Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Erythromyeloid progenitors give rise to a population of osteoclasts that contribute to bone homeostasis and repair

Nature Cell Biology volume 22, pages 49–59 (2020)Cite this article

Subjects

Abstract

Osteoclasts are multinucleated cells of the monocyte/macrophage lineage that degrade bone. Here, we used lineage tracing studies—labelling cells expressing Cx3cr1, Csf1r or Flt3—to identify the precursors of osteoclasts in mice. We identified an erythromyeloid progenitor (EMP)-derived osteoclast precursor population. Yolk-sac macrophages of EMP origin produced neonatal osteoclasts that can create a space for postnatal bone marrow haematopoiesis. Furthermore, EMPs gave rise to long-lasting osteoclast precursors that contributed to postnatal bone remodelling in both physiological and pathological settings. Our single-cell RNA-sequencing data showed that EMP-derived osteoclast precursors arose independently of the haematopoietic stem cell (HSC) lineage and the data from fate tracking of EMP and HSC lineages indicated the possibility of cell–cell fusion between these two lineages. Cx3cr1+ yolk-sac macrophage descendants resided in the adult spleen, and parabiosis experiments showed that these cells migrated through the bloodstream to the remodelled bone after injury.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Csf1r+ yolk-sac macrophages give rise to the neonatal osteoclasts.
Fig. 2: Cx3cr1+ yolk-sac macrophages give rise to the osteoclasts that survive in adulthood.
Fig. 3: EMPs and HSCs give rise to the postnatal osteoclast.
Fig. 4: EMP- and HSC-derived cells specify distinct subpopulations.
Fig. 5: Descendants of Cx3cr1+ yolk-sac macrophages provide osteoclasts after bone injury.
Fig. 6: Descendants of Cx3cr1+ yolk-sac macrophages migrated into a bone injury site through the blood circulation.

Similar content being viewed by others

Data availability

The scRNA-seq data that support the findings of this study have been deposited in the Gene Expression Omnibus (GEO) under accession code GSE125088. Source data are available online for Figs. 1–6 and Extended Data Figs. 3 and 5–7. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

References

  1. Frost, H. M., Vilanueva, A. R., Jett, S. & Eyring, E. Tetracycline-based analysis of bone remodelling in osteopetrosis. Clin. Orthop. Relat. Res. 65, 203–217 (1969).

    Article  CAS  PubMed  Google Scholar 

  2. Chan, C. K. F. et al. Identification of the human skeletal stem cell. Cell 175, 43–56 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Debnath, S. et al. Discovery of a periosteal stem cell mediating intramembranous bone formation. Nature 562, 133–139 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Yue, R., Zhou, Bo. O., Shimada, IsseiS., Zhao, Z. & Morrison, SeanJ. Leptin receptor promotes adipogenesis and reduces osteogenesis by regulating mesenchymal stromal cells in adult bone marrow. Cell Stem Cell 18, 782–796 (2016).

    Article  CAS  PubMed  Google Scholar 

  5. Udagawa, N. et al. Origin of osteoclasts: mature monocytes and macrophages are capable of differentiating into osteoclasts under a suitable microenvironment prepared by bone marrow-derived stromal cells. Proc. Natl Acad. Sci. USA 87, 7260–7264 (1990).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Takahashi, N. et al. Postmitotic osteoclast precursors are mononuclear cells which express macrophage-associated phenotypes. Dev. Biol. 163, 212–221 (1994).

    Article  CAS  PubMed  Google Scholar 

  7. Lacey, D. L. et al. Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation. Cell 93, 165–176 (1998).

    Article  CAS  PubMed  Google Scholar 

  8. Yasuda, H. et al. Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL. Proc. Natl Acad. Sci. USA 95, 3597–3602 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Jee, W. S. & Nolan, P. D. Origin of osteoclasts from the fusion of phagocytes. Nature 200, 225–226 (1963).

    Article  CAS  PubMed  Google Scholar 

  10. Horton, M. A., Pringle, J. A. & Chambers, T. J. When is it an osteoclast? J. Clin. Pathol. 38, 596–597 (1985).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Italiani, P. & Boraschi, D. Development and functional differentiation of tissue-resident versus monocyte-derived macrophages in inflammatory reactions. Results Probl. Cell Differ. 62, 23–43 (2017).

    Article  CAS  PubMed  Google Scholar 

  12. van de Laar, L. et al. Yolk sac macrophages, fetal liver, and adult monocytes can colonize an empty niche and develop into functional tissue-resident macrophages. Immunity 44, 755–768 (2016).

    Article  PubMed  CAS  Google Scholar 

  13. Moore, M. A. & Metcalf, D. Ontogeny of the haemopoietic system: yolk sac origin of in vivo and in vitro colony forming cells in the developing mouse embryo. Br. J. Haematol. 18, 279–296 (1970).

    Article  CAS  PubMed  Google Scholar 

  14. Hoeffel, G. & Ginhoux, F. Fetal monocytes and the origins of tissue-resident macrophages. Cell. Immunol. 330, 5–15 (2018).

    Article  CAS  PubMed  Google Scholar 

  15. Lee, C. Z. W., Kozaki, T. & Ginhoux, F. Studying tissue macrophages in vitro: are iPSC-derived cells the answer? Nat. Rev. Immunol. 18, 716–725 (2018).

    Article  CAS  PubMed  Google Scholar 

  16. Palis, J., Robertson, S., Kennedy, M., Wall, C. & Keller, G. Development of erythroid and myeloid progenitors in the yolk sac and embryo proper of the mouse. Development 126, 5073–5084 (1999).

    CAS  PubMed  Google Scholar 

  17. Ginhoux, F. & Guilliams, M. Tissue-resident macrophage ontogeny and homeostasis. Immunity 44, 439–449 (2016).

    Article  CAS  PubMed  Google Scholar 

  18. Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gomez Perdiguero, E. et al. Tissue-resident macrophages originate from yolk-sac-derived erythro-myeloid progenitors. Nature 518, 547–551 (2015).

    Article  PubMed  CAS  Google Scholar 

  20. Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012).

    Article  CAS  PubMed  Google Scholar 

  21. Mass, E. et al. Specification of tissue-resident macrophages during organogenesis. Science 353, aaf4238 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  22. Hoeffel, G. et al. C-Myb+ erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages. Immunity 42, 665–678 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Perdiguero, E. G. & Geissmann, F. The development and maintenance of resident macrophages. Nat. Immunol. 17, 2–8 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Plein, A., Fantin, A., Denti, L., Pollard, J. W. & Ruhrberg, C. Erythro-myeloid progenitors contribute endothelial cells to blood vessels. Nature 562, 223–228 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Wilson, C. H. et al. The kinetics of ER fusion protein activation in vivo. Oncogene 33, 4877–4880 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Romeo, S. G. et al. Endothelial proteolytic activity and interaction with non-resorbing osteoclasts mediate bone elongation. Nat. Cell Biol. 21, 430–441 (2019).

    Article  CAS  PubMed  Google Scholar 

  27. Stremmel, C. et al. Yolk sac macrophage progenitors traffic to the embryo during defined stages of development. Nat. Commun. 9, 75 (2018).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Goldmann, T. et al. Origin, fate and dynamics of macrophages at central nervous system interfaces. Nat. Immunol. 17, 797–805 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Mossadegh-Keller, N. et al. Developmental origin and maintenance of distinct testicular macrophage populations. J. Exp. Med. 214, 2829–2841 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Molawi, K. et al. Progressive replacement of embryo-derived cardiac macrophages with age. J. Exp. Med. 211, 2151–2158 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Hagemeyer, N. et al. Transcriptome-based profiling of yolk sac-derived macrophages reveals a role for Irf8 in macrophage maturation. EMBO J. 35, 1730–1744 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Jacome-Galarza, C. E. et al. Developmental origin, functional maintenance and genetic rescue of osteoclasts. Nature 568, 541–545 (2019).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Takayanagi, H. et al. Induction and activation of the transcription factor NFATc1 (NFAT2) integrate RANKL signaling in terminal differentiation of osteoclasts. Dev. Cell 3, 889–901 (2002).

    Article  CAS  PubMed  Google Scholar 

  34. Yagi, M. et al. DC-STAMP is essential for cell-cell fusion in osteoclasts and foreign body giant cells. J. Exp. Med. 202, 345–351 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Yang, M. et al. Osteoclast stimulatory transmembrane protein (OC-STAMP), a novel protein induced by RANKL that promotes osteoclast differentiation. J. Cell. Physiol. 215, 497–505 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Miyamoto, H. et al. Osteoclast stimulatory transmembrane protein and dendritic cell-specific transmembrane protein cooperatively modulate cell-cell fusion to form osteoclasts and foreign body giant cells. J. Bone Miner. Res. 27, 1289–1297 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Wu, H., Xu, G. & Li, Y. P. Atp6v0d2 is an essential component of the osteoclast-specific proton pump that mediates extracellular acidification in bone resorption. J. Bone Miner. Res. 24, 871–885 (2009).

    Article  CAS  PubMed  Google Scholar 

  38. Saftig, P. et al. Impaired osteoclastic bone resorption leads to osteopetrosis in cathepsin-K-deficient mice. Proc. Natl Acad. Sci. USA 95, 13453–13458 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Grigoriadis, A. E. et al. c-Fos: a key regulator of osteoclast-macrophage lineage determination and bone remodeling. Science 266, 443–448 (1994).

    Article  CAS  PubMed  Google Scholar 

  40. Ikeda, F. et al. Critical roles of c-Jun signaling in regulation of NFAT family and RANKL-regulated osteoclast differentiation. J. Clin. Invest. 114, 475–484 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. Teo, B. H., Bobryshev, Y. V., Teh, B. K., Wong, S. H. & Lu, J. Complement C1q production by osteoclasts and its regulation of osteoclast development. Biochem. J. 447, 229–237 (2012).

    Article  CAS  PubMed  Google Scholar 

  42. Kobayashi, K. et al. Tumor necrosis factor α stimulates osteoclast differentiation by a mechanism independent of the ODF/RANKL-RANK interaction. J. Exp. Med. 191, 275–286 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Kim, J. H. et al. The mechanism of osteoclast differentiation induced by IL-1. J. Immunol. 183, 1862–1870 (2009).

    Article  CAS  PubMed  Google Scholar 

  44. Pierce, A. M. & Lindskog, S. Evidence for capping of Fc gamma receptors on osteoclasts. Calcif. Tissue Int. 39, 109–116 (1986).

    Article  CAS  PubMed  Google Scholar 

  45. Seeling, M. et al. Inflammatory monocytes and Fcγ receptor IV on osteoclasts are critical for bone destruction during inflammatory arthritis in mice. Proc. Natl Acad. Sci. USA 110, 10729–10734 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hong, M. H., Williams, H., Jin, C. H. & Pike, J. W. The inhibitory effect of interleukin-10 on mouse osteoclast formation involves novel tyrosine-phosphorylated proteins. J. Bone Miner. Res. 15, 911–918 (2000).

    Article  CAS  PubMed  Google Scholar 

  47. Bendixen, A. C. et al. IL-4 inhibits osteoclast formation through a direct action on osteoclast precursors via peroxisome proliferator-activated receptor γ1. Proc. Natl Acad. Sci. USA 98, 2443–2448 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. He, Y. X. et al. Impaired bone healing pattern in mice with ovariectomy-induced osteoporosis: a drill-hole defect model. Bone 48, 1388–1400 (2011).

    Article  PubMed  Google Scholar 

  49. Conboy, I. M. et al. Rejuvenation of aged progenitor cells by exposure to a young systemic environment. Nature 433, 760–764 (2005).

    Article  CAS  PubMed  Google Scholar 

  50. Nakamichi, Y. et al. Spleen serves as a reservoir of osteoclast precursors through vitamin D-induced IL-34 expression in osteopetrotic op/op mice. Proc. Natl Acad. Sci. USA 109, 10006–10011 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. A-Gonzalez, N. & Castrillo, A. Origin and specialization of splenic macrophages. Cell. Immunol. 330, 151–158 (2018).

    Article  CAS  PubMed  Google Scholar 

  52. Kurotaki, D., Uede, T. & Tamura, T. Functions and development of red pulp macrophages. Microbiol. Immunol. 59, 55–62 (2015).

    Article  CAS  PubMed  Google Scholar 

  53. Blavier, L. & Delaisse, J. M. Matrix metalloproteinases are obligatory for the migration of preosteoclasts to the developing marrow cavity of primitive long bones. J. Cell Sci. 108, 3649–3659 (1995).

    CAS  PubMed  Google Scholar 

  54. Sabatel, C. et al. Exposure to bacterial CpG DNA protects from airway allergic inflammation by expanding regulatory lung interstitial macrophages. Immunity 46, 457–473 (2017).

    Article  CAS  PubMed  Google Scholar 

  55. Epelman, S. et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity 40, 91–104 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. Kawamoto, T. Use of a new adhesive film for the preparation of multi-purpose fresh-frozen sections from hard tissues, whole-animals, insects and plants. Arch. Histol. Cytol. 66, 123–143 (2003).

    Article  PubMed  Google Scholar 

  57. Macosko, E. Z. et al. Highly parallel genome-wide expression profiling of individual cells using nanoliter droplets. Cell 161, 1202–1214 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  58. van der Maaten, L. & Hinton, G. Visualizing data using t-SNE. J. Mach. Learn. Res. 9, 2579–2605 (2008).

    Google Scholar 

Download references

Acknowledgements

We thank A. J. Mirando, S. Ide, E. Hocke and K. Abramson for assistance and discussions; staff at Duke Molecular Physiology Institute Molecular Genomics Core for generation and analysis of 10x Genomics scRNA-seq libraries; T. Kimura for reading the manuscript and discussion; G. S. Baht for the instruction of the parabiosis surgery. This research was supported by a grant from the National Institute on Aging (NIA) of the National Institutes of Health (NIH) R01 AG049745 and NIH R01 AI088100. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH.

Author information

Authors and Affiliations

  1. Department of Orthopaedic Surgery and Regeneration Next Initiative, Duke University, Durham, NC, USA

    Yasuhito Yahara, Tomasa Barrientos, Yuning J. Tang, Vijitha Puviindran, Puviindran Nadesan, Hongyuan Zhang, Yarui Diao & Benjamin A. Alman

  2. Department of Orthopaedic Surgery, Faculty of Medicine, University of Toyama, Toyama, Japan

    Yasuhito Yahara

  3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada

    Yuning J. Tang

  4. Department of Cell Biology and Regeneration Next Initiative, Duke University School of Medicine, Durham, NC, USA

    Hongyuan Zhang, Yarui Diao & Yu Xiang

  5. Duke Molecular Physiology Institute, Duke University, Durham, NC, USA

    Jason R. Gibson & Simon G. Gregory

  6. Department of Anesthesiology, Duke University Medical Center, Durham, NC, USA

    Yawar J. Qadri

  7. Division of Nephrology, Department of Medicine, Duke University School of Medicine, Durham, NC, USA

    Tomokazu Souma

  8. Department of Immunology, Duke University School of Medicine, Durham, NC, USA

    Mari L. Shinohara

  9. Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA

    Mari L. Shinohara

Authors
  1. Yasuhito Yahara
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Tomasa Barrientos
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Yuning J. Tang
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Vijitha Puviindran
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Puviindran Nadesan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Hongyuan Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Jason R. Gibson
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Simon G. Gregory
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Yarui Diao
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yu Xiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yawar J. Qadri
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Tomokazu Souma
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Mari L. Shinohara
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Benjamin A. Alman
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

Conceptualization: B.A.A. and Y.Y. Methodology: B.A.A., Y.Y., Y.J.T. and Y.J.Q. Validation: Y.J.T., P.N., V.P., H.Z. and T.B. Formal analysis: B.A.A., Y.Y., V.P., J.G., S.G.G., Y.D., Y.X. and T.S. Investigation: Y.Y., Y.J.T., P.N., V.P., J.G. and S.G.G. Resources: B.A.A. and Y.Y. Data curation: Y.Y., Y.J.T., P.N., V.P., H.Z., J.G., S.G.G. and Y.J.Q. Writing, original draft: Y.Y.; reading and editing: B.A.A., Y.J.T., Y.J.Q., M.L.S., V.P., H.Z. and S.G.G. Visualization: Y.Y. Supervision: B.A.A., T.B. and M.L.S. Project administration: B.A.A. Funding acquisition: B.A.A.

Corresponding author

Correspondence to Benjamin A. Alman.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Non-leakiness of Csf1r-Mer-iCre-Mer mice.

a, Representative image of the postnatal day (P) 0 femur of Csf1r-Mer-iCre-Mer; R26tdTomato mice without 4 hydroxytamoxifen (4OHT) induction (n=3 mice). Scale bars, upper panel; 500 μm. Lower panels; 100 μm.

Extended Data Fig. 2 Cx3cr1+ yolk-sac macrophage descendant in several skeletal elements.

a, Representative image of the postnatal day (P) 3 femur of Cx3cr1creER; R26tdTomato mice without 4 hydroxytamoxifen (4OHT) induction (n=3 mice). Scale bar, 100 μm. b, Representative images of tdTomato+ cells in neonatal brain and calvaria of Cx3cr1creER; R26tdTomato mice treated with 4OHT at E9.5 (n=3 mice). Scale bars, 50 μm. c, Representative image of tdTomato and F4/80 expression in the scapula of 2-month-old (mo) Cx3cr1creER; R26tdTomato mice labeled at E9.5 (n=3 mice). Scale bar, 50 μm. d, Representative image of tdTomato and F4/80 expression in the vertebral body of 2 mo Cx3cr1creER; R26tdTomato mice labeled at E9.5 (n=3 mice). Scale bars, 50 μm.

Extended Data Fig. 3 Characterization of HSC- and EMP- derived cells.

a, Representative image of the femur of embryonic day (E) 17.5 R26tdTomato mice (Cre-negative littermate control of Flt3cre; R26tdTomato mice, n = 3 mice). Scale bar, 100 μm. b, Relative mRNA expression levels of Cre were analyzed by quantitative PCR. RNA was isolated from adult bone marrow cells (BMCs) and E8.5 whole body. Flt3cre; R26tdTomato mice and their littermate Cre-negative control were used. c, Gating strategy of CD45+tdTomato+ BMCs. d, Flow cytometry analysis of tdTomato+ cells from whole-body cell suspension of E14.5 Csf1r-Mer-iCre-Mer; R26tdTomato mice labeled at E8.5 (n = 6 embryos) and Cx3cr1creER; R26tdTomato mice labeled at E9.5 (n = 5 embryos). 4OHT, 4-hydroxytamoxifen. e, Quantitative visualization of percentage of tdTomato+ and RANK+ cells from whole-body cell suspension of E14.5 Csf1r-Mer-iCre-Mer; R26tdTomato mice labeled at E8.5 (n = 6 embryos) and Cx3cr1creER; R26tdTomato mice labeled at E9.5 (n = 5 embryos). Unpaired two-tailed t-test. Error bars denote means ± s.d. f, Percentage of F4/80highCD11bint macrophage and CD11bhighF4/80int monocyte isolated from E14.5 whole body lysate of Csf1r-Mer-iCre-Mer; R26tdTomato mice induced at E8.5 (n = 6 embryos) and Cx3cr1creER; R26tdTomato mice induced at E9.5 (n = 5 embryos). Statistics source data are provided in Source Data Extended Data Fig. 3.

Source Data

Extended Data Fig. 4 Single cell RNA-sequencing analysis of E14.5 embryo.

a, Schematic representation of sample preparation for the single cell RNA-sequencing. 4OHT, 4-hydroxytamoxifen. b, Cell sorting strategy for the single cell RNA-sequencing. tdTomato+eGFP-, tdTomato+eGFP+, and tdTomato-eGFP+ (red box) were isolated from whole-body cell suspension of Csf1r-Mer-iCre-Mer; Csf1reGFP; R26tdTomato mice induced at E8.5. tdTomato+CX3CR1-, tdTomato+CX3CR1+, and tdTomato-CX3CR1+ cell populations (green box) were also isolated from E14.5 Cx3cr1creER; R26tdTomato mice induced at E9.5. Two biological replicates of each genotype. c, Dot plot showing expression of the selected marker gene in each cluster of Csf1r-Mer-iCre-Mer; Csf1reGFP; R26tdTomato mice. The size of the dot encodes the percentage expression, and its color encodes the average expression level. d, Dot plot showing expression of the selected marker gene in each cluster of Cx3cr1creER; R26tdTomato mice. e, Scaled expression level and frequency (the number of cells) of tdTomato, Myb, and Flt3 were visualized. tdTomato+ cells from Csf1r-Mer-iCre-Mer; Csf1reGFP; R26tdTomato (n=10,336 cells) and Cx3cr1creER; R26tdTomato (n=6,706 cells) mice were evaluated.

Extended Data Fig. 5 Single cell RNA sequencing identified EMP-derived osteoclast and their precursor populations.

a, t-SNE plot of tdTomato+Tnfrsf11a+Ptprc+ cells (n=3,368 cells) from Csf1r-Mer-iCre-Mer; Csf1reGFP; R26tdTomato and Cx3cr1creER; R26tdTomato mice identifying 13 clusters. The right gray rectangle representing the normalized expression of indicated marker genes visualized onto t-SNE plots. Cells in cluster 9 expressed osteoclast-specific marker genes. EMP, erythromyeloid progenitor. b, Heatmap representing the top 10 significantly differentially expressed genes in each cluster. c, Violin plots showing mRNA expression levels of selected marker genes. Green color showing the expression levels of the cells from Cx3cr1creER; R26tdTomato mice. Blue color showing the expression levels of the cells from Csf1r-Mer-iCre-Mer; Csf1reGFP; R26tdTomato mice. The number shows cluster identity, and the number of cells in each cluster is provided in Statistical source data Extended Data Fig 5. d, Selected pathways significantly enriched in the cells belonging cluster 6 (n=274 cells). Hypergeometric probability mass function was used. Statistics source data are provided in source data Extended Data Fig 5.

Extended Data Fig. 6 The healing process of the bone injury.

a, Representative images of the bone injury site during the healing process. 2-month-old C57BL/6J mice received drill hole injury and were analyzed (n=3 mice per group). Hematoxylin and Eosin staining showing inflammatory cells (day 3), newly synthesized bone (day 7), and regenerated bone tissue (day 21). Scale bars, 100 μm. b, Immunohistochemical analysis for F4/80 expression. Representative images showing F4/80+ macrophages around the injury site at day 3 (n=3 mice). Scale bars, 100 μm. c, Representative images of tdTomato+ cells around the injury site. 2-month-old Csf1r-Mer-iCre-Mer; R26tdTomato mice induced with 4OHT at E9.5 received drill hole injury and were analyzed at day 3 (n=3), 7 (n=3), 14 (n=4), and 21 (n=5). n representing the number of independent animals. White dot lines represent the injury site. 4OHT, 4-hydroxytamoxifen. Scale bars, 100 μm. d, Representative visualization of tdTomato+ and TRAP+ cells showing less contribution of tdTomato+ cells to the bone remodeling at day 14 (n=4 mice). Scale bars, 50 μm. e, Percentage of tdTomato+TRAP+ to TRAP+ cells around the injury site of Csf1r-Mer-iCre-Mer; R26tdTomato mice at day 3 (n=3), 7 (n=3), 14 (n=4), and 21 (n=5). n representing the number of independent animals. Statistics source data are provided in Source Data Extended Data Fig. 6.

Source Data

Extended Data Fig. 7 Migration of EMP-derived osteoclast precursors through the blood circulation.

a, Immunohistochemical analysis for Ki67 expression. Representative images showing Ki67+ and tdTomato+ cells around the injury site and adjacent metaphysis at day 14 (n=3 mice per group). Scale bars, 50 μm. b, The number of single positive cells of Ki67 and tdTomato and double-positive cells per 0.3 mm2 were quantified (n=3 mice per group). Error bars denote means ± s.d. c, Representative images of the injured femur of Cx3cr1eGFP/+ mice after the parabiotic union. tdTomato+ cells migrated through the blood circulation at the site of a bone injury. Scale bars, 1 mm. Right panels showing enlarged images (n=4 independent parabiotic pairs). Scale bars, 100 μm. d, Representative images of uninjured side femur of Cx3cr1eGFP/+ mice. Scale bars, 1 mm. Right panels showing enlarged images (n = 4 independent parabiotic pairs). White triangles indicating tdTomato+ cells. Scale bars, 100 μm. e, The number of tdTomato+ cells around the injury site and contralateral side of the femur. tdTomato+ cells per 1.26 mm2 were counted and visualized by bar chart (n = 4 mice per group). Unpaired two-tailed t-test. Error bars denote means ± s.d. Statistics source data are provided in Source Data Extended Data Fig. 7.

Source Data

Extended Data Fig. 8 Schematic representation showing the differential origin of osteoclast precursors and their differentiation.

Myb-independent early erythromyeloid progenitors (EMPs) appear around E7-7.5 in the yolk-sac and differentiate into Csf1r+ yolk-sac macrophage (YS Mac) at E8.5 without passing through monocyte intermediates. YS Mac differentiates into CX3C chemokine receptor 1 (CX3CR1) positive premacrophage (pMac), resulting in a significant source of yolk-sac-derived macrophages. Late EMPs emerge in the yolk-sac at E8.5 and migrate to the fetal liver to produce Myb-expressing fetal liver (FL) myeloid progenitors (MP), resulting in FL monocyte (FL Mono). Hematopoietic stem cell precursors (pro-HSCs) emerge at E10.5. Pro-HSCs migrate to the fetal liver around E12 and turns to fetal HSCs, which later shift to the bone marrow. Bone marrow HSCs eventually can establish the circulating monocyte-derived macrophages. YS-derived macrophages differentiate into osteoclast (YS-derived Oc) in the neonatal bone with possible cell-cell fusion with HSC-derived Oc precursors. Cx3cr1+ yolk-sac macrophage descendants in the spleen can provide long-lasting Oc that contribute to the postnatal bone remodeling after injury via the bloodstream.

Supplementary information

Reporting Summary

Supplementary Tables 1–3

Supplementary Table 1: a list of differentially expressed genes in cluster 9 of tdTomato+Tnfrsf11a+Ptprc+ cells (total 94 cells) compared with all of the remaining cells. Non-parameteric Wilcoxon rank-sum test was used. Supplementary Table 2: a list of differentially expressed genes in cluster 6 of tdTomato+Tnfrsf11a+Ptprc+ cells (total 274 cells) compared with all of the remaining cells. Non-parameteric Wilcoxon rank-sum test was used. Supplementary Table 3: antibodies used for FACS.

Source data

Source Data Fig. 1

Statistical Source Data

Source Data Fig. 2

Statistical Source Data

Source Data Fig. 3

Statistical Source Data

Source Data Fig. 4

Statistical Source Data

Source Data Fig. 5

Statistical Source Data

Source Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 3

Statistical Source Data

Source Data Extended Data Fig. 5

Statistical Source Data

Source Data Extended Data Fig. 6

Statistical Source Data

Source Data Extended Data Fig. 7

Statistical Source Data

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yahara, Y., Barrientos, T., Tang, Y.J. et al. Erythromyeloid progenitors give rise to a population of osteoclasts that contribute to bone homeostasis and repair. Nat Cell Biol 22, 49–59 (2020). https://doi.org/10.1038/s41556-019-0437-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41556-019-0437-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing