Scleraxis-lineage cells are required for tendon homeostasis and their depletion induces an accelerated extracellular matrix aging phenotype
- Center for Musculoskeletal Research, Department of Orthopaedics & Rehabilitation, University of Rochester Medical Center, United States
- Department of Biomedical Engineering, University of Rochester, United States
Figures
Figure 1
Depletion of Scleraxis-lineage cell during long-term homeostasis significantly disrupts tendon structure and mechanical properties.
(A) 3 M old Scx-Cre+; Rosa- DTRF/+ (DTR) and Scx-Cre-; Rosa-DTRF/+ (WT) mice received five hind paw injections of DT and were harvested at 3, 6, and 9 M post-depletion. (B) Quantification of total …
Figure 2 with 4 supplements
Scleraxis-lineage cells maintain FDL tendon homeostasis by regulating the synthesis of high turnover rate ECM proteins.
(A) Volcano plot and heatmap visualizing the significantly different protein abundances between DTR and WT groups at 3 M post-depletion. (B) Classification of all downregulated proteins between the …
Figure 2—figure supplement 1
Scleraxis-lineage cells maintain long-term tendon homeostasis via synthesis of high turnover rate glycoproteins and proteoglycans.
(A) Volcano plot and (B) heatmap visualizing the significantly different protein abundances between DTR and WT groups at 9 M post-depletion. Blue indicates proteins that are decreased in DTR tendons …
Figure 2—figure supplement 2
The proteome between 12 M old WT and young (6 M old) DTR tendons is almost identical with no differences in high turnover rate glycoproteins and proteoglycans.
(A) Volcano plot and (B) heatmap visualizing the significantly different protein abundances between 12 M old WT and 6 M old DTR groups. Blue indicates proteins that are decreased in 12 M old WT vs. …
Figure 2—figure supplement 3
Significant decrease of Coch +and Chad + cells with Scleraxis-lineage cell depletion and natural aging.
(A) UMAP plots of Coch +tenocytes in the 6 M WT, DTR, and 21 M B6 groups. (B) Total tenocyte number of Coch + cells in each condition. (C) % of Coch +normalized by total tenocytes for each …
Figure 2—figure supplement 4
Depletion of Scleraxis-lineage cells significantly impairs tendon structural and material properties.
Timeline of DT injections and tissue harvesting (A). Quantification of CSA (B), stiffness (C), and elastic modulus (D) of WT and DTR tendons at 3, 6, and 9 months post-depletion. CSA, stiffness, and …
Figure 3 with 2 supplements
scRNAseq demonstrates broad cellular heterogeneity and intrinsic programmatic skewing of tenocytes with depletion and natural aging.
(A) UMAP dimensionality reduction revealed seven broad and distinct cell populations on clustering based on unbiased differential gene expression of the integrated dataset. (B) Annotation marker for …
Figure 3—figure supplement 1
Annotation of scRNAseq-based identified cell clusters in the integrated data.
(A) UMAP with all different clusters annotated based on (B). (B) Heatmap of top 15 DEGs for each cluster. (C) Feature plot of the epitenon marker Ccn3 being expressed by the epitenon sub-cluster. (D)…
Figure 3—figure supplement 2
Tendon resident macrophages exhibit significant age-related intrinsic programmatic shifts.
(A) UMAP plot of the overall tendon resident macrophages as well as separated based on age (6 M WT and 21 M old B6). (B) Heatmap with the top 50 DEG of tendon resident macrophages between the young …
Figure 4
DTR and aged tendons lose tenocytes associated with ECM biosynthesis and immune surveillance, while DTR tendons retain tenocytes associated with ECM organization and remodeling.
(A) UMAP plot of all three tenocytes subpopulations. (B) UMAP plot showing the shifts of tenocytes 1–3 with depletion and natural aging. (C–E) Quantification of % in tenocytes 1 (C), tenocytes 2 (D),…
Figure 5
Tenocytes 1 and 2 become more ECM organizational in DTR tendons while tenocytes 1 exhibit indications of aging hallmarks such as loss of proteostasis and inflammaging.
(A) UMAP plot of tenocytes 1 in 6 M WT and DTR groups. (B) Volcano plot visualizing all the significantly different genes in tenocytes 1 between 6 M WT vs. 6 M DTR. Blue dots indicate genes that are …
Figure 6 with 1 supplement
Tenocyte-tenocyte communication is impaired with both Scleraxis-lineage cell depletion and natural aging.
(A) UMAP plot of tenocytes subpopulations in the 6 M WT and DTR groups. (B) Differential number of cell-cell interactions of tenocytes 1–3 in the 6 M WT and DTR groups (red color indicates an …
Figure 6—figure supplement 1
Tenocyte-tenocyte communication in the young adult FDL tendons.
(A) UMAP plot visualizing the three tenocyte subpopulations that exist in the 6 M WT FDL tendons. (B) Circle plot visualizing the autocrine and paracrine communication strength of each tenocyte …
Figure 7
Schematic highlighting key findings and proposed models for divergent healing responses in young depleted vs aged tendons.
Both young DTR and aged WT tendons have similar decreases in tissue structure, organization, material quality, and total cell density. They also follow the same mechanism of ECM degeneration via a …
Author response image 1
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Genetic reagent (Mus. musculus) | Scx-Cre | Dr. Ronen Schweitzer | ||
| Genetic reagent (Mus. musculus) | C57BL/6-Gt(ROSA)26S ortm1(HBEGF)Awai/J (Rosa-DTRLSL) | Jackson Laboratory | Stock #: 007900 | Referred to as DTR in manuscript RRID:IMSR_JAX:007900 |
| Genetic reagent (Mus. musculus) | C57Bl/6 J | Jackson Laboratory | Stock #: 000664 | Referred to as B6 in manuscript RRID:MGI:2159769 |
| Antibody | Anti-rabbit Rhodamine- Red-X (donkey polyclonal) | Jackson ImmunoResearch | Catalog #: 711-296-152 | 1:200 RRID:AB_2340614 |
| Antibody | Anti-GBP2 (rabbit polyclonal) | Proteintech | Catolog #: 11854–1-AP | 1:500 RRID: AB_2109336 |
| Chemical Compound, Drug | Diphtheria Toxin (DT) | Millipore Sigma | Catalog #: D0564-1MG | 20 ng DT / injection |
| Software, algorithm | GraphPad Prism software | GraphPad Prism | https://graphpad.com | Version 9.5 |
| Software, algorithm | OlyVIA software | Olympus | https://www.olympus-lifescience.com/en/support/downloads/ | Version 2.9 |
| Software, algorithm | ImageJ software | ImageJ | http://imagej.nih.gov/ij/ | |
| Software, algorithm | R studio | R Studio | https://www.rstudio.com | |
| Software, algorithm | DAVID Gene Functional Classification Tool | Huang et al., 2009 | https://david.ncifcrf.gov/ | Version 6.8 |
| Software, algorithm | CellChat | Jin et al., 2021 | http://www.cellchat.org | Version 1.1.3 |
| Software, algorithm | Seurat R package | Stuart et al., 2019 | https://www.rdocumentation.org/packages/Seurat/versions/4.1.0 | Version 4.0 |
| Software, algorithm | Discoverer software platform | Thermo Fisher | Version 2.4 | |
| Software, algorithm | PANTHER classification system | Mi et al., 2021 | http://pantherdb.org/ | |
| Software, algorithm | Search Tool for Retrieval of Interacting Genes/ Proteins (STRING) | Szklarczyk et al., 2019 | v11.0 | |
| Software, algorithm | MatrisomeDB | Hynes and Naba, 2012. | https://web.mit.edu/hyneslab/matrisome/ | |
| Software, algorithm | CellMarker software | Zhang et al., 2019 | http://bio-bigdata.hrbmu.edu.cn/CellMarker/search.jsp | |
| Other | Gene Expression Omnibus (GEO) | Accession # GSE214929 | Single-cell RNA sequencing data | |
| Other | ProteomeXchange Consortium | Dataset Identifier: PXD037230 | Proteomics data |
