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. 2019 Apr;568(7753):541-545.
doi: 10.1038/s41586-019-1105-7. Epub 2019 Apr 10.

Developmental origin, functional maintenance and genetic rescue of osteoclasts

Christian E Jacome-Galarza #  1 , Gulce I Percin #  2   3 , James T Muller #  1 , Elvira Mass #  1   4 , Tomi Lazarov  1 , Jiri Eitler  2 , Martina Rauner  5 , Vijay K Yadav  6 , Lucile Crozet  1 , Mathieu Bohm  1 , Pierre-Louis Loyher  1 , Gerard Karsenty  6 , Claudia Waskow #  7   8   9 , Frederic Geissmann #  10
Affiliations

Developmental origin, functional maintenance and genetic rescue of osteoclasts

Christian E Jacome-Galarza et al. Nature. 2019 Apr.

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 failure1-9, whereas excess activity can contribute to bone loss and osteoporosis10. Osteopetrosis can be partially treated by bone marrow transplantation in humans and mice11-18, 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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Conflict of interest statement

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.

Figures

Fig. 1 |
Fig. 1 |. HSC-derived precursors are dispensable for osteoclasts and bone development.
a, Representative photographs of teeth of three-to-four-week-old Csf1rCre;Csf1rfl/fl mice (top left), Csf1rCre;Tnfrsf11afl/fl mice (top middle) and control littermates. Bottom left, representative photographs of leg bones from controls and Csf1rCre;Tnfrsf11afl/fl mice; white arrowhead highlights the lack of blood cells. Bottom middle, osteoclast numbers in E18.5, P7, and four-week-old Csf1rCre;Tnfrsf11afl/fl mice (n = 3) and control littermates (n = 3), and representative haematoxylin and TRAP staining of femur sections; the arrow indicates an osteoclast. b, Representative photographs of teeth of three-to-four-week-old Flt3Cre;Tnfrsf11afl/fl, Flt3Cre;Csf1rfl/fl, VavCre;Csf1rfl/fl mice and littermates. c, Bone marrow CD45+ cell numbers in Flt3Cre;Tnfrsf11afl/fl and control littermates at 4 (top left, n = 4 Flt3Cre;Tnfrsf11afl/fl, n = 3 control) and 22 weeks of age (top right; n = 4 Flt3Cre;Tnfrsf11afl/fl, n = 5 control) and VavCre;Csf1rfl/fl and control littermates at 4 (bottom left; n = 8 VavCre;Csf1rfl/fl, n = 8 control) and 62 weeks of age (bottom right; n = 5 VavCre;Csf1rfl/fl, n = 8 control), Cre- black Cre+ white. d, Osteoclast counts in femurs from Flt3Cre;Tnfrsf11afl/fl (left), Flt3Cre;Csf1rfl/fl (middle) and VavCre;Csf1rfl/fl (right) mice and control littermates of the indicated ages, n numbers indicated on individual bars. e, Representative haematoxylin and TRAP staining of femur sections from 22-week-old Flt3Cre;Tnfrsf11afl/fl mice (top), 62-week-old VavDD/Cre;Csf1rfl/fl mice (bottom) and control littermates. The white arrowhead points to trabecular bone. f, Quantitative analysis of bone volume/total volume of humerus or femurs from Flt3Cre;Tnfrsf11afl/fl (n = 4), Flt3Cre;Csf1rfl/fl (n = 5) and control littermates (n = 8), and VavCre;Csf1rfl/fl (n = 5) mice and control littermates (n = 5) as determined by micro-CT. P values were determined by ANOVA. g, Expression and mean fluorescence intensity (MFI) of YFP in TRAP+ multinucleated cells from Csf1rCre;Rosa26LSL-YFP mice at E16.5, E18.5, P7 and six months (n = 3 per time point). For MFI and percentages, at least 100 osteoclasts were quantified per time point and genotype. Box plots show the median, box edges show the first and third quartiles and whiskers show the minimum and maximum. h, Similar analysis as in g, for Flt3Cre;Rosa26LSL-YFP mice at E16.5, E18.5, P7, 4 weeks (4 wk), 3 months (3 m) and 6 months (6 m). Data are mean ± s.d.; dots in graphs represent individual independent biological replicates; n indicates the number of mice per group. Statistical significance was analysed with GraphPad Prism using unpaired two-tailed t-tests unless otherwise indicated. *P < 0.05, **P < 0.01, ***P < 0.001 and ****P < 0.0001.
Fig. 2 |
Fig. 2 |. EMP-derived osteoclasts are required for bone development.
a, MGC number in femur anlage ossification centres from E15.5–16.5 Myb−/− (n = 6) and littermate controls (n = 3). b, Representative confocal microscopy of frozen sections from the ossification centres in a, stained for TRAP and with TO-PRO-3 nuclear stain. c, Percentage of TRAP+ cells expressing YFP in femur anlage from E15.5 Csf1rMer-iCre-Mer;Rosa26LSL-YFP mice (n = 8) and Cre-negative controls (n = 5), pulsed at E8.5 with 4-OHT. d, Representative confocal microscopy of a sample from c. e, Representative teeth of cre+ Tnfrsf11aKoba-Cre;Csf1rfl/fl (top) and Tnfrsf11aWask-Cre;Csf1rfl/fl mice (bottom) and cre− control littermates. f, Leg bones from Tnfrsf11aCre;Csf1rfl/fl mice (cre+, n = 6) and control littermates (cre−, n = 6) at P7 and 4 weeks of age. Arrowhead highlights the colour of an area of bone. g, Representative micro-CT scans of long bones from mice in e (n = 6 per genotype). h, Skull length from three-week-old Csf1r−/− (n = 6) control littermates (n = 12) and Tnfrsf11aWask-Cre;Csf1rfl/fl mice (n = 4), as determined by micro-CT. i, Osteoclast counts in bone sections from E18.5, P7 and three-to-four-week old Tnfrsf11aCre;Csf1rfl/fl mice and littermate controls. j, Number of bone marrow CD45+ cells determined by flow cytometry of cells from four-week-old control (cre−) littermates, Tnfrsf11aKoba-Cre;Csf1rfl/fl (n = 9; n = 13 control) and Tnfrsf11aWask-Cre;Csf1rfl/fl (n = 23, n = 27 control) mice (cre+). 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 < 0.01, ***P < 0.001 and ****P < 0.0001.
Fig. 3 |
Fig. 3 |. In vivo dynamics of osteoclasts.
a, Parabiosis of Csf1rCre;Rosa26LSL-YFP mouse surgically paired with a Csf1rCre;Rosa26LSL-tdTomato partner for four-to-eight weeks. Representative confocal microscopy of frozen sections from the femur of a Csf1rCre;Rosa26LSL-YFP partner stained with antibodies for tdTomato (red) and YFP (green), ELF 97 (blue) and TOPRO-3 (grey). n = 3. b, Pie graphs showing the percentage of tdTomato+ (red), YFP+ (green) and tdTomato+YFP+ cells (yellow) among bone marrow mononuclear cells (MNC), megakaryocytes and multinuclear giant cells (MGC) from parabionts paired for the indicated time (n = 8). c, Similar analysis as in b for parabionts separated after four weeks and analysed 14 and 24 weeks after separation (n = 3). d, Bar graph showing number of nuclei per TRAP+ MGC in femurs from wild-type mice at one, three and six months of age (n = 3 mice per time point). e, Representative confocal microscopy of an EdU-labelled nucleus in a TRAP+ osteoclast (left) and histogram showing the percentage of TRAP+ osteoclasts with EdU-labelled nuclei and the number of labelled nuclei per cell 72 h after intravenous pulse-labelling with EdU (n = 5 mice). f, A model for development and maintenance of osteoclast syncytia. Data are mean ± s.d.; dots in graphs represent individual mice; n indicates the number of mice per group; two-way ANOVA with Tukey’s multiple comparisons test *P ≤ 0.05 and **P ≤ 0.005.
Fig. 4 |
Fig. 4 |. Rescue of osteopetrosis.
a, Bone volume/total volume for femurs from 10-week-old cathepsin K−/− mice (n = 3) after six weeks parabiosis with cathepsin K+/− mice and from positive (n = 4) and negative (n = 4) control parabionts, analysed by von Kossa staining. b, Monocyte transfer. Histograms represent percentage of tdTomato+ cells among bone TRAP+ MGCs from Csf1rCre;Rosa26LSL-YFP recipients analysed by confocal microscopy (left) 11 days (n = 2) and 60 days (n = 5) after intravenous transfer at six weeks of age of 3 × 106 Ly6C+ bone marrow cells from Csf1rCre;Rosa26LSL-tdTomato donors, and percentages of tdTomato+ cells among bone marrow precursors and blood leukocytes, analysed by flow cytometry after 60 days (n = 5, right). c, Representative high-power confocal microscopy of the femur of a recipient mouse 60 days after intravenous transfer (from b), stained with antibodies for tdTomato and YFP, ELF97 phosphatase substrate and TOPRO-3. d, Representative photographs of teeth (top) and CT scan of leg bones from Csf1rcre;Csf1rfl/fl mice (n = 3) transferred with monocytic cells from Csf1rCre;Rosa26LSL-YFP donors at P5, P8 and P11, and from wild-type and non-transferred Csf1rcre;Csf1rfl/fl controls. Arrows indicate the presence of teeth eruption (top panels) and bone marrow cavity (bottom panels). e, Representative confocal microscopy of a femur from mouse no. 3 in d, stained with YFP antibody and with ELF97 and TOPRO-3. f, Number of TRAP+ osteoclasts in bone sections from mice in d and non-transferred controls (left), and percentage of YFP+ TRAP+ cells in transferred mice. The different symbols represent individual mice. Mean values for three sections per mouse. At least 100 osteoclasts were quantified per mouse. g, Percentages of YFP+ cells among bone marrow precursors and blood leukocytes in the recipient mice (from d) at the time of analysis. Data are mean ± s.d.; dots in graphs represent individual mice; n indicates the number of mice per group; ANOVA with Tukey’s multiple comparisons test. ***P < 0.001 and ****P < 0.0001.
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