Letter | Published:

Developmental origin, functional maintenance and genetic rescue of osteoclasts

Abstract

Osteoclasts are multinucleated giant cells that resorb bone, ensuring development and continuous remodelling of the skeleton and the bone marrow haematopoietic niche. Defective osteoclast activity leads to osteopetrosis and bone marrow failure1,2,3,4,5,6,7,8,9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11,12,13,14,15,16,17,18, consistent with a haematopoietic origin of osteoclasts13,16,19 and studies that suggest that they develop by fusion of monocytic precursors derived from haematopoietic stem cells in the presence of CSF1 and RANK ligand1,20. However, the developmental origin and lifespan of osteoclasts, and the mechanisms that ensure maintenance of osteoclast function throughout life in vivo remain largely unexplored. Here we report that osteoclasts that colonize fetal ossification centres originate from embryonic erythro-myeloid progenitors21,22. These erythro-myeloid progenitor-derived osteoclasts are required for normal bone development and tooth eruption. Yet, timely transfusion of haematopoietic-stem-cell-derived monocytic cells in newborn mice is sufficient to rescue bone development in early-onset autosomal recessive osteopetrosis. We also found that the postnatal maintenance of osteoclasts, bone mass and the bone marrow cavity involve iterative fusion of circulating blood monocytic cells with long-lived osteoclast syncytia. As a consequence, parabiosis or transfusion of monocytic cells results in long-term gene transfer in osteoclasts in the absence of haematopoietic-stem-cell chimerism, and can rescue an adult-onset osteopetrotic phenotype caused by cathepsin K deficiency23,24. In sum, our results identify the developmental origin of osteoclasts and a mechanism that controls their maintenance in bones after birth. These data suggest strategies to rescue osteoclast deficiency in osteopetrosis and to modulate osteoclast activity in vivo.

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Data generated in this study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by a NIH/NCI P30CA008748 MSKCC core grant, NIH/NIAID 1R01AI130345 and NIH/NHLBI R01HL138090 to F.G. and by the German Research Foundation (DFG) through FOR2033-A03, TRR127-A5, WA2837/6-1 and WA2837/7-1 to C.W. The authors thank Y. Kobayashi, J. Pollard, T. Graf, R. Stanley, J. Frampton, T. Boehm and J. Penninger for providing mouse strains, and the MSKCC molecular cytology core for preparation of histological samples. The authors are indebted to R. O’Reilly and F. Boulad for helpful suggestions. F.G. is grateful to G. Ruth for support. This study is dedicated to the memory of Lucile Crozet.

Reviewer information

Nature thanks Roland Baron, Irving L. Weissmann and Mone Zaidi for their contribution to the peer review of this work.

Author information

F.G. and C.W. designed the study, supervised experiments and analysed data. F.G. wrote the draft of the manuscript. C.E.J.-G. performed histology and immunofluorescence analyses. E.M. and C.E.J.-G. supervised or performed fate-mapping and genetic deletion experiments with Csf1rMer-icre-Mer, Flt3cre, Csf1ricre and Tnfrsf11acre, EdU incorporation studies and adoptive transfer studies. G.I.P. performed lineage tracing and genetic deletion experiments with Tnfrsf11acre and Vavcre mice and histomorphometry studies. J.T.M. performed and analysed rescue experiments in Catk-deficient mice and Csf1rcre;Csf1rf/f mice. P.-L.L. assisted with parabiosis surgeries. V.K.Y. and G.K. analysed Catk parabiosis rescue experiments. J.E. performed inducible genetic deletion experiments in R26-creERT2+;Csf1rF/F embryos. T.L., L.C., G.I.P., M.B. and E.M. performed flow cytometry analyses. M.R. scanned bones using micro-CT, and M.R. and G.I.P. analysed micro-CT data. All authors contributed to the manuscript.

Competing interests

F.G. is a consultant and principal investigator on a Sponsored Research Agreement with Third Rock Venture (TRV). The other authors declare no competing interests.

Correspondence to Claudia Waskow or Frederic Geissmann.

Extended data figures and tables

Extended Data Fig. 1 Csf1rcre;Tnfrsf11afl/fl, Csf1rfl/fl;Csf1rcre and Tnfrsf11acre;Csf1rfl/fl mice are osteopetrotic.

a, b, Representative CT scans (nanospect CT) of four-week-old Csf1rcre;Tnfrsf11afl/fl mice. Arrows indicate skull deformation and absence of bone marrow cavity in mutant mice. c, Haematoxylin and TRAP staining of bone sections from four-week-old Csf1rcre;Tnfrsf11afl/fl mice showing closure of the bone marrow. Histology of paraffin sections (5-μm thickness) corroborates the phenotype from mice in b. d, Inguinal lymph nodes from Csf1rcre;Tnfrsf11afl/fl mice. e, A representative Csf1rcre;Tnfrsf11afl/fl and a littermate control. f, Representative CT scan reconstructions (nanospect CT) of four-week-old Tnfrsf11aKoba-cre;Csf1rfl/fl mice. Arrows indicate skull deformation, but with presence of a bone marrow cavity in mutant mice, in contrast to those in a and b. g, Representative CT scans of Csf1rcre;Csf1rfl/fl mice. Arrows indicate skull deformation and absence of bone marrow cavity in mutant mice as in a and b. CT scans and photographs are representative of >10 litters.

Extended Data Fig. 2 Bone histology and flow cytometry analysis of bone marrow phenotypic LSK, long-term HSCs, short-term HSCs and multipotent progenitors in mice of indicated genotypes.

a, Young Flt3cre;Csf1rfl/fl and Flt3cre;Tnfrsf11afl/fl mice have normal long bones. Haematoxylin and TRAP staining of bone sections from four-week-old Flt3cre;Tnfrsf11afl/fl mice, showing normal bone structure and bone marrow cavity. b, LSK cell numbers in bone marrow from three-to-four-week-old Flt3cre;Tnfrsf11afl/fl (n = 3) and Flt3cre;Tnfrsf11afl/+ mice (n = 2) and littermate controls (n = 4) and from 22-week-old Flt3cre;Tnfrsf11afl/fl (n = 5) and Flt3cre;Tnfrsf11afl/+ mice (n = 4) and littermate controls (n = 4). c, Haematoxylin and TRAP staining of bone sections from four-week-old Flt3cre;Csf1rfl/fl mice showing normal bone structure and bone marrow cavity. d, e, Phenotypic long-term HSCs (LT-HSCs) are reduced in aged wild-type mice but not in young Vavcre;Csf1rfl/− mice. f, g, LT-HSCs are reduced in in young Csf1r−/− mice, and to a lesser extent, in young Tnfrsf11aWask-cre;Csf1rfl/− mice. Cell counts for two femurs are shown. h, Flow cytometry analysis of F4/80+ cells in brain (microglia) and epidermis (Langerhans cells) in E18.5 Tnfrsf11acre;Csf1rfl/fl embryos and littermate controls (n = 3 per group). i, Haematoxylin and TRAP staining of bone sections from P7 Tnfrsf11acre;Csf1rfl/fl mice and littermate controls, showing absence of the bone marrow cavity. j, Flow cytometry of fetal liver at E15.5 (representative results of three experiments). k, LSK numbers in bone marrow of three-to-four-week-old Tnfrsf11aWask-cre;Csf1rfl/fl mice (n = 24) and littermate controls (n = 20), and Tnfrsf11aKoba-cre;Csf1rfl/fl (n = 7) and Tnfrsf11aKoba-cre;Csf1rfl+l mice (n = 6) and littermate controls (n = 8). l, For comparison, LSK numbers in bone marrow of three-to-four-week-old Csf1r−/− mice (n = 22) and littermate controls (n = 21). m, Representative micrographs of femur sections from four-week-old Tnfrsf11acre;Csf1rfl/fl mice and littermate controls, stained with haematoxylin and TRAP. n, Blood leukocytes numbers in four-week-old Tnfrsf11acre;Csf1rfl/fl mice (n = 5), Tnfrsf11acre;Csf1rfl/+ mice (n = 6) and littermate controls (n = 12). Points represent individual mice; results from three independent experiments. Data are mean ± s.d.; n indicates the number of mice per group; unpaired two tailed t-tests. *P < 0.05, **P < 0.005, ***P < 0.0005 and ****P < 0.0001. LT-HSC, LinKIT+SCA1+;KIT+SCA1+CD34FLT3. Phenotypic short-term HSCs (ST-HSC), LinKIT+SCA1+CD34+FLT3. MPP, multipotent progenitors; LinKIT+SCA1+CD34+FLT3+. Lin, CD3+CD19+NK1.1+TER119+CD11b+GR1+B220+. Source data

Extended Data Fig. 3 Bone histomorphometry in aged Flt3cre;Tnfrsf11afl/fl, Flt3cre;Csf1rfl/fl and Vavcre;Csf1rfl/fl mice and control littermates.

a, Representative Micro-CT of humeri (top) and femora (bottom) of mice of the indicated age and genotype. b, Bone length, connectivity density (conn density), trabecular number (Tb.N.) and trabecular spacing (Tb.Sp.) analysed by micro-CT in aged Flt3cre;Tnfrsf11afl/fl (n = 4) and Flt3cre;Csf1rfl/fl (n = 4) mice and control littermates (n = 7). c, Bone histomorphometry as in b, for Vavcre;Csf1rfl/fl mice and control littermates (n = 5). d, e, Dynamic bone histomorphometry in aged Flt3cre;Tnfrsf11afl/fl and Flt3cre;Csf1rfl/fl mice using in vivo calcein labelling. d, Representative micrographs of calcein labelling (green) of femora of mice from the indicated genotypes and ages. Scale bars: 200 μm (top); 50 μm (bottom). e, Quantification of calcein labelling by fluorescence microscopy of mineralized surface/bone surface (MS/BS), mineral apposition rate (MAR) and bone formation rate/bone surface (BFR/BS) in aged Flt3cre;Tnfrsf11afl/fl (n = 5), Flt3cre;Csf1rfl/fl (n = 3) and control littermates (n = 10) Data are mean ± s.d.; dots in graphs represent individual mice; n indicates the number of mice per group; unpaired two tailed t-tests. *P < 0.05, **P < 0.005, ***P < 0.0005 and ****P < 0.0001. Source data

Extended Data Fig. 4 Colonization of the bone marrow by Csf1r+ and Flt3+ haematopoietic cells.

a, Representative confocal microscopy of frozen sections from Flt3cre;Rosa26LSL-YFP and Csf1rcre;Rosa26LSL-YFPmice analysed at E16.5 (n = 3). b, YFP-labelling efficiency in Csf1rcre;Rosa26LSL-YFP mice analysed by flow cytometry in the indicated cell populations (left), and by confocal microscopy on frozen bone sections at the indicated age (right). Magnified regions (bottom right) show YFP expression in individual osteoclasts. YFP, YFP antibody; TRAP, ELF97 fluorescent substrate; TO-PRO-3, nuclear stain. c, YFP-labelling efficiency in Flt3cre;Rosa26LSL-YFP mice analysed as in b. Data in b and c are representative of at least three experiments per time point and genotype. Points represent individual mice. d, Genetic lineage tracing of osteoclasts in ossification centres using Csf1rMer-icre-Mer;Rosa26LSL-YFP mice. Representative high-power confocal microscopy of embryonic femurs showing MGCs in primary ossification centres from Csf1rMer-icre-Mer;Rosa26LSL-YFP E18.5 embryos pulsed with 4-OHT at E8.5, showing YFP expression in MGCs after Cre recombination (left) and quantified as MFI (right) from Cre+ (n = 8) and Cre (n = 4) (d), and unpulsed controls (e), showing the lack of YFP in Cre+ (n = 4) and Cre (n = 4). Sections were labelled with antibodies against YFP, TRAP (ELF97 substrate) and TO-PRO-3. Source data

Extended Data Fig. 5 Tnfrs11aWask-cre knock-in mice enable deletion of target genes in fetal macrophages, but not in definitive HSCs and their progeny in blood and tissues, whereas Vavcre mice enable deletion of target genes in definitive HSCs, but not in fetal macrophages.

a, Bar graphs indicate percentage of cells expressing eYFP obtained by flow cytometry of Tnfrsf11acre;Rosa26LSL-YFP cells from the indicated cell types, organs and time points. Data represent three independent experiments; n, number of mice per group indicated on x axis. b, Lineage tracing in the fetal liver of Vavcre+;tdRFPwt/ki mice. n, number of mice per group indicated on x axis. c, Representive molecular analysis of Csf1r deletion in purified bone-marrow haematopoietic stem and progenitor cells (HSPC) from 62-week-old Vavcre;Csf1rfl/fl mice and controls (n = 5). d, Representative photograph of teeth from three-week-old Rosa26-creERT2+;Csf1rfl/− pulsed with tamoxifen at E10.5 (n = 3 mice from 3 independent litters). FL, fetal liver; MP, myeloid progenitor; PMN, polymorphonuclear cells; mono, monocytes; T, T cells; B, B cells; PEC, peritoneal exudate cells. Data are mean ± s.d.; points represent individual mice. Source data

Extended Data Fig. 6 Bone morphometric and dynamic histomorphometry effects of Csf1r deletion in P21 Tnfrsf11acre;Csf1fl/fl mice.

ad, Bone volume/total volume (BV/TV, a), bone length (b), connectivity density (c), and trabecular number (d) were analysed by micro-CT in 21-day-old mice. Csf1r−/− (n = 4), control littermates (n = 7); Tnfrsf11aWask-cre+;Csf1rfll−(n = 8), Tnfrsf11aWask-cre+;Csf1rfll+(n = 7), Tnfrsf11aWask-cre+;Csf1rfll−(n = 3) and control littermates (n = 5). e, Representative micrographs of calcein labelling (green) of femur of mice from the indicated genotypes and ages (n = 4). Scale bar, 50 μm. f, Quantification of calcein labelling by fluorescence microscopy: mineralized surface/bone surface, mineral apposition rate and bone formation rate/bone surface in Tnfrsf11aWask-cre+;Csf1rfll− Csf1r−/− (n = 4) and control littermates (n = 15). Data are mean ± s.d.; dots in graphs represent individual mice; n indicates the number of mice per group; unpaired two-tailed t-test. *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005 and ****P ≤ 0.0001. Source data

Extended Data Fig. 7 EdU labelling of bone marrow myeloid cells.

a, Short-term kinetics: EdU (20 μg g−1) was injected intraperitoneally in C57Bl6/N mice at t = 0. Mice were euthanized at the indicated time points and the percentage of EdU+ cells (blue) and the geometric MFI of EdU+ cells (red) were determined by flow cytometry, showing rapid EdU incorporation. Percentage of EdU+ cells plateaus at ~30 min, and geometric MFI plateaus at ~75 min. Following a first round of cell division, ~50% of monocytic cells are labelled after 8–12 h (n = 3–8 mice, see Source Data). b, Long-term kinetics: (1–240 h) EdU was injected as in a and percentage of EdU+ monocytic cells in bone marrow (top) and blood (bottom) was determined by flow cytometry, showing labelling of ~50% of monocytic cells for ~2 days. Labelled cells were not detectable after three days. Points represent values from individual mice; data for each time point are pooled from two to three independent experiments (see Source Data). c, Parabiosis between Csf1rcre;Rosa26LSL-YFP and Csf1rcre;Rosa26LSL-tdTomato pairs as described in Fig. 3, paired for 1–8 weeks, and from Csf1rcre;Rosa26LSL-YFP partners separated after 4 weeks of parabiosis and analysed 4 weeks, 14 weeks and 24 weeks after separation. Scatter plots represent the MFI of individual TRAP+ MGCs for YFP (y axis) and tdTomato (x axis), and histograms represent the overlaid distribution of the MFI values for YFP and tdTomato in TRAP+ MGCs at the indicated time points. Data are mean ± s.d.; dots in graphs represent individual mice; n indicates the number of mice per group. Source data

Extended Data Fig. 8 FACS analysis of monocyte purification, and blood and bone marrow from transferred Csf1rcre;Csf1rfl/fl mice.

a, Representative flow cytometry plots of purified bone marrow monocytes from magnetic-bead based enrichment; percentage of live YFP+ monocytes is indicated. b, c, Representative flow cytometry plots from blood (b) and bone marrow (c) of 14-day-old mice transferred with 1 × 106 YFP+ monocytes on day 5, 8 and 11; the percentage of YFP+ cells is indicated. Results shown in ac are representative of three independent experiments.

Extended Data Fig. 9 Rescue of osteoclasts by monocyte transfer in Csf1rcre;Csf1rfl/fl mice.

High-power confocal microscopy images of frozen sections from Csf1rcre;Csf1rfl/fl mice transferred with monocytes from Csf1rcre;Rosa26LSL-YFP and controls, stained with YFP antibody, TRAP substrate ELF97 and TO-PRO-3 nuclear stain. Examples of multinucleated TRAP+YFP+ cells (osteoclasts) are indicated with dotted lines. n = 3 mice from independent litters. Numbers 1–3 correspond to the mice in Fig. 4d.

Supplementary information

Reporting Summary

Supplementary Table

Supplementary Table 1: Mouse Genotyping, primers, program, and expected band sizes.

Supplementary Table

Supplementary Table 2: Antibodies for Bone Histology, listed clones, company and dilutions.

Supplementary Table

Supplementary Table 3: Antibodies for FACS, listed clones, company and dilutions.

Source data

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Fig. 1: HSC-derived precursors are dispensable for osteoclasts and bone development.
Fig. 2: EMP-derived osteoclasts are required for bone development.
Fig. 3: In vivo dynamics of osteoclasts.
Fig. 4: Rescue of osteopetrosis.
Extended Data Fig. 1: Csf1rcre;Tnfrsf11afl/fl, Csf1rfl/fl;Csf1rcre and Tnfrsf11acre;Csf1rfl/fl mice are osteopetrotic.
Extended Data Fig. 2: Bone histology and flow cytometry analysis of bone marrow phenotypic LSK, long-term HSCs, short-term HSCs and multipotent progenitors in mice of indicated genotypes.
Extended Data Fig. 3: Bone histomorphometry in aged Flt3cre;Tnfrsf11afl/fl, Flt3cre;Csf1rfl/fl and Vavcre;Csf1rfl/fl mice and control littermates.
Extended Data Fig. 4: Colonization of the bone marrow by Csf1r+ and Flt3+ haematopoietic cells.
Extended Data Fig. 5: Tnfrs11aWask-cre knock-in mice enable deletion of target genes in fetal macrophages, but not in definitive HSCs and their progeny in blood and tissues, whereas Vavcre mice enable deletion of target genes in definitive HSCs, but not in fetal macrophages.
Extended Data Fig. 6: Bone morphometric and dynamic histomorphometry effects of Csf1r deletion in P21 Tnfrsf11acre;Csf1fl/fl mice.
Extended Data Fig. 7: EdU labelling of bone marrow myeloid cells.
Extended Data Fig. 8: FACS analysis of monocyte purification, and blood and bone marrow from transferred Csf1rcre;Csf1rfl/fl mice.
Extended Data Fig. 9: Rescue of osteoclasts by monocyte transfer in Csf1rcre;Csf1rfl/fl mice.

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