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

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.

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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.

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. 16 and Extended Data Figs. 3 and 57. All other data supporting the findings of this study are available from the corresponding author on reasonable request.

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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.

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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.

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Correspondence to Benjamin A. Alman.

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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.

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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

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