Hypertrophic chondrocytes serve as a reservoir for marrow-associated skeletal stem and progenitor cells, osteoblasts, and adipocytes during skeletal development
- Department of Cell Biology, Duke University School of Medicine, United States
- Department of Orthopaedic Surgery, Duke University School of Medicine, United States
- Department of Pharmacology and Cancer Biology, Duke University School of Medicine, United States
- Program of Developmental and Stem Cell Biology, Duke University School of Medicine, United States
- Division of Laboratory Animal Resources, Duke University School of Medicine, United States
- School of Biomedical Sciences, University of Hong Kong, Hong Kong
Figures
Figure 1 with 1 supplement
Hypertrophic chondrocytes are the primary source of osteoblasts/osteocytes in trabecular bone.
Tibia sections of Col10a1CreERT2; Rosa26fs-tdTomato mice injected with tamoxifen at E13.5, 14.5, and 15.5 and subsequently sectioned at E16.5 and stained for (a) DAPI/RFP. (b–c) Higher magnification …
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Figure 1—source data 1
Osteoblast and osteocyte quantification on Col10a1Cre;Rosa26fs-tdTomato 2M old mice.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Contribution of hypertrophic chondrocytes to the bone marrow are observed into skeletal maturity and CRE expression is restricted to hypertrophic chondrocytes in Col10a1Cre mice.
(a) Tibia sections of Col10a1CreERT2; Rosa26fs-tdTomato mice injected with tamoxifen at 2 M of age and subsequently sectioned at 2-week chase. (b) Tibia sections of Col10a1CreERT2; Rosa26fs-tdTomato …
Figure 2 with 2 supplements
Single cell transcriptomics at E16.5 captures hypertrophic chondrocytes and osteoblasts.
(a) UMAP shown in two-dimensional space produced using Seurat 4 package of R from single-cell RNA-sequencing of cleaned skeletal rudiments from Col10Cre; Rosa26fs-tdTomato mice at E16.5. (b–d) …
Figure 2—figure supplement 1
FACS plots for isolation of tdTOMATO+ cells at E16.5 and 2 M for scRNA-sequencing.
(a) E16.5 FACS plot identified tdTOMATO+ expressing cells isolated by total bone digestion and utilized for 10 X Genomics scRNA-sequencing represented in Figure 2. (b) 2 M FACS plot identified …
Figure 2—figure supplement 2
Additional genes of interest expressed in hypertrophic chondrocytes and descendants at E16.5.
(a) Feature plot of Cre expression was enriched in clusters 1–2 at E16.5. (b–c) Feature plot of genes associated with chondrocytes were observed enriched in clusters 1–7. (d-g) Feature plot of genes …
Figure 3 with 1 supplement
Single-cell transcriptomics of E16.5 hypertrophic chondrocytes and descendants reveals an intermediate SSPC upstream of osteoblasts.
(a–d) Feature plot of SSPC-associated genes identified between hypertrophic chondrocyte and osteoblast clusters (e) Violin plot representing the relative level of SSPC associated gene expression (a–d…
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Figure 3—source data 1
PDGFRa+ TOMATO+ quantification at e16.5 of Col10a1Cre; Rosa26-fs-tdTomato; PDGFRa-H2B-GFP.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
SSPC associated proteins are detected in hypertrophic chondrocyte-derived marrow-associated cells.
(a) Immunostaining of SSPC associated proteins in both bone sections from Col10a1CreERT2; Rosa26fs-tdTomato mice injected with tamoxifen at P6 and chased for 2 weeks and (b) 2 M old Col10a1Cre; …
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Figure 3—figure supplement 1—source data 1
LEPR+ TOMATO+ quantification of Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig3-figsupp1-data1-v2.xlsx
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Figure 3—figure supplement 1—source data 2
Flow cytometric analysis of PDGFRa-H2B-GFP and tdTOMATO on Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig3-figsupp1-data2-v2.xlsx
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Figure 3—figure supplement 1—source data 3
Flow cytometric analysis of PDGFRb and tdTOMATO on Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig3-figsupp1-data3-v2.xlsx
Figure 4 with 4 supplements
Single cell transcriptomics of hypertrophic chondrocyte descendants following FACS and 10 X Genomics sequencing at 2 M of age.
(a) UMAP shown in two-dimensional space produced using Seurat 3 package of R from single-cell RNA-sequencing of bone marrow digest from Col10Cre; Rosa26fs-tdTomato mice at 2 M. (b–f) Feature plots …
Figure 4—figure supplement 1
Additional osteoblast associated genes observed in clusters 1 and 2 at 2 months of age.
(a–g) Feature plots of genes associated with osteoblasts were observed in clusters 1 and 2 with minimal expression in clusters 3–5. (h) Violin plots representing relative gene expression levels in (a…
Figure 4—figure supplement 2
Differentially expressed genes mostly unique to clusters 3, 4, and 5.
(a–j) Feature plots of genes identified by differential gene expression in cluster 3 (a–d), cluster 4 (e–h), and cluster 5 (i–l). (m) Violin plots of differentially expressed genes represented in (a–…
Figure 4—figure supplement 3
Hypertrophic chondrocyte-derived cells can associate with blood vessels; however, do not express genes associated with pericytes or vascular smooth muscle cells.
(a) Immunostaining for endothelial-cell-associated protein, ENDOMUCIN, on 2-month-old Col10a1Cre; Rosa26fs-tdTomato bone sections show adjacent tdTOMATO+ cells indicated by orange arrows. Feature …
Figure 4—figure supplement 4
LeprCre; R26-tdTomato and AdipoqCre; R26-tdTomato mice exhibit reporter expression in marrow associated cells, osteoblasts, and adipocytes with age.
(a) Immunostaining of bone sections from LeprCre; Rosa26fs-tdTomato mice exhibit tdTOMATO+ marrow-associated cells; however, do not display tdTOMATO+, OCN+ osteoblasts at P0. Immunostaining at 2 …
Figure 5 with 1 supplement
Many hypertrophic chondrocyte derived cells express genes associated with SSPCs.
(a–f) Feature plots of genes previously identified as SSPC markers in genetic mouse models. (g–i) Feature plots of genes previously identified as SSPC markers for use in flow cytometry and FACS. (j) …
Figure 5—figure supplement 1
Contribution of hypertrophic chondrocyte descendants to total SSPC populations.
(a) Quantitation of immunostaining for LEPR on bone sections from 2 M old Col10a1Cre; Rosa26fs-tdTomato mice exhibit 37.4% LEPR+; tdTOMATO+ as compared to total LEPR+ marrow-associated cells (Figure …
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Figure 5—figure supplement 1—source data 1
LEPR+ TOMATO+ quantification on Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig5-figsupp1-data1-v2.xlsx
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Figure 5—figure supplement 1—source data 2
Flow cytometric analysis of PDGFRa-H2B-GFP and TOMATO on Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig5-figsupp1-data2-v2.xlsx
Figure 6
Hypertrophic chondrocyte derived SSPCs exhibit osteogenic and adipogenic differentiation capacities.
(a) Feature plot and violin plot of the osteoblast specification gene, Runx2. (b–c) Feature plots and violin plots of the adipogenic specification gene, Pparg, and adipogenic associated gene, …
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Figure 6—source data 1
PERILIPIN+ TOMATO+ quantifications on Col10a1Cre;Rosa26fs-tdTomato at 2M.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig6-data1-v2.xlsx
Figure 7
CFU-Fs derived from hypertrophic chondrocytes are similar to CFU-Fs derived from other cell sources, but contain SSPCs with enhanced proliferative capacities.
(a–c) CFU-Fs derived from hypertrophic chondrocytes (tdTOMATO+) (a) and those derived from other cell sources (tdTOMATO-)(b) are established and develop at similar frequencies at 2- and 8 months of …
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Figure 7—source data 1
Tomato+ colony unit formation quantifications at 2 and 8M of Col10a1Cre;Rosa26fs-tdTomato.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data1-v2.xlsx
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Figure 7—source data 2
Bulk RNA-sequencing total gene list of 3 TOMATO+ and 3 TOMATO- colonies from Col10a1Cre;Rosa26fs-tdTomato.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data2-v2.xlsx
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Figure 7—source data 3
Gene ontology analysis of genes enriched in TOMATO+ colonies.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data3-v2.xlsx
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Figure 7—source data 4
Gene ontology analysis of genes enriched in TOMATO- colonies.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data4-v2.xlsx
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Figure 7—source data 5
EDU+ quantifications of TOMATO+ and TOMATO- colonies.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data5-v2.xlsx
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Figure 7—source data 6
Colony size of TOMATO+ and TOMATO- colonies.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig7-data6-v2.xlsx
Figure 8 with 1 supplement
Kidney capsule transplantations of tdTOMATO+ cells exhibit complete ossicle formation with bone, adipocytes, and bone marrow compared to tdTOMATO- resulting in only bone formation.
(a) Immunofluroescent stain for bone (osteopontin), adipocytes (perilipin), and vessels (endomucin) for tomato+ transplant (top) and tomato- transplant (bottom). (b) Hematoxylin and Eosin stain for …
Figure 8—figure supplement 1
In vitro differentiation of colonies reveals relatively similar osteogenic and adipogenic differentiation capacities.
(a–b) CFUs from Col10a1Cre; Rosa26fs-tdTomato bone marrow differentiate into osteoblasts stained with alkaline phosphatase (a) and von kossa (b) (CFU-OB). alkaline phosphatase: SD tdTOMATO+ (T) ± …
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Figure 8—figure supplement 1—source data 1
Differentiation assay quantifications of TOMATO+ and TOMATO- colonies.
- https://cdn.elifesciences.org/articles/76932/elife-76932-fig8-figsupp1-data1-v2.xlsx
Figure 9
Revised model for the generation of trabecular/endocortical bone associated osteoblasts/osteocytes from multiple sources.
(a) Perichondrial or periosteal SSPCs/osteoprogenitors (pSSPC/pOP) migrate into the marrow utilizing blood vessels and possess the ability to further differentiate into osteoblasts/osteocytes …
Tables
Appendix 1—key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 J | Jackson Laboratory | RRID:IMSR_JAX:000664 | |
| Genetic reagent (Mus musculus) | Col10a1Cre | DOI: 10.1242/bio.201411031 | Dr. Klaus von der Mark | |
| Genetic reagent (Mus musculus) | Col10a1CreERT2 | This paper | Dr. Kathryn Cheah | |
| Genetic reagent (Mus musculus) | Rosa26fs-tdTomato | Jackson Laboratory | RRID:IMSR_JAX:007909 | |
| Genetic reagent (Mus musculus) | PdgfraH2B-GFP | Jackson Laboratory | RRID:IMSR_JAX:007669 | |
| Genetic reagent (Mus musculus) | LeprCre | Jackson Laboratory | RRID:IMSR_JAX:008320 | |
| Genetic reagent (Mus musculus) | AdipoqCre | Jackson Laboratory | RRID:IMSR_JAX:028020 | |
| Sequence-based reagent | Col10a1Cre/ ERT2_F | DOI: 10.1242/bio.201411031 | TTTAGAGCATTATT TCAAGGCA GTTTCCADr. Klaus von der Mark | |
| Sequence-based reagent | Col10a1Cre/ ERT2_R | DOI: 10.1242/bio.201411031 | AGGCAAATCTT GGTGTACGGDr. Klaus von der Mark | |
| Antibody | ENDOMUCIN (rat mAb) | Abcam | Cat# ab106100, RRID:AB_10859306 | IF (1:100), 0.3% Triton X-100 in 1 x PBS |
| Antibody | PERILIPIN (rabbit mAb) | Cell Signaling | Cat# 9349, RRID:AB_10829911 | IF (1:100) |
| Antibody | OSTEOCALCIN (rabbit pAb) | Millipore | Cat# AB10911, RRID:AB_1587337 | IF (1:200), 10 ug/mL Proteinase K |
| Antibody | OSTERIX(Rabbit pAb) | Abcam | Cat# ab22552, RRID:AB_2194492 | IF (1:400) |
| Antibody | CRE(Rabbit mAb) | Cell Signaling | Cat# 15036, RRID:AB_2798694 | IF (1:100), 0.3% Triton X-100 in 1 x PBS |
| Antibody | RFP (rabbit pAb) | Abcam | Cat# ab62341, RRID:AB_945213 | IF (1:100) |
| Antibody | LEPR (goat pAb) | R&D Systems | Cat# AF497, RRID:AB_2281270 | IF (1:50), 0.3% Triton X-100 in 1 x PBS |
| Antibody | PDGFRa (goat mAb) | R&D Systems | Cat# AF1062, RRID:AB_2236897 | IF (1:50), 0.3% Triton X-100 in 1 x PBS |
| Antibody | PDGFRb (rat mAb) | Invitrogen | Cat# 14-1402-81, RRID:AB_467492 | IF (1:50), 0.3% Triton X-100 in 1 x PBS |
| Antibody | Osteopontin (goat pAb) | R&D Systems | Cat# AF808, RRID:AB_2194992 | IF (1:100), 0.3% Triton X-100 in 1 x PBS |
| Antibody | PDGFRb (rat mAb) | Invitrogen | Cat# 17-1402-80, RRID:AB_1548752 | Flow Cytometry (1:25) |
| Antibody | CD45-FITC (rat mAb) | Biolegend | Cat# 103107, RRID:AB_312972 | Flow Cytometry (1:1000) |
| Antibody | CD45-APC (rat mAb) | Biolegend | Cat# 103111, RRID:AB_312976 | Flow Cytometry (1:1000) |
| Antibody | CD31-FITC (rat mAb) | Biolegend | Cat# 102405, RRID:AB_312900 | Flow Cytometry (1:1000) |
| Antibody | CD31-APC (rat mAb) | Biolegend | Cat# 102409, RRID:AB_312904 | Flow Cytometry (1:1000) |
| Antibody | TER119-FITC (rat mAb) | BD Biosciences | Cat# 561032, RRID:AB_10563083 | Flow Cytometry (1:500) |
| Antibody | TER119-APC (rat mAb) | Biolegend | Cat# 116211, RRID:AB_313712 | Flow Cytometry (1:250) |
| Antibody | DAPI (FLOW) | ThermoFisher | Cat# D1306, RRID:AB_2629482 | Flow Cytometry (1:1000) |
| Antibody | Goat anti-Mouse Alexa Fluor 488 pAb | Invitrogen | Cat# A-11001, RRID:AB_2534069 | IF (1:200) |
| Antibody | Donkey anti-Goat Alexa Fluor 488 pAb | Invitrogen | Cat# A-11055, RRID:AB_2534102 | IF (1:200) |
| Antibody | Goat anti-Rat Alexa Fluor 488 pAb | Invitrogen | Cat# A-11006, RRID:AB_2534074 | IF (1:200) |
| Antibody | Goat anti-Rabbit Alexa Fluor 594 pAb | Invitrogen | Cat# A-11037, RRID:AB_2534095 | IF (1:200) |
| Antibody | Goat anti-Rabbit Alexa Fluor 488 pAb | Invitrogen | Cat# A-11034, RRID:AB_2576217 | IF (1:200) |
| Antibody | Goat anti-Rat Alexa Fluor 647 pAb | Invitrogen | Cat# A-21247, RRID:AB_141778 | IF (1:200) |
| Antibody | Goat anti-Rabbit Alexa Fluor 488 pAb | Invitrogen | Cat# A-11008, RRID:AB_143165 | For LepRCre;tdTomato stained PERILIPIN only - IF (1:200) |
| Chemical compound, drug | Collagenase II | Gibco | 17101–015 | |
| Chemical compound, drug | Collagenase D | Roche | 11088866001 | |
| Chemical compound, drug | Polyvinylpyrrolidone | Sigma | P5288 | |
| Chemical compound, drug | Glutamax 100 x | Gibco | 35050–061 | |
| Chemical compound, drug | L-Ascorbic Acid | Sigma | A4544 | |
| Chemical compound, drug | B-glycerophosphate | Sigma | G9422 | |
| Chemical compound, drug | Insulin Human | Sigma | I3536 | |
| Chemical compound, drug | IBMX | Sigma | I7018 | |
| Chemical compound, drug | Dexamethasone | Sigma | D2915 | |
| Chemical compound, drug | Antigen Unmasking solution, Citrate Acid Based | Vector | H3300 | |
| Commercial assay, kit | Click-iT Plus EdU Cell Proliferation Kit | Invitrogen | C10637 | |
| Software, algorithm | FIJI | doi:10.1038/ nmeth.2019 | Fiji, RRID:SCR_002285 | |
| Software, algorithm | Seurat | CRAN | Seurat, RRID:SCR_007322 | |
| Software, algorithm | Monocle3 | Github | Monocle3, RRID:SCR_018685 | |
| Software, algorithm | Trim Galore | Babraham Bioinformatics | Trim Galore,RRID:SCR_011847 | Felix Krueger |
| Software, algorithem | Cutadapt | Github | cutadapt, RRID:SCR_011841 | |
| Software, algorithm | STAR RNA-seq Alignment | Github | STAR, RRID:SCR_004463 | |
| Software, algorithm | HTSeq tool | Github | HTSeq, RRID:SCR_005514 | |
| Software, algorithm | DESeq2 | Bioconductor | DESeq2, RRID:SCR_015687 | |
| Software, algorithm | Gene Ontology | Geneontoloy.org | Gene Ontology, RRID:SCR_002811 | |
| Other | Silver Nitrate 10% | Ricca | 6830–4 | |
| Other | Oil Red O | Sigma | O0625 | |
| Other | Alizarin Red | Sigma | A5533 | |
| Other | Mayer’s Hematoxylin solution | Electron Microscopy Sciences | 26043–06 | |
| Other | Eosin Y with Phloxine staining solution | Electron Microscopy Sciences | 26051–21 |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/76932/elife-76932-transrepform1-v2.pdf
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Source code 1
Code used for generation of Single Cell datasets and analysis at e16.5/2M of Col10a1Cre;Rosa26fs-tdTomato.
- https://cdn.elifesciences.org/articles/76932/elife-76932-code1-v2.zip
