Author response: Osteocytes regulate senescence of bone and bone marrow

Abstract

Article Figures and data Abstract Editor's evaluation eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The skeletal system contains a series of sophisticated cellular lineages arising from the mesenchymal stem cells (MSCs) and hematopoietic stem cells (HSCs) that determine the homeostasis of bone and bone marrow. Here, we reasoned that osteocyte may exert a function in regulation of these lineage cell specifications and tissue homeostasis. Using a mouse model of conditional deletion of osteocytes by the expression of diphtheria toxin subunit α in dentin matrix protein 1 (DMP1)-positive osteocytes, we demonstrated that partial ablation of DMP1-positive osteocytes caused severe sarcopenia, osteoporosis, and degenerative kyphosis, leading to shorter lifespan in these animals. Osteocytes reduction altered mesenchymal lineage commitment, resulting in impairment of osteogenesis and induction of osteoclastogensis. Single-cell RNA sequencing further revealed that hematopoietic lineage was mobilized toward myeloid lineage differentiation with expanded myeloid progenitors, neutrophils, and monocytes, while the lymphopoiesis was impaired with reduced B cells in the osteocyte ablation mice. The acquisition of a senescence-associated secretory phenotype (SASP) in both osteogenic and myeloid lineage cells was the underlying cause. Together, we showed that osteocytes play critical roles in regulation of lineage cell specifications in bone and bone marrow through mediation of senescence. Editor's evaluation The work provides a new understanding of the role of osteocytes in regulating other lineage cells in bone, bone marrow, and skeletal muscle. The set of data from the genetic mouse model, bone phenotypic analyses, and scRNA-seq analysis supports the conclusion. This is an important and logically presented study that offers new insight into the biology of osteocytes. https://doi.org/10.7554/eLife.81480.sa0 Decision letter Reviews on Sciety eLife's review process eLife digest A hallmark of aging is the weakening of our muscles and bones, which become more fragile as we get older. These gradual changes can result in a humpback and muscle shrinking among other conditions. At the same time little is known about what role osteocytes – the most abundant type of bone cell – play in the process of bone and muscle aging. One way to investigate the role of osteocytes in aging is to remove them and observe what happens to nearby cells as they age. To achieve this Ding, Gao, Gao et al. genetically altered mice so that they would carry and activate a gene called DTA in their osteocytes. DTA is a gene derived from the bacterium that causes diphtheria, and when it is activated, it produces a toxin that accumulates in cells, eventually killing them. In the mice line developed by Ding, Gao, Gao et al. DTA slowly killed osteocytes, leading to adult mice lacking most of their osteocyte population that have a normal embryonic development. This is important because the fact that the mice develop normally before birth allowed the team to rule out embryonic defects when looking at their results. Ding, Gao, Gao et al. found that, without enough osteocytes, the nearby bone and bone marrow cells aged faster than expected. Indeed, the skeleton and muscles of adult mice was severely affected by the loss of osteocytes, leading to fragile bones with lower mass and muscle shrinking. These mice looked old in their young age and died earlier. At the cellular level, the removal of osteocytes impaired the formation of osteoblasts, the cells that are responsible for making bones. It also led to an increase in the numbers of osteoclasts – the cells that destroy bone tissue to repair it and maintain it – and fat tissue cells. Furthermore, cells in the bone marrow, which go on to make white blood cells, were also affected. The mechanisms through which osteocytes affect the growth of these other cells is yet to be fully understood. However, Ding, Gao, Gao et al. did observe that these cells acquired traits characteristic of aging cells, implying that osteocytes have a role in regulating cellular aging or senescence. Among these senescence traits is the increased production and secretion of molecules that interact with the immune system, a feature known as the ‘senescence-associated secretory phenotype’. Overall, the results of Ding, Gao, Gao et al. suggest that reducing the number of osteocytes in mice leads to faster bone aging and affects the balance of the different cell types required for healthy bone and bone marrow growth. Future research could focus on finding drugs that allow osteocytes to keep performing their role during aging, and thus help maintain bone health. The findings of Ding, Gao, Gao et al. also suggest that osteocytes may be playing a previously underappreciated role in age-related diseases, which warrants further investigation. Introduction The skeletal system is an elaborate organ mainly containing bone, bone marrow, and other connective tissues, whose function includes movement, support, hematopoiesis, immune responses, and endocrine regulation (Karsenty and Ferron, 2012; Katsnelson, 2010; Quarles, 2011). The skeletal system hosts at least more than 12 types of cell lineages arising from hematopoietic stem cells (HSCs) and mesenchymal stem cells (MSCs) (Méndez-Ferrer et al., 2010). During hematopoiesis, HSCs give rise to lymphoid and myeloid lineage cells, including B cells, neutrophils, monocytes, as well as osteoclasts. Meanwhile, MSCs differentiate into osteoblastic lineage cells, bone marrow adipocytes, and form fibroconnective tissues. The sophisticated processes of differentiation and interaction of these cell lineages are critical not only to skeletal development, but also to the integrity of hematopoietic, immune, and endocrine systems (Méndez-Ferrer et al., 2010; Le et al., 2018; Yu and Scadden, 2016). During aging, these cell lineage commitments change rigorously and cause imbalance between myeloid–lymphoid hematopoiesis and adipo-osteogenic differentiation (Chen et al., 2016; Sinha et al., 2022), which lead to the increased myelopoiesis and adipogenesis as opposed to lymphopoiesis and osteogenesis. While the complex communications between these cell lineages have been documented, it is still unclear what determines these cell lineages to survive and how their cell fates are maintained during development and aging. It has been speculated that cellular senescence, characterized by cell proliferation arrest, altered metabolism, and apoptosis resistance (Gorgoulis et al., 2019; Tchkonia et al., 2013), may be responsible for the regulation of lineage cell fates. However, the precise role in aging and age-related diseases remains unclear. Osteocytes, as the long-living terminally differentiated cells and the most abundant cells within the bone matrix (Tresguerres et al., 2020), play vital roles in maintaining the skeletal homeostasis. Apart from mechanical transduction (Long, 2011; Sato et al., 2020), osteocytes have been shown to regulate bone formation, bone resorption, bone marrow hematopoiesis (Asada et al., 2013; Azab et al., 2020; Fulzele et al., 2013; Xiao et al., 2021), and generate endocrine signals to mediate function of other organs (Razzaque, 2009; Fulzele et al., 2017; Cain et al., 2012). Osteocytes regulate both the osteoblast and osteoclast activities during bone remodeling (Delgado-Calle and Bellido, 2022; Tresguerres et al., 2020). Sclerostin, one of the key inhibitors of Wnt signaling pathway, is mainly produced by osteocytes (Tresguerres et al., 2020). NO and PGE2 secretion by osteocytes in response to mechanical stimulation have anabolic effects on osteoblasts (Rochefort et al., 2010). Receptor activation of nuclear factor-κ B ligand (RANKL), the osteoclast differentiation factor, is mainly produced by osteocytes (Nakashima et al., 2011). Osteocytes regulate neutrophil development through secretion of soluble factors like IL19 (Xiao et al., 2021) and can also regulate myelopoiesis through Gsα-dependent and -independent pathways (Fulzele et al., 2013; Azab et al., 2020). In addition, studies have shown that aging is associated with dysfunction of osteocytes. Degeneration of osteocytes lacuna-canalicular network had been observed in older adults (Busse et al., 2010) and the aging animal model (Tiede-Lewis et al., 2017). Senescent osteocytes and their senescence-associated secretory phenotype (SASP) have been shown to contribute to age-related bone loss (Farr et al., 2016; Kim et al., 2020). Together, current data suggest that osteocyte is a singling cell that coordinates activities of bone and bone marrow during skeletal aging (Sfeir et al., 2022). Here, we hypothesize that coordination of bone and bone marrow homeostasis requires the presence of functional osteocytes. Reduction of osteocytes and their function may result in the detrimental impact in altering lineage cell fates and specifications in bone marrow. Using a mouse model of conditional deletion of osteocytes by the expression of diphtheria toxin subunit α (DTA) in dentin matrix protein 1 (DMP1)-positive osteocytes, we showed that osteocytes regulated bone and bone marrow lineage cell specification. Ablation of osteocytes in these mice caused impairment of osteogenesis and lymphopoiesis, and increased osteoclastogenesis and mobilization of myelopoiesis toward myeloid lineage differentiation with expanded myeloid progenitors, neutrophils, and monocytes. These have resulted in the induction of accelerated skeletal aging. Mice with osteocyte ablation have severe sarcopenia, osteoporosis, and kyphosis at the early stage of 13 weeks, resulting in shorter lifespan. Together, we demonstrated that osteocytes play a critical role in regulation of the HSC and MSC lineage cell differentiations by mediation of senescence. Results Mice with fewer osteocytes have severe osteoporosis, kyphosis, sarcopenia, and shorter lifespan To delineate the role of osteocyte in skeletal tissue development and maturation, we established a mouse model based on DTA-mediated cell knockout using the promoter of DMP1 (Breitman et al., 1990). The latter is a protein highly expressed in late-stage osteocytes but has been shown not to be essential for early skeletal development (Feng et al., 2003). The results showed that complete ablation of DMP1-positive osteocytes (osteocyteDMP1) in Dmp1cre Rosa26em1Cin(SA-IRES-Loxp-ZsGreen-stop-Loxp-DTA) homozygotes (DTAho) caused lethality of mice before birth. This has led us to investigate the impact of partial ablation of osteocytes using Dmp1cre Rosa26em1Cin(SA-IRES-Loxp-ZsGreen-stop-Loxp-DTA) heterozygotes (DTAhet). Interestingly, Alizarin red/Alcian blue staining of whole-mount skeleton at E19.0 showed no apparent differences of craniofacial, long bones or spines between WT and DTAhet mice (Figure 1—figure supplement 1A). As shown in Figure 1A and B, DTAhet mice had more empty lacunae without the presence of osteocytes within cortical and trabecular bone matrix compared to WT mice. Further, reduced dendrites were also observed in residual osteocytes of DTAhet mice (Figure 1C and D), indicating the impairment of osteocyte network. To define how osteocyte partial ablation was achieved, we performed the quantification of empty lacunae ratio of DTAhet mice at 13 weeks. About 80% empty lacunae was observed in DTAhet mice at 13 weeks, which increased by about 20% compared to 4 weeks (Figure 1—figure supplement 1B and C), indicating that diphtheria toxin (DT) had an accumulative effect with age in DTAhet mice. Together, these results indicated that although there was partial ablation of osteocyteDMP1 in DTAhet mice, the embryonic development of skeletal tissue appeared to be normal. Figure 1 with 1 supplement see all Download asset Open asset DTAhet mice display partial osteocyte ablation. (A, B) Hematoxylin–eosin staining of WT and DTAhet mice femur at 4 weeks (A) and quantification of the ratio of empty lacunae (arrows) (B) (n = 8–12 per group), indicating reduced osteocyte number in DTAhet mice. Scare bar, 20 µm. (C, D) Immunofluorescence staining of femoral cortical bone of 4-week-old WT and DTAhet mice (C) and quantification of dendrites per osteocyte based on the images (D) (n = 152 osteocytes in WT group and n = 64 osteocytes in DTAhet group). Scare bar, 20 µm. Error bar represents the standard deviation. Figure 1—source data 1 DTAhet mice display partial osteocyte ablation. https://cdn.elifesciences.org/articles/81480/elife-81480-fig1-data1-v2.xlsx Download elife-81480-fig1-data1-v2.xlsx Next, we investigated whether reduction of osteocyteDMP1 in DTAhet mice had an impact on postnatal maturation of bone tissue. Micro-computed tomography (μCT) examination of the appendicular skeleton revealed a significant decrease in femur bone mineral density (BMD), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), as well as greater trabecular separation (Tb.Sp) in DTAhet mice compared to those in WT mice at 4 weeks (Figure 2A and B). Moreover, ablation of osteocytes also led to cortical bone loss with decreased cortical thickness (Ct.Th) and increased cortical porosity (Ct.Po) (Figure 2A and C). At 13 weeks, DTAhet mice exhibited more bone loss in both trabecular and cortical bone compared to those in WT mice (Figure 2D–G). The progressive bone loss was observed through the life of DTAhet mice. The phenotype observed is unique and gender-insensitive (Figure 2—figure supplement 1A–C). Similarly, μCT observation of axial skeleton also revealed the significant bone loss in vertebral bodies (Figure 2H and I, Figure 2—figure supplement 1D and E). Furthermore, there was no increase of bone mass of vertebral bodies from 4 to 13 weeks in DTAhet mice (Figure 2J and K), suggesting the retardation of vertebral body maturation. At 13 weeks, obvious kyphosis occurred in DTAhet mice (Figure 2L) due to serve osteoporosis and vertebral body compression. Whole-body μCT scan revealed that there was a giant increase of thoracic and lumbar curvature of DTAhet mice (Figure 2M). At the age of 30 weeks, almost all of DTAhet mice developed severe kyphosis (Figure 2N). Consistent with the development of kyphosis, gait analysis revealed that DTAhet mice at 4 weeks had abnormal steps when running (Figure 2—figure supplement 2A and B). The front and hind stride length were much shorter in DTAhet mice (Figure 2—figure supplement 2C). Also, the swing speed of DTAhet mice was much slower than WT mice (Figure 2—figure supplement 2D, Videos 1–6). Figure 2 with 2 supplements see all Download asset Open asset Osteocyte ablation induces severe osteoporosis and kyphosis. (A–C) Representative micro-computed tomography (µCT) reconstructive images of male WT and DTAhet mice femur at 4 weeks (A) and trabecular microstructural parameters (BMD, bone mineral density; BV/TV, bone volume fraction; Tb.N, trabecular number; Tb.Sp, trabecular separation; , Tb.Th, trabecular thickness) (B) and cortical microstructural parameters (Ct.Th, cortical thickness; Ct.Po, cortical porosity) (C) derived from µCT analysis (n = 4–7 per group). (D–G) Representative µCT reconstructive images of male WT and DTAhet mice femur at 13 weeks (D) and trabecular microstructural parameters (BMD, BV/TV, Tb.N, Tb.Sp, and Tb.Th) (E, F) and cortical microstructural parameters (Ct.Th and Ct.Po) (G) derived from µCT analysis (n = 3 per group), demonstrating severe bone loss in DTAhet mice. (H, I) Representative µCT reconstructive images of male WT and DTAhet mice third lumbar at 4 weeks (H) and trabecular microstructural parameters (BMD, BV/TV, Tb.N, Tb.Sp, and Tb.Th) (I) derived from µCT analysis (n = 4–7 per group). (J, K) Representative µCT reconstructive images of male WT and DTAhet mice third lumbar at 13 weeks (J) and trabecular microstructural parameters (BMD, BV/TV, Tb.N, Tb.Sp, and Tb.Th) (K) derived from µCT analysis (n = 3 per group), showing vertebral body bone loss in the spine of DTAhet mice. (L) Gross images of male WT and DTAhet mice at 13 weeks. (M) Representative whole-body µCT reconstructive and sagittal images of male WT and DTAhet mice at 13 weeks. (N) Representative whole-body µCT reconstructive and sagittal images of male DTAhet mice at 37 weeks, noting that severe kyphosis occurred in DTAhet mice. Error bar represents the standard deviation. Figure 2—source data 1 Osteocyte ablation induces severe osteoporosis in male mice. https://cdn.elifesciences.org/articles/81480/elife-81480-fig2-data1-v2.xlsx Download elife-81480-fig2-data1-v2.xlsx Video 1 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement in WT mice at 4 weeks. Video 2 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement defects in DTAhet mice at 4 weeks. Video 3 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement in WT mice at 13 weeks. Video 4 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement defects in DTAhet mice at 13 weeks. Video 5 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement in WT mice at 37 weeks. Video 6 Download asset This video cannot be played in place because your browser does support HTML5 video. You may still download the video for offline viewing. Download as MPEG-4 Download as WebM Download as Ogg Representative movie showing movement defects in DTAhet mice at 37 weeks. Whole-body examination of DTAhet mice revealed there was a continual body weight loss and muscle weight loss (Figure 3A–C) from 4 weeks. Histology examination of gastrocnemius muscles revealed focal muscle atrophy with mild inflammation at 4 weeks (Figure 3D and E). Many myonuclei were mispositioned and became centralized in contrast to those in WT mice. No muscle fibrosis was observed. At 13 weeks, there was continual muscle atrophy, rimmed vacuoles, and inclusion bodies seen within the muscle fibers (Figure 3F and G). To preclude the direct target of DMP1 on muscle, we quantified the number of muscle fibers and the results showed that there was no reduction of numbers of muscle fibers after osteocyte ablation at 4 weeks (Figure 3—figure supplement 1A) and 13 weeks in DTAhet mice compared to WT mice (Figure 3—figure supplement 1B). Measurement of Dmp1 expression in WT muscle showed that the level of Dmp1 expression in muscle was very weak and far less than bone (Figure 3—figure supplement 1C). Together, these results suggested that DTAhet mice had systemic muscle atrophy and sarcopenia. It is most likely that sarcopenia was caused by the impairment of osteocyte-muscle crosstalk. Analysis of lifespan in these mice further revealed the average lifespan of DTAhet mice was about 20–40 weeks, which was much shorter than WT mice (Figure 3H). Together, these data demonstrated that osteocytes ablation caused severe osteoporosis and kyphosis, as well as sarcopenia, which occurred at the very early stage compared to normal aging process. These age-related skeletal phenotypes combined with shortened lifespan demonstrated that osteocyte ablation led to the accelerated skeletal aging. Figure 3 with 1 supplement see all Download asset Open asset Osteocyte ablation leads to severe sarcopenia and shorter lifespan. (A, B) Gross images (A) and weight (B) of male WT and DTAhet mice at 4 weeks (n = 5–8 per group). (C) The ratio of gastrocnemius muscle weight in WT and DTAhet mice at 4 weeks (n = 3 per group). (D, E) Hematoxylin–eosin staining of WT and DTAhet mice gastrocnemius muscle at 4 weeks (D) and quantification of myonuclear number per area fiber (n = 11 per group) and centralized nucleus per field (E) (n = 5 per group), showing focal muscle atrophy, increased centralized myonuclei, and mild inflammation in DTAhet mice. Scale bar, 20 µm. (F, G) Hematoxylin–eosin staining of WT and DTAhet mice gastrocnemius muscle at 13 weeks (F) and quantification of myonuclear number per area fiber (n = 11 per group) and centralized nucleus per field (G) (n = 6 per group), noting muscle atrophy, rimmed vacuoles, and inclusion bodies within the muscle fibers in DTAhet mice. Scale bar, 20 µm. (H) Kaplan–Meier survival curve of WT and DTAhet mice (n = 4–5 per group), showing that DTAhet mice had shorter lifespan than that of WT mice. Error bar represents the standard deviation. Figure 3—source data 1 Osteocyte ablation leads to severe sarcopenia and shorter lifespan. https://cdn.elifesciences.org/articles/81480/elife-81480-fig3-data1-v2.xlsx Download elife-81480-fig3-data1-v2.xlsx Ablation of osteocytes alters mesenchymal lineage commitment and promotes osteoclastogensis To explore the potential mechanism of why reduction of osteocytes has caused severe osteoporosis and kyphosis, RNA sequencing was performed on whole bone with bone marrow flushed out from DTAhet and WT mice at 4 weeks. Selected skeleton-related Gene Ontology (GO) analysis revealed that downregulated genes by osteocyte ablation were enriched in ossification, osteoblast differentiation, positive regulation of osteoblast differentiation, endochondral ossification, and bone morphogenesis (Figure 4—figure supplement 1A and Supplementary file 1). Heatmap of significantly differentiated genes (fold change >2.0-fold, WT average FPKM > 10, false discovery rate [FDR] < 0.05) and subsequent RT-qPCR verified that genes that are critical for osteogenesis, including Alpl, Bglap, Col1a1, Spp1, Sp7, and Runx2, were affected by the ablation of osteocytes (Figure 4—figure supplement 1B and C). Also, gene set enrichment analysis (GSEA) revealed that osteogenesis-related pathways, including Wnt signaling pathway, Hedgehog signaling pathway, and Notch signaling pathway, were downregulated (Figure 4—figure supplement 1D–F). In addition, the number of osteoblasts (N.Ob/BS) and osteoid-covered surface (OS/BS) was remarkably reduced in DTAhet mice compared to WT mice (Figure 4A and B). Also, bone marrow fat accumulation in DTAhet mice was observed (Figure 4C and D). Together, these results suggested that DTAhet mice displayed increased adipogenesis and decreased osteogenesis. To further evaluate the dynamics of bone formation in DTAhet mice, a 7-day dynamic histomorphometric analysis using calcein labeling was performed. The result showed that mineral surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) were significantly decreased in DTAhet mice (Figure 4E and F). Serum procollagen type 1 N-terminal propeptide (P1NP), a bone formation index, was also reduced after osteocyte ablation (Figure 4G). Intriguingly, in vitro osteogenesis showed that there were also decreased osteogenesis and mineralization in DTAhet mice compared to WT mice at both time points of 4 and 13 weeks and the impairment of osteogenesis was greater in DTAhet mice at 13 weeks compared to 4 weeks (Figure 4H and I). And the mRNA level of osteogenic markers at 4 weeks, including Alpl, Bglap, and Runx2, was also decreased (Figure 4J). Figure 4 with 1 supplement see all Download asset Open asset Ablation of osteocytes alters mesenchymal lineage commitment and promotes osteoclastogensis. (A, B) Goldner trichrome staining of male WT and DTAhet mice femur at 4 weeks (A) and histomorphometry analysis of osteoblast numbers (N.Ob/BS) (arrows) and osteoid-covered surface (OS/BS) (B) (n = 6 per group). Scale bar, 20 µm. (C, D) Hematoxylin–eosin staining of WT and DTAhet mice femur at 4 weeks (C) and histomorphometry analysis of adipocyte (arrows) volume (Ad.V/TV) (D) (n = 6 per group). Scale bar, 50 µm. (E, F) Representative images of calcein double labeling of the mineral layers of male WT and DTAhet mice femur at 4 weeks (E) and histomorphometry analysis of the mineral surface (MS/BS), mineral apposition rate (MAR), and bone formation rate (BFR/BS) (F) (n = 4 per group). Scale bar, 50 µm. (G) ELISA of the concentration of bone formation index P1NP in the serum (n = 6–7 per group). (H, I) Alizarin red staining of osteogenesis from 4-week (H) and 13-week mice (I). Scale bar, 250 µm. (J) RT-qPCR analysis of osteoblast signature genes expression at the mRNA levels of osteogenesis from 4-week mice (n = 3 per group from three independent experiments), indicating impaired osteogenesis in DTAhet mice. (K, L) Tartrate-resistant acid phosphatase (TRAP) staining of WT and DTAhet mice femur at 4 weeks (K) and histomorphometry analysis of osteoclast (arrows) surface (Oc.S/BS) and osteoclast numbers (N.Oc/BS) (L) (n = 6 per group). Scale bar, 20 µm. (M) ELISAs of the concentration of receptor activation of nuclear factor-κ B ligand (RANKL), osteoprotegrin (OPG), and the ratio of RANKL/OPG in the serum (n = 6–7 per group). (N) ELISA of the concentration of bone resorption index CTX in the serum (n = 6–7 per group). (O, P) TRAP staining of osteoclastogenesis from 4-week (O) and 13-week mice (P) and quantitative analysis (Q) of TRAP-positive cells (nucleus > 3) per well (n = 3 per group from three independent experiments). Scale bar, 250 µm. (R) RT-qPCR analysis of osteoclast signature genes expression at the mRNA level of osteoclastogenesis from 4-week mice (n = 3 per group from three independent experiments), showing increased osteoclastogensis in DTAhet mice. Error bar represents the standard deviation. Figure 4—source data 1 Ablation of osteocytes alters mesenchymal lineage commitment and promotes osteoclastogensis. https://cdn.elifesciences.org/articles/81480/elife-81480-fig4-data1-v2.xlsx Download elife-81480-fig4-data1-v2.xlsx In the aspect of osteoclastogenesis, histomorphometry analysis revealed that osteoclast surface (Oc.S/BS) and numbers (N.Oc/BS) were significantly increased after osteocytes deletion (Figure 4K and L). Circulatory RANKL was also increased in DTAhet mice (Figure 4M). In contrast, circulatory osteoprotegrin (OPG), a decoy receptor of RANKL, was decreased (Figure 4M), leading to the elevated ratio of RANKL/OPG (Figure 4M). Serum collagen type I c-telopeptide (CTX), a bone resorption index, was also significantly augmented in DTAhet mice compared to WT mice (Figure 4N), which implicated a high level of osteoclast activity of DTAhet mice in vivo. Also, flow cytometry analysis revealed that there was a slight increase (less than 1%) of osteoclast progenitors (B220-CD11bloLy-6Chi) in DTAhet mice at 4 weeks compared to WT mice (Figure 4—figure supplement 1G and H). To assess the effects of osteocyte ablation on osteoclastogenesis, bone marrow-derived macrophages (BMMs) from WT and DTAhet mice at both time points of 4 and 13 weeks were collected and plated at the same density for the examination of osteoclastogenesis in vitro. The results showed that osteoclastogenesis was increased in DTAhet mice compared to WT mice at both time points (Figure 4O and Q). Interestingly, the induction of osteoclastogenesis was greater in DTAhet mice at 13 weeks compared to 4 weeks (Figure 4P and Q), suggesting the time-dependent accumulative effect of osteoclastogenesis in DTAhet mice. Also, the expression of the signature genes of osteoclasts, including Acp5, Calcr, and Ocstamp, at the mRNA level was significantly upregulated in DTAhet mice (Figure 4Q). Together, osteocytes ablation impaired osteogenesis and promoted osteoclastogenesis. Alteration of hematopoietic lineage commitment by osteocyte ablation As a part of the skeletal system, bone marrow has its vital functions in maintaining bone homeostasis (Divieti Pajevic and Krause, 2019; Fulzele et al., 2013; Asada et al., 2013). HSCs give rise to lymphoid and myeloid lineage cells to establish the hematopoietic and immune system. To gain a full insight into the role of osteocyte in bone marrow homeostasis, single-cell RNA sequencing (scRNA-seq) was performed using 10X Genomics Chromium platform. After rigorous quality control, gene expression data from 26,562 cells (13,835 and 12,727 cells from 4-week littermate WT and DTAhet mice, respectively) were compiled for clustering analysis and revealed 10 distinct populations visualized with Uniform Manifold Approximation and Projection (UMAP) embeddings (Figure 5A–C). These 10 distinct populations included B cell, hematopoietic stem cell and progenitor cell (HSPC), megakaryocyte, neutrophil, erythrocyte, monocyte, dendritic cell (DC), macrophage, T cell, and MSC (Figure 5A and C). Proportion analysis revealed a significant expansion of neutrophils in DTAhet mice (Figure 5D and E). Also, the number of B cells was significantly less in DTAhet mice than that in WT mice (Figure 5D and E), which implicated that osteocytes ablation induced lymphoid–myeloid malfunction in the bone marrow. To further dissect the differences in the bone marrow d