A latent Axin2+/Scx+ progenitor pool is the central organizer of tendon healing – npj Regenerative Medicine

Axin2
TdTom cells are major contributors to tendon healing

The Wnt responsive lineage-tracing mouse line, Axin2CreERT227,28, was found to label a subset of cells in adult tendons using either the ROSALSLmTmG or ROSALSLTdTomato (abbreviated hereafter as Axin2TdTom) reporter lines (Supplementary Figs. 1 and 2A, B). As there are only a few markers of tendon cell heterogeneity and since Axin2 marks stem and progenitor cells in other tissues29,30,31, we sought to further define this cell population in the tendon. Multiphoton microscopy allowed us to simultaneously visualize collagen organization and density via second harmonic generation imaging (SHG). Multiphoton imaging of adult tendons in Axin2TdTom mice revealed TdTom+ cells with elongated tenocyte morphology embedded in the collagen matrix, in the peritendinous tissue or sheath, as well as in surrounding non-tendinous regions predominantly along the vasculature, likely pericytes (Supplementary Fig. 1A–E’ and Supplementary Movies 1 and 2). We next compared Axin2TdTom-labeling with the well-known tendon cell labeling line, ScxCreERT2; ROSALSLTdTomato mice (abbreviated as ScxTdTom)32. ScxTdTom cells were found within the main tendon body, referred to as the fascicles in larger vertebrates33 and a smaller subset in peritendinous regions. Quantification of TdTom-stained cryosections showed labeling in 10.16 ± 2.56% of fascicular tenocytes, 24.30 ± 3.22% of peritendinous cells, and 18.55 ± 2.72% of surrounding non-tendinous cells of Axin2TdTom limbs (Supplementary Fig. 1F, G), and 31.48 ± 4.97% of fascicular tenocytes, 8.05 ± 3.57% of peritendinous cells, and 0.66 ± 0.3% of surrounding non-tendinous cells of ScxTdTom limbs (Supplementary Fig. 1A, H–L and Supplementary Movies 3 and 4).

To determine if the Axin2TdTom cells co-express Scx, we examined Axin2TdTom cells for expression of ScxGFP by flow cytometry and multiphoton microscopy to verify that Axin2TdTom labels adult tendon cells. After treating the mice with Tamoxifen (TAM) at postnatal day (P) 60, we found that a subset of Scx-GFP cells were Axin2TdTom-positive. Double Scx-GFP+/Axin2TdTom cells were observed in the main tendon body, entrenched between highly SHG-positive collagen fibers at P80 and comprised approximately 10% of the total analyzed cell population (Fig. 1A–D, Supplementary Fig. 2C, and Supplementary Movie 5). Therefore, Axin2TdTom marks a subset of Scx-GFP cells in the adult tendon during homeostasis.

Fig. 1: Axin2TdTom cells are major contributors to tendon healing.
figure 1

AD Sagittal view of Axin2TdTom cells co-expressing Scx-GFP (arrows) in the main Achilles tendon body (n = 20 Achilles tendons examined) (SHG). Cartoon indicates mouse genotype and TAM was given at P60 with imaging at P80. EO Mice of the indicated genotype were given TAM at 3 months of age and Achilles tendon injury was performed at 4 months. Two-photon microscopy images were taken of contralateral uninjured Achilles tendons of Axin2TdTom; Scx-GFP mice (E, E’) and of injured tendons at 10 dpi, 20 dpi, and 30 dpi (FH’). Two-photon fluorescent and SHG images of contralateral uninjured (I, I’) and injured Achilles tendons from ScxTdTom; Scx-GFP mice at 10 dpi, 20 dpi, and 30 dpi (JL’). Two-Photon images show Axin2TdTom cells at the injury site with more rounded morphology at 10 dpi (M) and co-expressing Scx-GFP with elongated morphology at 20 dpi and 30 dpi (MO). EO Circle indicates the site of injury; n > 6 mice were examined per time point and genotype. EO are sagittal views; E’–L’ are transverse views digitally resliced from sagittal sections of 0.4 μm.

To understand if Axin2TdTom cells are involved in the tendon healing response, we performed genetic lineage tracing of Axin2TdTom cells using a mouse partial acute excisional injury model of the Achilles tendon. We treated 3-month-old mice with TAM to lineage trace Axin2TdTom cells and at 4 months, performed a biopsy punch injury in the midbody of the Achilles tendon (Supplementary Fig. 2D, E). We examined cell lineage and collagen organization and density in healing tendons using 2-photon microscopy and SHG imaging. At 10 days post-injury (dpi), the wound site was infiltrated with Axin2-lineage cells, and the SHG signal was impaired compared with uninjured contralateral control tendons (Fig. 1B, E–F’). Axin2-lineage cells did not express Scx-GFP at 10 dpi (Fig. 1F, F’, M), suggesting Scx-GFP is downregulated upon injury. At 20 dpi and 30 dpi, the Axin2TdTom cells co-expressed Scx-GFP and their cell morphology changed from round to elongated, indicative of tenocyte differentiation (Fig. 1G, H’, N, and O and Supplementary Movies 68). Importantly, we found that the behavior of Axin2-lineage cells in response to injury mirrored that of Scx-lineage tendon cells, which have been shown to infiltrate healing sites in injuries in other tendons34. Lineage tracing of Scx-labeled tendon cells after injury revealed that similar to Axin2TdTom cells, ScxTdTom cells were observed throughout the healing site and were negative for Scx-GFP at 10 dpi (Fig. 1I, J”,). By 20 dpi, ScxTdTom cells became elongated in appearance and expressed Scx-GFP (Fig. 1K, L”).To determine if Axin2TdTom cells proliferate after injury, BrdU was administered continuously during healing. We found that Axin2TdTom cells incorporate BrdU at the injury site significantly more than the contralateral uninjured tendons (Supplementary Fig. 2F–H). Quantification of cells showed that Axin2TdTom cells comprise a major portion of the total proliferating cells at 20 dpi and of the total cells in the healing region per defined area at 10 dpi, 20 dpi, and 30 dpi (Supplementary Fig. 2I, J). Together, these data show that Axin2TdTom cells are the major cell population that infiltrates, proliferates, and differentiates into tenocytes after injury.

Adult Axin2 cells proliferate less frequently since birth than non-Axin2
TdTom cells

To understand the formation and activity of Axin2TdTom cells in adult vs growing tendons, first, we examined Axin2 cells and Wnt signaling from stages of postnatal tendon growth to physiological homeostasis. Our previous work showed that a significant decrease in tendon cell proliferation occurs after P21. Therefore, TAM treatment was performed at the neonatal stage P2, when the tendon is growing longitudinally and has significant cell proliferation, and at P60 during homeostasis, when longitudinal tendon growth and cell proliferation have slowed17. As there is no published methodology to prospectively isolate tendon cells, we devised a negative sorting strategy to remove blood (CD45+) and endothelial (CD31+) cells. Using flow cytometry and our negative sorting strategy, we found that TAM treatment at P2 resulted in 28 ± 6% of the total cells being labeled by Axin2TdTom at P80 (Fig. 2A, C and Supplementary Fig. 3A) whereas Tam at P60 showed that 10 ± 2% of the cells were labeled by Axin2TdTom at P80 (Fig. 2B, C and Supplementary Fig. 3B). These results could indicate that the number of Axin2TdTom cells in the tendon decreases with age or that early Axin2TdTom cells at P2 proliferate and expand during growth. To explore this possibility further, we found decreased qualitative expression of Axin2 by single-molecule fluorescent in situ hybridization (smFISH) at P28 compared to P0 (Supplementary Fig. 3C, D). In addition, we observed decreased expression of Wnt ligands and associated pathway genes and less accessible associated genomic regions over time upon integrated analysis of RNA-seq and ATAC-seq datasets of tendon cells from P0 to P35 (Supplementary Fig. 3E, F). Together, this analysis suggests that Wnt signaling decreases as the tendon shifts from periods of growth (P0–P21) to physiological homeostasis (>P35)17 and that the early P2 labeled Axin2TdTom cells may proliferate in growth during periods of increased Wnt signaling.

Fig. 2: Axin2TdTom marks a quiescent subpopulation of tendon cells with stem/progenitor cell activity.
figure 2

AC TAM was given to Axin2TdTom mice at (P2) or adults (P60). Tissue was collected and analyzed at P80. Increased numbers of Axin2TdTom cells are observed upon TAM at P2 (A) compared with P60 (B) in P80 sagittal Achilles tendon sections. C Flow cytometry at P80 shows a greater percentage of Axin2TdTom cells per total tendon cells when TAM labeling occurs at P2 vs P60 (P2 cohort, n = 6 mice; Adult/P60 cohort, n = 8 mice, two-tailed unpaired T-test; ****p < 0.0001). D The DOX-inducible H2B-GFP system was used to evaluate Axin2TdTom cell proliferation from birth. DOX was administered to timed pregnant females from Embryonic day 10 (E) to birth. At birth, DOX was removed. For Axin2TdTom labeling, TAM was given at P60 and cells were analyzed at P80. CD34+/CD45+ cells were removed to enrich for tendon cells. Axin2TdTom and Axin2(Neg) cells from the same mouse were compared. Axin2TdTom cells had significantly higher H2B-GFP intensity compared to Axin2(Neg) cells at P80 (n = 4 mice/time point, one-way ANOVA, F = 148; n.s. not significant; *p < 0.05) (D). EH Achilles tendons from 4-month-old mice with TAM at 3 months were collected and 100–500 Axin2TdTom (CD31/CD45/TER119) cells/mouse were isolated by FACS. Cells from each mouse (n = 5) were expanded separately in-vitro for 60 days and a small punch of alginate-based hydrogel cell constructs were transplanted into injured tendons of immunodeficient mice (Foxn1nu/nu). The injury site was analyzed at 10 days and 20 days after injury/transplantation (DPT) (n = 9 mice, 18 injured/transplanted tendons). IL smFISH for Tnmd and Axin2 with co-immunofluorescence staining for TdTom shows co-expression in transplanted Axin2TdTom cells at 20 DPT in sagittal Achilles tendon sections (arrowheads; n = 9 mice, 18 tendons, quantification of at least three sections/mouse). M Quantification of total cells (Dapi+) or TdTom+ cells per 200 × 200 μm area in the host injury site at 10 and 20 DPT. N Quantification of smFISH and immunostaining shows the percentage of Tnmd+ TdTom+ or Axin2+ TdTom+ cells per total TdTom+ cells in the injury area. Bars show S.D. (quantification was made using at least three locations in each section in at least three sections/tendon).

To define the proliferative activity of the adult labeled Axin2TdTom cells, we used the doxycycline (DOX) inducible histone 2B-green fluorescent protein (H2B-GFP) reporter mouse model (Col1a1:tetO-H2B-GFP;ROSA-rtTA). This system is used to quantify cell proliferation and identify slowly cycling label-retaining cell populations based on the stability and dilution of the H2B-GFP protein in each cell35,36. We activated H2B-GFP expression by administering DOX from E10.5 to P0 to timed-pregnant females. We removed DOX at birth and allowed the cells to dilute their label based on their proliferation rates as previously reported17. After administering TAM at P60, we compared Axin2TdTom to Axin2(Neg) (non-Axin2TdTom) cells from the same mouse at P80 and used GFP beads to calibrate measurements of GFP intensity and decay between experiments. We were unable to use Scx-GFP to select for tendon cells as it would interfere with H2B-GFP measurements. Therefore, to enrich the sample for tenocytes, we examined H2B-GFP presence and intensity after excluding CD31+ endothelial and CD45+ blood cells. This allowed us to determine how P60-labeled Axin2TdTom tendon cells replicated since birth in comparison to non-Axin2TdTom cells. Surprisingly, we found that almost all Axin2TdTom cells were H2B-GFP+ (95% ± 3%; n = 4 mice) compared with only 46.1% ± 16.5% Axin2(Neg) cells being H2B-GFP+at P80. Axin2TdTom cells also had significantly higher intensity than that of Axin2(Neg) cells with 5200 ± 300 vs 1716 ± 904 fluorescence value at P80 (Fig. 2D). Taken together, this indicates that the Axin2TdTom cells at P60 are derived from a cell population that divided less frequently since birth than the Axin2(Neg) cells, and suggests they are a distinct quiescent cell subset.

Axin2
TdTom cells differentiate and self-renew in a transplantation assay

As latency or quiescence is a characteristic of other tissue-resident stem or progenitor cells (reviewed in ref. 37), we next sought to test the potential of Axin2TdTom cells by examining their capacity to proliferate, differentiate, and self-renew using a cell transplantation assay into injured host tendons. After administering TAM to label Axin2TdTom cells at 3 months, approximately 100–500 Axin2TdTom cells were isolated from Achilles tendons at 4 months and cells from individual mice were cultured separately. After expanding the cells for 60 days, approximately 4 million Axin2TdTom cells per mouse were harvested, embedded in alginate gels, and biopsy punch gel constructs were transplanted into excisional defect injuries in the Achilles tendons of Foxn1nu/nu mice38. After 10 and 20 days, we examined the site of transplantation and injury for the presence of Axin2TdTom cells by immunostaining for TdTomato (Fig. 2E–H, J, and L and Supplementary Movie 9). After quantifying the number of TdTom+ and total cells per area at the injury site (Fig. 2M, N), we observed similar numbers of total cells per area at 10 and 20 days post injury and transplantation with approximately 21% ± 9% of them being TdTom+ (Fig. 2M). To determine if the expanded Axin2TdTom cells differentiated and/or retained Axin2+ identity, we performed smFISH for Tnmd, an ECM component of differentiating tendon cells, and Axin2 and co-immunostained for TdTom. We found that 90% ± 2% (standard error of means (SEM)) of the TdTom+ transplanted cells also expressed Tnmd, and 38% ± 7% (SEM) expressed Axin2 (Fig. 2N). Since Axin2TdTom cells demonstrate the ability to differentiate into Tnmd-expressing tenocytes and retain their Axin2+ state after significant expansion, we conclude that this latent tendon cell population may also have self-renewal potential.

Axin2
TdTom cells readily expand, are enriched for stem/progenitor cell markers, and undergo multilineage differentiation in vitro

As Axin2TdTom cells show potent expansion capabilities following injury and upon transplantation in vivo, we sought to further define Axin2TdTom cells using in vitro assays. We TAM-treated 3-month-old mice, harvested and isolated Axin2TdTom and Axin2(Neg) cells by flow cytometry at 4 months (isolation strategy shown in Supplementary Fig. 4), and co-cultured them for 10 days. In culture, the Axin2TdTom cells changed morphology, transitioning from thin spindle-shaped cells with long cytoplasmic extensions to more rounded cells (Fig. 3A–C). Notably, Axin2TdTom cells comprised 9% ± 2% of the cell population prior to plating, and by day 10 of culture they were 47% ± 5% of the cells (Fig. 3D, E), which represents an almost five-fold increase from their abundance in tendons. To compare the gene expression of Axin2TdTom and Axin2(Neg) cells, the cells were harvested as described but plated separately on day 0 of culture. After 10 days, RT-qPCR analysis revealed that Axin2TdTom cells had increased levels of Axin2 relative to Axin2(Neg) cells (Fig. 3F), suggestive of a persistent response to canonical Wnt signaling. We also observed greater relative expression of the tendon genes, Scx, Mohawk (Mkx), and Collagen 1a1, and of Ki67 (Fig. 3F), consistent with their tendon identity and ability to readily expand in vitro. As TDSPCs have primarily been characterized ex vivo using cell culture and transplantation assays14, the identity of the resident cell population has been elusive. To test if the Axin2TdTom cells are enriched for markers of previously described TDSPCs, we analyzed the surface marker expression of CD44, CD90.2, and Sca-1 comparing Axin2TdTom and Axin2(Neg) cells by flow cytometry after 10 days in culture, prior to passaging. We found that Axin2TdTom cells had a significantly higher percentage of CD44, CD90.2, Sca-1, and double positive CD90.2/CD44 expressing cells compared with Axin2(Neg) cells (Fig. 3G–K). The Axin2TdTom cells also were negative for CD31 and CD45 at day 10 in culture (Supplementary Fig. 4A). Together, these data show that a higher percentage of Axin2TdTom cells express the surface markers that were previously characterized for TDSPCs and are suggestive that Axin2TdTom cells could have been responsible for the activities of TDSPCs14. As the presence of Axin2 transcripts suggests the cells are actively experiencing Wnt signaling, we next tested if Wnt signaling is necessary for the proliferation and gene expression of Axin2TdTom cells. We added Wnt974, a Porcupine inhibitor, which blocks Wnt secretion, to isolated and cultured Axin2TdTom cells, and administered BrdU. Compared to DMSO treated controls, we observed decreased BrdU-incorporation and decreased Axin2 and Scx expression by RT-qPCR in Axin2TdTom cells (Fig. 3L and Supplementary Fig. 4B, C), suggesting that Wnt signaling is important for proliferation and maintenance of Scx expression in vitro. Finally, we examined the ability of Axin2TdTom cells to undergo multilineage differentiation in vitro by culturing the cells in osteogenic, adipogenic, and chondrogenic induction media. To perform these assays, we isolated and expanded Axin2TdTom cells for 60 days to obtain sufficient cell numbers. Notably, Axin2(Neg) cells did not readily expand to sufficient numbers in culture so multilineage differentiation assays were not performed. Axin2TdTom cells from individual mice were cultured separately in induction media, stained, and the number of differentiated (positively stained cells) per total cells in a defined area per well was quantified. We observed Alizarin red, Oil red O, and Alcian blue stained Axin2TdTom cells, but at differing frequencies with a higher ratio of osteogenic differentiation (Fig. 3M and Supplementary Fig. 4D). In addition, treatment with Wnt974 significantly increased the ratio of Alcian blue-stained cells (Fig. 3M), suggesting that Wnt signaling antagonizes differentiation towards the chondrogenic lineage. Together, our results show that the Axin2TdTom cells in vitro can readily expand, better retain tendon identity, are enriched for markers of TDSPCs, and have multilineage differentiation potential.

Fig. 3: Axin2TdTom cells readily expand, are enriched for progenitor/stem cell markers, and undergo multilineage differentiation in vitro.
figure 3

AM TAM was given to 3-month-old mice and at 4 months, Axin2TdTom cells were extracted from the limb tendons and analyzed or plated in cell culture. AC Images are focused on Axin2TdTom cells to show changing cell morphology at 4 days, 7 days, and 10 days in culture. D, E Representative flow cytometry plots showing Axin2TdTom and Axin2(Neg) cells prior to (0 days) and at 10 days in culture. F Axin2TdTom cells express higher relative amounts of Scx, Sox9, Col1a2, Axin2, Mkx and mKI67 transcripts by RT-qPCR relative to Axin2(Neg) cells after 10 days in culture (n = 3 mice; Multiple T-test with two-stage step-up method of Benjamini, Krieger and Yekutieli; *p < 0.05). Flow cytometry histograms show Axin2TdTom cells are enriched for CD44/CD90.2 double positive (G) vs Axin2(Neg) cells at 10 days of culture (H). I, J A higher percentage of Axin2TdTom cells express Sca-1 compared with Axin2(Neg) cells at day 10 in culture. K Quantification of CD 90.2, CD 44, Sca-1, and double-positive CD90.2/CD44 Axin2TdTom cells vs Axin2(Neg) cells shows the cells are significantly enriched for the markers (n > 4 mice per experimental group; Multiple T-test with Welch correction *p < 0.05, ****p < 0.0001). L The Porcupine and Wnt secretion inhibitor Wnt 974 or vehicle (DMSO) were added to a final concentration of 2.5 μM to cultured Axin2TdTom cells. BrdU was added to both conditions for 5 days and cells were analyzed for TdTom expression and BrdU incorporation (n ≥ 5 with each n representing cells from an individual mouse per condition were analyzed; unpaired T-test with Welch’s correction *p < 0.05). M Axin2TdTom cells from 4-month-old mice were cultured and expanded separately for 60 days. After obtaining sufficient Axin2TdTom cell numbers, adipogenic, osteogenic, and chondrogenic assays were performed and cells were stained (examples shown in Supplementary Fig. 4). Stained vs total cells were quantified per 200 × 200 μm area with at least four measurements per well, in at least three wells per treatment, and each well-represented cells from a different mouse (n = 5). Addition of Wnt974 yielded a higher percentile of chondrogenic cells (T-test with Welch’s correction *p < 0.05; each dot represents a 200 × 200 μm quantification of cells).

A single cell atlas of the adult tendon reveals activation of Scx+/Axin2
+ cells during healing

We next sought to better define Axin2TdTom cells in the context of all the cells in the tendon. Since Axin2TdTom cells and ScxTdTom cells are both found to infiltrate the injury site (Fig. 1F–L’ and Supplementary Fig. 2J), we believe the target cells overlap the original populations that are labeled by Axin2CreERT2 and ScxCreERT2 in our lineage tracing strategy. To better characterize Axin2TdTom cells as Scx-GFP+ tendon cells, we performed single-cell RNA-sequencing on 4-month-old Achilles tendons during homeostasis and at 10 dpi. Our analysis identified distinct tendon and connective tissue cell clusters, as well as other cell types, including Schwann cells, endothelial cells, skeletal muscle, and pericytes (Fig. 4A). Blood cells were present but removed from subsequent analysis. Sub-clustering of tendon and connective tissue cells revealed distinct populations that we characterized based on the enrichment of specific markers (Supplementary Table 1). The clusters include midbody tenocyte (MBT) (e.g., Scx+, Tnmd+, Thbs4+, and Fmod+), the myotendinous junction (MTJ)-associated tenocyte (e.g., Col22a1+, Chodl, and Scx+)39, osteotendinous or enthesis-associated (Ent) tenocyte (e.g., Pthlh+, Inhbb+, and Scx+)40, and osteoblast-like cells (Ost; e.g., Sox9+, Lum+, Osr2+, Smoc2+, Gli1+, and Scx)41,42,43 in addition to four tendon fibroblast clusters (TF1–4). We did not detect Scx in TF1–4 (Fig. 4B, C), but these clusters were identified as tendon or tendon-associated by their enriched expression of Col1a1, Col3a1, and other ECM components. Two additional clusters were named injury-responsive cell states (IRC-1 and IRC-2), as they were found in homeostasis and expanded significantly upon injury.

Fig. 4: A single cell and lineage tracing analysis reveals Axin2-descendant cells express IRC markers and are distinct from pericytes during healing.
figure 4

A UMAP representation of single cell RNA-seq data from Achilles tendons showing total cells excluding blood cell clusters. B UMAP of sub-clustered tendon cells in homeostasis and 10 dpi. For homeostasis, Achilles tendons from 4 month-old Axin2TdTom mice (n = 4, 8 tendons) with Tam at 3 months. For 10 dpi, three Achilles tendons were harvested (n = 3 mice) after tendon injury at 4 months of age. Ten tendon clusters were identified: MBT, MTJ, Ent, Ost, TF1–, IRC-1, and IRC-2. C A cartoon model depicting detected clusters in the tendon. D, E Slingshot pseudotemporal reconstruction analysis inferred trajectory from the MBT to IRC-1 and IRC-2 clusters. UMAP feature plots for Acta2 (F) and Sox9 (G) are shown for homeostasis and 10 dpi. Tenocyte clusters (MBT, MTJ, and Ent) are outlined in red dotted lines for homeostasis, and injury-responsive cell states (IRC-1 and IRC-2) are outlined for 10 dpi. Acta2 and Sox9 are expressed in cells in IRC-1 and IRC-2, respectively, at 10 dpi. H, I Quantification of cells co-expressing Sox9 or αSMA with Axin2TdTom per 200 × 200 μm square area of the injury site at 10 dpi, 20 dpi, and 30 dpi. Quantification was done on transverse Achilles tendon sections from mice TAM treated at 3 months, injured at 4 months, and stage of analysis post injury is indicated (representative examples shown in Supplementary Fig. 5; n > 3 mice per group per timepoint were analyzed with three 200 × 200 μm square areas quantified per section, >4 sections per mouse; one-way ANOVA, F = 436; ****p < 0.0001; n.s. not significant). J Dot plot showing the average and percent expression of tendon and pericyte markers in our scRNA-seq at 10 dpi. IRC-1 and IRC-2 show similarities in tendon gene expression (such as TnC, Col12a1, and Egr1) with the MBT cluster and the other tendon clusters compared with other cell types. Notably, few genes are shared between IRC-1, IRC-2, and the pericyte cluster.

To determine which cluster contained Axin2+ cells, we examined our single cell RNA-seq datasets for Axin2 and TdTomato transcripts. Axin2+ and TdTomato+ cells were predominantly found in the Scx+ MBT cluster during homeostasis in concordance with our histological and 2-photon microscopy observations (Fig. 5A–D). Axin2+ cells were also found in the Ost and Ent clusters (Figs. 3I and 5B, C), which is consistent with a previous lineage tracing study of enthesis injury44. Although MTJ- and Ent appear similar to the MBT, these distinct cell clusters should be located at the myotendinous and osteotendinous (enthesis) junctions, respectively, and not near the injury site, which is in the middle of the tendon body (for reference please see Supplementary Fig. 2D, E).

Fig. 5: Axin2TdTom cell identity is maintained through autocrine Wnt signaling.
figure 5

A Cartoon showing the experimental set-up used for the analysis with TAM treatment at 3 months and scRNA-seq of total Achilles tendon cells in homeostasis at 4 months. Single cell RNA-seq dot plot of canonical Wnt pathway components expressed in the different cell clusters during homeostasis; Scx, Tnmd, and TdTom expression is shown. BE UMAP feature plots of Axin2, TdTom, Scx, and Wnt9A show that these genes are expressed in the MBT cluster during homeostasis (MBT, MTJ, and Ent clusters are outlined in red). F, H smFISH images of one cell containing Axin2 (green), Wnt9a (white), and PolyA (red) puncta in 4-month-old Achilles tendons from wild type (WT) (F) and Axin2:CreERT2; Porcnfl/fl mutants (H; note scale bars are 2 μm). G Quantification of Axin2, Wnt9a, and PolyA RNA puncta per cell in Achilles tendons shows expression of Axin2 and Wnt9A in WT and a decrease in RNA puncta in Axin2:CreERT2; Porcnfl/fl and in Scx:CreERT2; Porcnfl/fl mutants at 4 months of age (n > 8 Achilles tendon per group with n > 20 tendon cells analyzed per tendon; groups consisted of 4-month-old WT, and Axin2TdTom; Porcnfl/fl, ScxTdTom; Porcnfl/fl with TAM given at 3 months; multiple T-test with Holm–Sidak correction was used for statistical analysis; n.s. not significant; ****p < 0.0001). I Representative cell co-expressing AXIN2 (green) and WNT9A (white) in human semitendinosus (hamstring) tendon (I). PolyA (red) was used as an internal expression control (FI).

Because of the high proportion of Scx+, Axin2+, and TdTomato+ cells in the MBT, we focused our analysis on the MBT cluster. Pseudotemporal reconstruction analysis of the UMAP representation using Slingshot45, inferred a trajectory from the MBT to IRC-1 and IRC-2 clusters (Fig. 4D, E), suggesting that MBT could give rise to cells in these injury-responsive states. IRC-1 and IRC-2 expressed genes known to be upregulated after tendon injury and associated with myofibroblast and mesenchymal identities, including Acta2 and Sox97 (Fig. 4F, G). We focused on the expression of Acta2, which encodes αSMA and is expressed in myofibroblasts, as well as in growing and healing tendons7,46, and Sox9, a gene expressed in skeletal progenitors and chondrocytes47. These genes are expressed in IRC-1 and IRC-2 cells at 10 dpi, respectively (Fig. 4F, G). To determine if Axin2TdTom cells can acquire expression of genes found in IRC-1 and IRC-2 during healing, we used genetic lineage tracing. Immunofluorescent staining of injured Achilles tendons showed a majority of Axin2TdTom cells co-expressing Sox9 and αSMA at 10 dpi, 20 dpi, and 30 dpi whereas uninjured tendons expressed limited amounts of Sox9 and αSMA (Fig. 4H, I and Supplementary Fig. 5A–H). As Acta2 is highly associated with myofibroblasts, which in some studies originate from pericytes or vascular smooth muscle cells48, and because we found Axin2TdTom in vascular-associated cells outside the tendon, we examined the expression of additional genes to determine if IRC-1 and IRC-2 could represent a pericyte-like cell responding to injury rather than a tendon-derived cell. Analysis of gene expression in the different cell clusters after tendon injury shows that IRC-1 and IRC-2 share similarities in gene expression with the tendon clusters, and the MBT in particular (Egr1, TnC, and Col12a1) but do not express several markers that are enriched in pericytes, including Notch3 and Myh11 (Fig. 4J). These results are consistent with Axin2-lineage tendon cells transitioning to IRC-1 and IRC-2 states in healing and support a model in which Axin2+ cells are a major responding cell population in tendon injury. While our pseudotemporal reconstruction analysis was able to infer a trajectory between the MBT and the IRC clusters, it is also possible that other cell populations such as the Axin2+/Scx+ cells from peritendinous regions contribute to the IRC states.

Axin2
TdTom tendon cells are a unique tendon cell population expressing stem/progenitor cell markers

To molecularly examine the Axin2TdTom cells before and after injury, we performed bulk RNA-seq analysis and compared Axin2TdTom to non-Axin2TdTom tendon cells during homeostasis and at 10 dpi. As the total RNA quantity in tendon cells is limiting and to avoid pooling samples or multiple tendon types which individually have a limited number of Axin2TdTom cells, we chose to amplify cohorts of pooled 4-month-old Achilles tendon cells using SMART-seq2 and perform differential expression analysis (Supplementary Fig. 5L, M)49. During homeostasis, Axin2TdTom cells have significantly higher expression of several genes including CD201 (ProCR), Fam102a, Robo2, LepR, and Egfl6 (Supplementary Fig. 5N). CD201 marks stem/progenitor cell populations and is a target of Wnt signaling50. Robo2, LepR, and Egfl6 are expressed in different stem cell populations in the intestine, bone, and epidermis51,52,53, whereas Fam102a has been identified as a late iPSC signature gene54,55 Immunostaining and flow cytometry analysis confirmed enriched expression of CD201 in Axin2TdTom cells (Supplementary Fig. 6B). After injury, Axin2TdTom cells were significantly enriched for Sox9 and Acta2, consistent with our single cell RNA-sequencing and lineage tracing results, and for several tendon genes including Col1a2, Tnmd, and Mkx (Supplementary Fig. 5N). Of note, the previously described tendon sheath stem cell marker Tppp3 was enriched in non-Axin2TdTom cells at homeostasis (lfc = −1.906, p-value = 1.49e-9) (Supplementary Fig. 5N, O), indicating Axin2TdTom cells are distinct from Tppp3+ cells, which have been shown to contribute to patellar tendon healing9. Examination of our single cell data set revealed Tppp3 was expressed in several tendon cell clusters and did not appear restricted to a specific cluster (Supplementary Fig. 5K). This result is consistent with another tendon single cell RNA-seq analysis13. Further expression analysis by smFISH revealed Tppp3-expressing cells throughout the main tendon body and in peritendinous regions (Supplementary Fig. 5P–R) but these cells did not significantly express Axin2, confirming our RNA-seq results. Therefore, Axin2+ cells represent a unique population of injury-responsive cells in the adult tendon that does not overlap with other previously described populations.

Autocrine Wnt regulation of Axin2
TdTom cell state

To interrogate how Axin2TdTom cells are regulated, we examined our RNA-seq datasets and as expected, we found expression of several canonical Wnt signaling components in the Axin2+ cell-containing MBT cluster (Fzd1, Wnt9a, Dkk3, Ctnnnb1, Wls) (Fig. 5A) and enrichment for the Wnt pathway in Axin2TdTom cells (Supplementary Fig. 6A). As Axin2 is a direct target and negative regulator of canonical Wnt signaling56, these data are consistent with the notion that Axin2+ cells are regulated via canonical Wnt signaling. Intriguingly, we identified Wnt9a, a Wnt ligand that promotes the formation of synovial connective tissue cells57, as enriched in the MBT cluster during homeostasis in our single-cell data (Fig. 5A, E) and in Axin2TdTom cells in our bulk RNA-seq data (Supplementary Fig. 5N). Using smFISH, we confirmed that Wnt9a is expressed in Axin2+ cells (Fig. 5F). Based on the co-expression of Wnt9a and Axin2, we sought to test if Wnt signaling from the Axin2TdTom cells themselves is required for their identity in homeostasis. As the MBT cluster is enriched for Scx+ cells, we first tested if Wnt signals originating from a Scx+ tendon cell population are necessary for Axin2+ cell identity. We deleted Porcupine (Porcn), a gene required for Wnt secretion, in Scx-expressing cells using ScxCreERT2-TdTom; Porcnfl/fl mice by giving TAM at 3 months and examining RNA transcripts per cell by smFISH in sectioned tendons at 4 months. We found decreased transcript puncta per cell for Axin2 and Wnt9a (Fig. 5G), suggesting that Wnt ligands originating from Scx+ tendon cells are required to maintain Axin2 expression during homeostasis. To specifically test if Wnt secretion from Axin2TdTom cells is necessary to maintain their identity, we TAM-treated Axin2CreERT2-TdTom; Porcnfl/fl mice at 3 months and examined their tendons at 4 months. We found decreased expression of both Axin2 and Wnt9a upon Porcn deletion in Axin2-expressing cells (Fig. 5G, H). We confirmed that the cells were viable in Porcn mutants as we detected a strong PolyA RNA signal in each cell analyzed, which also standardized our staining assay (Fig. 5F–H). In addition, we found a decrease by flow cytometry in the percentage of Axin2TdTom tendon cells co-expressing ProCR/CD201 from Axin2CreERT2-TdTom; Porcnfl/fl mice compared to wild-type cells (Supplementary Fig. 6B–D). Despite the appearance of similar numbers of Axin2TdTom cells in our tendon sections of Porcn conditional knockout mice, we detected a decrease in total Axin2TdTom cells in mutant compared with control tendons by flow cytometry (Supplementary Fig. 6E, F), suggesting the loss of Wnt signaling affected Axin2TdTom cell survival. Notably, we found that some populations of non-Axin2-expressing cells expressed other Wnt ligands during homeostasis (e.g,. TF2–4; Fig. 5A), suggesting that Wnt secretion from Axin2TdTom and ScxTdTom cells is uniquely necessary for maintaining Axin2 and Wnt9a expression. Together, these data indicate that active Wnt secretion from Axin2TdTom tendon cells is required to maintain their cell state and Wnt9a expression, suggesting a positive feedback loop maintains their own identity.

Next, we sought to determine if this unique Axin2 + /Wnt9a+ cell is present in human tendons. We obtained healthy human hamstring tendons from patients ranging in age from 18 to 22 years and examined AXIN2 and WNT9A expression. Within the main tendon body, we observed a subset of cells expressing AXIN2 and WNT9A (11.7% ± 3.48% and 12.6% ± 4.7% cells per total nuclei per 200 µm2 area, respectively; n = 4), and a majority of the AXIN2+ cells (86.6% ± 7.6) co-expressed WNT9A (Fig. 5I). Together, these findings suggest that a similar AXIN2+ cell population exists in human tendons.

Loss of Wnt signaling from Axin2
TdTom cells impairs healing

As loss of Porcn either in Scx– or Axin2-expressing cells negatively affects the ability of the Axin2 cells to maintain their identity, we next tested if this loss affects their ability to respond to tendon injury. First, we deleted Porcn from the Scx-expressing cells (ScxCreERT2-TdTom; ; Porcnfl/fl) by giving TAM at 3 months and performing partial excisional tendon injuries at 4 months. Examination of ScxCreERT2-TdTom; Porcnfl/fl mice revealed a severely impaired healing response with virtually no Scx-lineage cells, reduced Scx-GFP expression, and a lack of matrix re-organization in the injured area at 30 dpi compared with wild-type injured tendons (Fig. 6A, B, and E; Supplementary Fig. 8A, and Supplementary Movie 10). During the healing process in wild-type tendons, we observed increased gene expression of Axin2, Scx, Col1a2, and Ki67 by RT-qPCR relative to uninjured contralateral control Achilles tendons (Supplementary Fig. 7). Genes associated with a mature tendon matrix, such as Fibromodulin (Fmod) and Connexin 43 (Cx43)58 were significantly upregulated at later healing time points, 20 dpi and 30 dpi, likely signifying cell differentiation and matrix maturation events (Supplementary Fig. 7A). Unlike the control injured tendons, ScxCreERT2-TdTom; Porcnfl/fl injured Achilles tendons did not display dynamic changes in the expression of tendon transcription factors (Scx, Mkx), matrix molecules (Col1a2, Cola3a1), proteins found in mature tendons (Fibromodulin, Connexin 43), proliferation (MKi67), and genes associated with mesenchymal progenitors or differentiation towards other cell fates (Sox9, Runx2) at 10 dpi, 20 dpi, and 30 dpi (Supplementary Fig. 7B), suggesting a severely blunted healing response.

Fig. 6: A cell-autonomous requirement for Porcn in Axin2TdTom cell injury response.
figure 6

Cartoon representations of the experimental design and mouse genotypes with TAM treatment at 3 months and tendon injury at 4 months are shown. AD Whole-mount fluorescent images of the whole Achilles tendon at 30 dpi (outlined by dotted line; muscle attachment is towards the top and bone attachment at bottom) ScxTdTom and Axin2TdTom cells localize to the injury site (arrows) in WT controls (A, C), but not in Scx:CreERT2TdTom; Porcnfl/fl or Axin2:CreERT2TdTom; Porcnfl/fl mutants (B, D). Two-photon fluorescent and SHG images of contralateral uninjured Achilles tendons (E, left) and injured 30 dpi Scx:CreERT2-TdTom; Porcnfl/fl ; Scx-GFP mutant Achilles tendons (E, right), and of uninjured (F, left) and injured 30 dpi Axin2-CreERT2; TdTom; Porcnfl/fl; Scx-GFP mutant tendons (F, right). AF (n = 6 mice per group per time point). G Quantification of total cells and Axin2TdTom (Axin2 cells) cells per 200 × 200 μm square area in the injury site in WT and in Axin2-CreERT2-TdTom; Porcnfl/fl mutant Achilles tendons at 30 dpi (n = 4 mice per group, with three 200 × 200 μm square areas quantified per section and n > 3 sections analyzed per mouse per time point; Unpaired T-test with Welch correction was used; n.s., not significant; ****p < 0.0001). H Quantification of Axin2TdTom and BrdU+ cells per 200 × 200 µm square area in injured and uninjured Achilles tendons from Axin2TdTom WT compared with Axin2:CreERT2-TdTom; Porcnfl/fl mutants at 30 dpi (n = 5 mice per group with quantifications from three 200 × 200 µm square areas per section, and three sections per tendon; unpaired T-test with Welch correction was used; ****p < 0.0001). IK Samples were analyzed for directionality using Fourier spectrum analysis and the FIJI directionality plugin was used to compute the histograms indicating the amount of structures and its direction in each image. The X-axis shows the direction of the structure in degrees, and the Y shows the sum of directions from each slide of the 2P images. I Axin2TdTom mice, there was more alignment into one coherent direction, whereas in J ScxCreERT2-TdTom; Porcnfl/fl and in F Axin2CreERT2-TdTom; Porcnfl/fl mice, the directionality was mostly un-orientated (n > 3).

To test if Wnt secretion specifically from the Axin2-expressing cells is required for tendon healing, we analyzed Axin2CreERT2-TdTom; Porcnfl/fl Achilles tendons at several stages after injury using the same TAM treatment and injury strategy. Similar to ScxCreERT2-TdTom; Porcnfl/fl injured tendons, the healing response was significantly impaired with disruption to the SHG signal, reduced cell proliferation, and abnormal morphology in healing Axin2CreERT2-TdTom; Porcnfl/fl Achilles tendons compared to controls at 30 dpi (Fig. 6C, D, and F–H, Supplementary Fig. 8A, and Supplementary Movie 11). Within the injury site, we observed a virtual loss of all Axin2TdTom cells, as well as significantly fewer total cells and cells incorporating BrdU in Axin2CreERT2-TdTom; Porcnfl/fl tendons compared to injured controls (Fig. 6G, H). Further examination revealed an increase in CD45+ cells at the healing site of Axin2CreERT2-TdTom; Porcnfl/fl tendons compared to wild type (WT) tendons at 30 dpi (Supplementary Fig. 8D, E,), suggesting that some of the cells present in Porcn mutants are blood cells. In addition, there was no significant increase in Scx, Mkx Col1a2, MKi67, Fmod, and Sox9 expression along with several other genes in the injured compared to contralateral uninjured Achilles tendons of Axin2CreERT2-TdTom; Porcnfl/fl mice (Supplementary Fig. 7C). Similarly, we observed significantly reduced numbers of Axin2TdTom cells that expressed Sox9 and αSMA in injured Axin2CreERT2-TdTom; Porcnfl/fl Achilles tendons compared to controls (Supplementary Fig. 8B, C), suggesting the IRC-1 and IRC-2 cell types are not present in Porcn mutants. In addition, Fourier transform methods were used to quantify matrix organization by assessing angles of deviation from the main direction of alignment59,60. We found disorganized matrix orientation upon loss of Porcn by Axin2CreERT2 or ScxCreERT2 compared with controls (Fig. 6I–K and Supplementary Fig. 9). The similarities in the severity of the healing phenotype observed in the Scx- and Axin2-mediated Porcn conditional loss-of-function supports a model in which Axin2TdTom cells are the major Wnt-secreting subset of the Scx cells and that they regulate their injury response in an autocrine manner. As the Axin2TdTom cell identity is affected in Porcn mutants, we cannot rule out that their inability to mount a healing response results from their loss of competence to respond to injury. Moreover, the lack of other Axin2(Neg) cell populations responding to injury in Axin2CreERT2-TdTom; Porcnfl/fl indicates the Axin2+/Scx+ cells through their secretion of Wnt signals, are central to the recruitment of virtually all tendon cells in healing.