁺ cells in MSC cluster 0 expressing Sox9, Runx2, Sox9 and Runx2, or neither. (F) Confocal microscopy images of the hindlimb 1 week following BT injury with tile scan (n = 2 mice/antigen of interest, n = 3 images/mouse) (left). Dotted line shows the HO site while dashed line shows labeled calcaneus and tibia bones. The yellow box highlights region of 63× total zoom inset images (right). Sections were immunolabeled with PDGFRα, SOX9, and pSMAD3 (bottom). 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.

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.