Osteocalcin expressing cells from tendon sheaths in mice contribute to tendon repair by activating Hedgehog signaling
- The Chinese University of Hong Kong, Hong Kong
- The Chinese University of Hong Kong, Prince of Wales Hospital, Hong Kong
Figures
Figure 1 with 1 supplement
Bglap as a tendon sheath specific marker.
(A) Fluorescent immunohistochemistry of mouse sheath tissues of the adult Tibialis anterior tendon at 2 months old (n = 5 mice per group). Scale bar, 40 μm. (B) QRT-PCR analysis of sheath-specific …
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Figure 1—source data 1
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- https://doi.org/10.7554/eLife.30474.005
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Figure 1—source data 2
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- https://doi.org/10.7554/eLife.30474.006
Figure 1—figure supplement 1
Fluorescence-assisted cell-sorting (FACS) analysis of sheath-derived cells.
(A) Cells dissociated from the Tibialis anterior sheath tissues of the BGLAP-Cre;Rosa26mT/mG mice after cell-sorting for GFP and tdTomato signals. (B) The number of colonies was counted in a colony-f…
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Figure 1—figure supplement 1—source data 1
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- https://doi.org/10.7554/eLife.30474.004
Figure 2 with 1 supplement
Multipotent differentiation potential of sheath-derived cells.
(A) Colony formation assay using sheath-derived cells (GFP+) or tendon-derived cells (tdTomato+) isolated from the adult tendon tissues of BGLAP-Cre;Rosa26mT/mG mice. Colonies were stained with …
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Figure 2—source data 1
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- https://doi.org/10.7554/eLife.30474.012
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Figure 2—source data 2
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Figure 2—source data 3
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- https://doi.org/10.7554/eLife.30474.014
Figure 2—figure supplement 1
Multipotent differentiation potential of sheath-derived cells in vitro and in vivo.
(A–C) Gene expression profiles of lineage markers using primary cells isolated from the BGLAP-Cre;Rosa26mT/mG Tibialis anterior tendon sheath tissues treated with 100 ng/ml BMP2 (CST, USA) or 2 …
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Figure 2—figure supplement 1—source data 1
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- https://doi.org/10.7554/eLife.30474.009
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Figure 2—figure supplement 1—source data 3
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Figure 3 with 2 supplements
Tendon sheath tissues contribute to tendon repair.
(A) Fluorescence analysis in cross- and longitudinal sections of the Tibialis anterior tendon of BGLAP-Cre;Rosa26mT/mG mice at Day 0 (Control) and Day 14 after injury(without grafting) (n = 3 mice …
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Figure 3—source data 1
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- https://doi.org/10.7554/eLife.30474.018
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Figure 3—source data 5
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- https://doi.org/10.7554/eLife.30474.022
Figure 3—figure supplement 1
BGLAP-Cre/GFP+cells contribute to tendon repair in BGLAP-Cre; Rosa26mT/mG mice.
(A) Fluorescence analysis of all channels in cross- and longitudinal sections of the Tibialis anterior tendon of the BGLAP-Cre;Rosa26mT/mG mice at Day 0 (Control) and Day 14 after injury (without …
Figure 3—figure supplement 2
GFP+sheath cells differentiate into Mkx-expressing cells and contribute to tendon repair.
(A) Co-localization of Mkx and BGLAP-Cre/GFP at the injured sites of the nude mouse tendon tissues with or without the sheath transplantation at Day 14 after injury for all channels (n = 5 mice per …
Figure 4
Activation of Hh signaling in sheath tissues upon injury.
(A, B) Gene expression profiles of (A) tendon progenitor markers and (B) ECM components in the tendon sheath tissues of wild-type mice two weeks after injury. Relative expression levels were …
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Figure 4—source data 1
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- https://doi.org/10.7554/eLife.30474.024
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Figure 4—source data 4
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Figure 5 with 1 supplement
Activated Hh signaling increases cell proliferation in sheath tissues.
(A) Histologic analysis of the sheath tissues of the Tibialis anterior tendon of different mice genotypes at multiple time points. Scale bar, 20 μm. (B) Statistical analyses of the sheath thickness …
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Figure 5—source data 1
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- https://doi.org/10.7554/eLife.30474.031
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Figure 5—source data 2
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Figure 5—source data 3
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- https://doi.org/10.7554/eLife.30474.033
Figure 5—figure supplement 1
Hh signaling increases sheath cell proliferation.
(A) Fluorescent immunohistochemistry of pH3 in wild-type adult tendons. Sheath-derived cells displayed a higher cell proliferation rate than tendon fibers. T: tendon fibers; S: sheath tissues. …
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Figure 5—figure supplement 1—source data 1
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- https://doi.org/10.7554/eLife.30474.030
Figure 6
Hh signaling is necessary for sheath cell proliferation.
(A) Histologic analysis of the sheath tissues of the Tibialis anterior tendon of mice with genotypes as shown. Scale bar, 100 μm. Higher magnification is shown on the right. Scale bar, 20 μm. (B) …
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Figure 6—source data 1
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- https://doi.org/10.7554/eLife.30474.035
Figure 7 with 1 supplement
Hh signaling is required for sheath-mediated tendon repair.
(A) Histologic analysis of the Tibialis anterior tendon of mice with genotypes as shown 4 weeks after injury (n = 4 mice per group). Scale bar, 400 μm. Higher magnifications of the boxed regions are …
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Figure 7—source data 1
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- https://doi.org/10.7554/eLife.30474.040
Figure 7—figure supplement 1
Tendon repair is significantly impaired in the Smoc/c;BGLAP-Cre mutant mice after injury.
(A, B) Gene expression profiles of the injured Tibialis anterior tendon fibers of (A) tendon progenitor markers and (B) tendon ECM components of mice (n = 4 mice per group) with genotypes as shown. …
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Figure 7—figure supplement 1—source data 1
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- https://doi.org/10.7554/eLife.30474.039
Figure 8
Hh activation upregulates Mkx and Collagen I via TGFβ/Smad3 signaling.
(A, C) Gene expression profiling using primary Ptch1c/c sheath cells infected with Cre-adenovirus for 48 hr. Relative expression levels were normalized to β-tubulin and the Ad-GFP group. Data are …
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Figure 8—source data 1
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Figure 8—source data 3
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Figure 8—source data 4
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- https://doi.org/10.7554/eLife.30474.045
Figure 9
Hh activation upregulates Mkx and Collagen I via TGFβ/Smad3 signaling.
(A) (Upper panel) Schematic diagram of putative Smad-binding element (SBE) motifs in the promoter region of Mkx. (TSS, transcription start site; the canonical Smad-binding conserved sequence …
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Figure 9—source data 1
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- https://doi.org/10.7554/eLife.30474.047
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Figure 9—source data 2
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- https://doi.org/10.7554/eLife.30474.048
Figure 10
Upregulation of GLI1 and MKX expression in human tendinopathy specimens.
(A, B) Fluorescent immunohistochemistry of GLI1 and MKX in (A) healthy patellar tendon tissues or (B) patellar tendinopathy tissues. The bottom images show two regions adjacent to the injured …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers |
|---|---|---|---|
| Genetic reagent (Mus musculus) | BGLAP-Cre;Rosa26mT/mG | This paper | NA |
| Genetic reagent (M. musculus) | Ptch1c/c;BGLAP-Cre | Mak et al. (2006) | PMID: 16936073; DOI: 10.1242/dev.02546 RRID:MGI:3687744 |
| Genetic reagent (M. musculus) | Smoc/c;BGLAP-Cre | Mak et al. (2008a) | PMID: 18434416; DOI: 10.1242/dev.018044 RRID:MGI:5311416 |
| Genetic reagent (M. musculus) | Ptch1LacZ/+ | This paper | NA |
| Recombinant DNA reagent | pGL3-Mkxp8K | This paper | NA |
| Recombinant DNA reagent | pGL3-Mkx4K | This paper | NA |
| Recombinant DNA reagent | pCMV5 SMAD2-HA | Addgene, USA | plasmid # 14930 |
| Recombinant DNA reagent | pCMV5B-Flag-SMAD3 | Addgene, USA | plasmid # 11742 |
| Antibody | Osteolcalcin/Bglap | Abcam, USA | ab93876, 5 ug/ml RRID: AB_10675660 |
| Antibody | Tppp3 | Abcam, USA | ab150998, 1:50 RRID: AB_2716739 |
| Antibody | Mkx | Lifespan Biosciences Inc., USA | LS-B8063, 1 ug/ml RRID: AB_2716740 |
| Antibody | Mkx | Abcam, USA | ab179597, 1 ug/ml RRID: AB_2716741 |
| Antibody | Gli1 | R&D, USA | MAB3324, 10 ug/ml RRID:AB_2111775 |
| Antibody | Gli2 | R&D, USA | AF3635, 5 ug/ml RRID:AB_2111902 |
| Antibody | pH3 | SantaCruz, USA | sc-8656-R, 1:200 RRID:AB_653256 |
| Antibody | Collagen I | Abcam, USA | ab292, 1:1000 RRID:AB_303415 |
| Antibody | TGF-b1 | Abcam, USA | ab64715, 1:500 RRID:AB_1144265 |
| Antibody | Smad3 | CST, USA | #9523, 1:1000 RRID:AB_2193182 |
| Antibody | p-Smad3 | CST, USA | #9520, 1:1000 RRID:AB_2193207 |
| Antibody | β-Actin | CST, USA | #8457, 1:10000 RRID:AB_10950489 |
| Commercial assay or kit | Apoptosis Fluorescein Detection Kit | Millipore, USA | s7111 |
| Commercial assay or kit | Hydroxyproline Assay Kit | Sigma-Aldrich, USA | MAK008 |
| Commercial assay or kit | Dual-luciferase reporter assay system | Promega, USA | E1980 |
| Chemical compound, drug | BMP2 | CST, USA | #4697 |
| Chemical compound, drug | TGF-β1 | Peprotech, USA | 100–21 |
| Chemical compound, drug | Purmorphamine | Millipore, USA | 540220 |
| Chemical compound, drug | SB431542 | Sigma-Aldrich, USA | S4317 |
Additional files
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Supplementary file 1
Primer sequences for QRT-PCR.
- https://doi.org/10.7554/eLife.30474.050
