Bone marrow Adipoq-lineage progenitors are a major cellular source of M-CSF that dominates bone marrow macrophage development, osteoclastogenesis, and bone mass

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1. 

   eLife

   Feb 16, 2023

   eLife assessment

   This paper is of interest for skeletal biologists studying bone marrow stem/progenitor cells and bone remodeling. Using Adipoq-Cre-driven conditional deletion of Csf1 that encodes M-CSF and reanalyzing publicly available scRNAseq data, the authors recognize a subpopulation of bone marrow cells (i.e. AdipoQ-lineage progenitors) as an important source of M-CSF. The authors found that M-CSF production from these bone marrow cells influences the development of macrophages and osteoclasts as well as bone mass, including the bone loss associated with estrogen deficiency. This is a clearly written and nicely presented study that has potential to offer important new information regarding the source of M-CSF in the bone marrow.

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2. 

   eLife

   Feb 16, 2023

   Reviewer #1 (Public Review):

   The authors' conclusions presented herein are supported by a well-established mouse genetic conditional approach and an extensive array of phenotypic analyses.

   Strengths:

   1. The authors utilized well-described genetic tools, AdipoQCre, to target preadipocyte-like progenitor cell populations in bone marrow, as well as Csf1 floxed alleles. They further sifted through the cell population by showing that mature lipid-laden adipocytes express Csf1 at a much lower level, and determined that AdipoQCre-marked progenitor cell population presents a major cellular source of M-CSF,

   2. The reanalysis of published scRNAseq datasets in Figure 1, as well as the following phenotypic analyses of the mutant mice are well-conducted. The analyses include a broad range of experiments both in vivo (3DmicroCT, histology, flow …

   Reviewer #1 (Public Review):

   The authors' conclusions presented herein are supported by a well-established mouse genetic conditional approach and an extensive array of phenotypic analyses.

   Strengths:

   1. The authors utilized well-described genetic tools, AdipoQCre, to target preadipocyte-like progenitor cell populations in bone marrow, as well as Csf1 floxed alleles. They further sifted through the cell population by showing that mature lipid-laden adipocytes express Csf1 at a much lower level, and determined that AdipoQCre-marked progenitor cell population presents a major cellular source of M-CSF,

   2. The reanalysis of published scRNAseq datasets in Figure 1, as well as the following phenotypic analyses of the mutant mice are well-conducted. The analyses include a broad range of experiments both in vivo (3DmicroCT, histology, flow cytometry) and ex vivo (osteoclastogenesis assay in bone marrow cell culture). The confidence of the reported findings is high.

   3. The data presented in this manuscript are of very high quality.

   Weaknesses:

   1. The role of AdipoQ-lineage progenitors as a source of M-CSF is overstated. The authors claim in many instances that "mature bone adipocytes do not express M-CSF", "These cells however do not produce Csf1", "...these peripheral AdipoQ+ cells nearly do not produce M-CSF". However, the authors' qPCR experiments only show four times differences in Csf1 expression. Therefore, the claim that AdipoQ-lineage progenitors are an exclusive source of M-CSF is not well substantiated. In line with this, some of the recent literature reporting conditional deletion of M-CSF in other bone cells (JBMR Plus. 4:e10080., Nature. 590:457-462) are not included.

   2. Some of the phenotypic analyses are still incomplete. The authors did not report whether CHet (AdipoQCre Csf1(flox/+)) showed any bone phenotype. Further, the authors did not show that Csf1 mRNA or M-CSF protein is expressed in AdipoQ-lineage progenitors using histological methods. Current evidence is only based on scRNAseq and qPCR of isolated cells. Whether there was any change in circulating bone resorption markers in CKO mice was not shown. Cortical bone parameters were not included in the 3D-microCT analyses. These missing pieces of information would be important to correctly interpret the phenotypes.

   3. Which bone marrow cell population(s) are marked by AdipoQCre remain largely unclear. It is possible that AdipoQCre also marks at least part of MSPC-osteo cluster in addition to MSPC-adipo. Adipo-lineage progenitors may not stay entirely as adipoprogenitors and drift toward osteoblasts or their precursor cells.

   4. The OVX data in Figure 5 are not very well explained. The data do not seem to support the authors' conclusion that M-CSF deficiency in AdipoQ-lineage progenitors alleviates estrogen-deficiency induced osteoporosis. The CKO mice lose bone mass almost to the same extent as WT mice upon OVX.

   Read the original source
3. 

   eLife

   Feb 16, 2023

   Reviewer #2 (Public Review):

   This study demonstrates that AdipoQ+ cells, which constitute approximately 0.8% of bone marrow mesenchymal cells, are major producers of M-CSF (Csf1) in murine bone marrow. The initial finding was discovered in scRNA seq datasets and studied in depth here with animal models and cellular assays. Deletion of Csf1 with AdipoQ-Cre increased trabecular bone mass in long bones and reduced the number of osteoclasts on trabecular bone surfaces. Cd11b+ F4/80+ macrophage numbers were also reduced in bone marrow. Bone loss from ovariectomy was prevented in Csf1∆AdipoQ female mice. Strengths of this study include use of a tissue-directed knock out (Adipo-Cre) model system to understand the relative contribution of AdipoQ+ cells to Csf1 levels and trabecular bone mass, careful examination of other adipose tissues for …

   Reviewer #2 (Public Review):

   This study demonstrates that AdipoQ+ cells, which constitute approximately 0.8% of bone marrow mesenchymal cells, are major producers of M-CSF (Csf1) in murine bone marrow. The initial finding was discovered in scRNA seq datasets and studied in depth here with animal models and cellular assays. Deletion of Csf1 with AdipoQ-Cre increased trabecular bone mass in long bones and reduced the number of osteoclasts on trabecular bone surfaces. Cd11b+ F4/80+ macrophage numbers were also reduced in bone marrow. Bone loss from ovariectomy was prevented in Csf1∆AdipoQ female mice. Strengths of this study include use of a tissue-directed knock out (Adipo-Cre) model system to understand the relative contribution of AdipoQ+ cells to Csf1 levels and trabecular bone mass, careful examination of other adipose tissues for Csf1 expression, challenging bone responses in Csf1∆AdipoQ female mice with ovariectomy, and studying the effect of Csf1 deletion in macrophage levels. Mechanical studies of bone strength were not included but would be necessary to determine if deletion of Csf1 in AdipoQ+ cells is sufficient to cause osteopetrosis as concluded by the authors. Additional information on other molecular changes Csf1∆AdipoQ mice would provide insights into how deletion of Csf1 in AdipoQ+ cells affects bone remodeling. Overall, this is a very important study that has a lot of merit. It's impact on the field will be high because it is challenging the paradigm that osteoblasts and osteocytes are the major sources of M-CSF in the bone marrow.

   Read the original source
4. 

   eLife

   Feb 16, 2023

   Reviewer #3 (Public Review):

   Macrophage colony-stimulating factor (M-CSF) plays key roles in the differentiation of myeloid-lineage cells, including monocytes, macrophages and osteoclasts. The latter mediate bone resorption, which is important for physiological bone remodelling but, unrestrained, contributes to bone loss in conditions such as in post-menopausal osteoporosis. M-CSF production within the bone marrow is implicated in the maintenance of myeloid and skeletal homeostasis, but the cellular source of bone marrow M-CSF has remained elusive. In this study, Inoue et al address this issue through advanced transcriptomic and gene targeting approaches. They conclude that a population of Adipoq-expressing progenitors within the bone marrow, designated "AdipoQ-lineage progenitors", is the key cellular source of M-CSF. Consistent with …

   Reviewer #3 (Public Review):

   Macrophage colony-stimulating factor (M-CSF) plays key roles in the differentiation of myeloid-lineage cells, including monocytes, macrophages and osteoclasts. The latter mediate bone resorption, which is important for physiological bone remodelling but, unrestrained, contributes to bone loss in conditions such as in post-menopausal osteoporosis. M-CSF production within the bone marrow is implicated in the maintenance of myeloid and skeletal homeostasis, but the cellular source of bone marrow M-CSF has remained elusive. In this study, Inoue et al address this issue through advanced transcriptomic and gene targeting approaches. They conclude that a population of Adipoq-expressing progenitors within the bone marrow, designated "AdipoQ-lineage progenitors", is the key cellular source of M-CSF. Consistent with this, they find that transgenic deletion of M-CSF from these cells disrupts macrophage and osteoclast development, leading to osteopetrosis and possibly preventing bone loss following ovariectomy. However, they have not adequately addressed the possibility that M-CSF production from other cell types, particularly adipocytes in peripheral adipose tissues, may also be influencing these phenotypes. Specific strengths and weaknesses are as follows:

   Strengths:

   1. The manuscript is written in a clear, succinct manner and the data are generally nicely presented. It is therefore a pleasure to read.

   2. The analysis of single-cell transcriptomic data is clear and convincing, and the skeletal phenotyping has been done to a high standard.

   Weaknesses:

   1. The authors underplay the potential contribution of M-CSF production from other cell types, particularly from adipocytes in peripheral adipose tissues. They show that M-CSF expression from these cells is lower than from the bone marrow progenitors that they focus on; however, based on this they allude to "no expression" of M-CSF from these other adipocytes. This overlooks the findings of other studies showing that peripheral adipocytes produce M-CSF and that this has biological functions. Whether their knockout model alters M-CSF expression in peripheral adipose tissue, whether for whole tissue or for isolated adipocytes, has not been tested.

   2. The decreases in M-CSF have been assessed at the transcript level, but not for M-CSF protein. Whether their knockout model

   3. It is also unclear if the Adipoq-lineage progenitors consist exclusively of adipogenic cells, or if osteogenic progenitors are also part of this population.

   If these weaknesses are addressed then this work has potential to yield firm conclusions and new insights into the regulation of myeloid and skeletal homeostasis, both in normal physiology and in clinically relevant conditions.

   Read the original source
5. 

   eLife

   Feb 16, 2023

   Author Response:

   Reviewer #1 (Public Review):

   The authors' conclusions presented herein are supported by a well-established mouse genetic conditional approach and an extensive array of phenotypic analyses.

   Strengths:

   1. The authors utilized well-described genetic tools, AdipoQCre, to target preadipocyte-like progenitor cell populations in bone marrow, as well as Csf1 floxed alleles. They further sifted through the cell population by showing that mature lipid-laden adipocytes express Csf1 at a much lower level, and determined that AdipoQCre-marked progenitor cell population presents a major cellular source of M-CSF,

   2. The reanalysis of published scRNAseq datasets in Figure 1, as well as the following phenotypic analyses of the mutant mice are well-conducted. The analyses include a broad range of experiments both in vivo (3DmicroCT, …

   Author Response:

   Reviewer #1 (Public Review):

   The authors' conclusions presented herein are supported by a well-established mouse genetic conditional approach and an extensive array of phenotypic analyses.

   Strengths:

   1. The authors utilized well-described genetic tools, AdipoQCre, to target preadipocyte-like progenitor cell populations in bone marrow, as well as Csf1 floxed alleles. They further sifted through the cell population by showing that mature lipid-laden adipocytes express Csf1 at a much lower level, and determined that AdipoQCre-marked progenitor cell population presents a major cellular source of M-CSF,

   2. The reanalysis of published scRNAseq datasets in Figure 1, as well as the following phenotypic analyses of the mutant mice are well-conducted. The analyses include a broad range of experiments both in vivo (3DmicroCT, histology, flow cytometry) and ex vivo (osteoclastogenesis assay in bone marrow cell culture). The confidence of the reported findings is high.

   3. The data presented in this manuscript are of very high quality.

   Weaknesses:

   1. The role of AdipoQ-lineage progenitors as a source of M-CSF is overstated. The authors claim in many instances that "mature bone adipocytes do not express M-CSF", "These cells however do not produce Csf1", "...these peripheral AdipoQ+ cells nearly do not produce M-CSF". However, the authors' qPCR experiments only show four times differences in Csf1 expression. Therefore, the claim that AdipoQ-lineage progenitors are an exclusive source of M-CSF is not well substantiated. In line with this, some of the recent literature reporting conditional deletion of M-CSF in other bone cells (JBMR Plus. 4:e10080., Nature. 590:457-462) are not included.

   We thank the reviewer for this important question. We have performed the below experiments to further clarify and support our conclusion:

   1. We increased the replicates of each group cells in Fig. 3A (the old Fig. 1E) to five/group and based on reviewer 3’ recommendation on housekeeping gene usage, we found that the mRNA expression of Csf1 in bone marrow AdipoQ-lineage progenitor cells is 20-30 fold higher than those in mature adipocytes. This result has been updated in Fig. 3A.

   2. We further performed immunofluorescence staining of M-CSF on bone slices, and found that the majority of bone marrow AdipoQ-expressing progenitor cells express M-CSF (Fig. 3B, 1865 cells out of 2001 cells counted, n=3 mice, 93.2%). In contrast, M-CSF expression was not detected in mature bone marrow adipocytes (Perilipin1+) (Fig. 3C, 0 cells out of 115 cells counted, n=3 mice, 0%), indicating that mature bone marrow adipocytes are unlikely a significant source of M-CSF.

   3. We performed western blot to analyze M-CSF protein expression in peripheral adipose. As shown in Fig. 3D, the stromal vascular fraction (SVF) cells in adipose, which contain multiple cell populations including adipogenic progenitors, express M-CSF. On the contrary, M-CSF was nearly undetectable in the peripheral mature adipocytes isolated from adipose (Fig. 3D).

   These data collectively support that mature adipocytes are not a significant source of M-CSF as evidenced by nearly undetectable M-CSF expression compared to the Adipoq-lineage progenitors. The results were described on pg. 5. However, the reviewer’s comment on ‘exclusive source’ is well taken as osteocytes and osteo lineage also express certain levels of M-CSF. We deleted ‘exclusive source’ in the manuscript, have added relevant literature and discussion in the Results and Discussion section on pp. 5 and 9.

   2. Some of the phenotypic analyses are still incomplete. The authors did not report whether CHet (AdipoQCre Csf1(flox/+)) showed any bone phenotype. Further, the authors did not show that Csf1 mRNA or M-CSF protein is expressed in AdipoQ-lineage progenitors using histological methods. Current evidence is only based on scRNAseq and qPCR of isolated cells. Whether there was any change in circulating bone resorption markers in CKO mice was not shown. Cortical bone parameters were not included in the 3D-microCT analyses. These missing pieces of information would be important to correctly interpret the phenotypes.

   The het mice (Csf1f/+;AdipoQ Cre) do not show abnormal bone phenotype, which is now shown in Fig. 4-figure supplement 4. We performed immunofluorescence staining of M-CSF on bone slices, and found that the majority of bone marrow AdipoQ-expressing progenitor cells express M-CSF (Fig. 3B, 1865 cells out of 2001 cells counted, n=3 mice, 93.2%). We tested serum TRAP level in mice, and found that the Csf1 deficiency in Csf1∆AdipoQ mice significantly decreased the TRAP level in serum, compared to that in the WT control mice (Fig. 5B). Csf1∆AdipoQ mice do not exhibit abnormal cortical bone phenotype. The cortical bone parameters are now included in Fig. 4G.

   3. Which bone marrow cell population(s) are marked by AdipoQCre remain largely unclear. It is possible that AdipoQCre also marks at least part of MSPC-osteo cluster in addition to MSPC-adipo. Adipo-lineage progenitors may not stay entirely as adipoprogenitors and drift toward osteoblasts or their precursor cells.

   We thank the reviewer for the insightful comment on this interesting mystery and complicated question, which is drawing more attention in the field.

   In addition to Adipoq-lineage progenitors, Adipoq Cre also labels other clusters. However, the expression levels of Adipoq and frequency of Adipoq+ cells in other cell populations are relatively low. For example, the integrated scRNAseq dataset we analyzed shows that Adipoq is expressed at a low level (with scaled mean expression at 0.68, (27)) in a small proportion of MSPC-osteo cells (Fig. 1), and small amounts (31, 37) (about 4%) of osteoblasts in 8 or 12-week-old mice are Adipoq-lineage. A recent report found that in 24-week-old mice, about 15-40% of osteoblasts are marked with Adipoq Cre (37). This raises a few important possibilities that will need to be distinguished in future work. One possibility is that the Adipoq-lineage cells (adipo-CAR cells/MALPs) have minor or latent osteogenic potential that may become more evident under specific conditions, such as in older animals. However, balanced against this is the alternative that Adipoq-cre could primarily target a population of solely adipogenic adipo-CAR cells but that its specificity is imperfect, leading to progressive low levels of deletion in a separate population expressing very low levels of Adipoq, such as osteo-CAR cells. An additional possibility is that the Adipoq-lineage cells may themselves actually be further subdivided into multiple component cell types, including a major adipogenic and a separate minor osteogenic subpopulation. Ultimately, at the root of these issues is that Adipoq cre primarily defines one or possibly more lineages of cells rather than a cell type within those lineages. Therefore, application of further markers to fractionate the adipoq-lineage into its component cell types will be needed to resolve these possibilities, focusing on whether any potential osteogenic activity present can be fractionated away from the primary adipogenic activity present.

   Of note, the Adipoq expression level and positive cell proportion are much higher in bone marrow Adipoq lineage progenitors than the levels seen in osteoblast lineage (Fig.1, Fig.2, (22, 27, 31)) or endothelial cells in bone marrow (38, 39). For example, the MSPC-Adipo cluster (Adipoq-lineage progenitors) has 6441 cells with the highest level (scaled mean expression level at 3.01 per (27) at Single Cell Portal) of Adipoq seen among bone marrow cells analyzed. In contrast, the MSPC-osteo cluster consists of 2247 cells with a very low Adipoq expression level (scaled mean expression level at 0.68 per (27) at Single Cell Portal). Taken together with both average expression level and cell numbers in each cluster, the relative overall contribution to Adipoq expression by MSPC-osteo vs the Adipoq-lineage progenitors is 7.8% ((2247 x 0.68)/(6441 x 3.01)). Therefore, the expression of Adipoq in MSPC-osteo cluster is marginal compared to that in the Adipoq-lineage progenitors. These data make Adipoq as an important marker to identify bone marrow Adipoq lineage progenitors. Overall, our work not only validates prior research identifying adipoq-lineage cells, identified as MALPs (22, 31), as a key osteoclast regulatory population, but also further extends the scope of their functions to encompass M-CSF production and regulation of macrophages.

   These points have been added to the Discussion sections on pp. 9-10.

   4. The OVX data in Figure 5 are not very well explained. The data do not seem to support the authors' conclusion that M-CSF deficiency in AdipoQ-lineage progenitors alleviates estrogen-deficiency induced osteoporosis. The CKO mice lose bone mass almost to the same extent as WT mice upon OVX.

   To address the reviewer’s question, we calculated the changes of the uCT parameter values between Sham and OVX groups in the WT control and Csf1∆AdipoQ mice. Significant changes were identified between the control and Csf1∆Adipoq mice in several μCT parameters. For example, a decrease in trabecular BV/TV after OVX: 35.1% in the control vs 20.9% in Csf1∆Adipoq mice; a decrease in Tb. N after OVX:11.34% in the control vs 7.97% in Csf1∆Adipoq mice; a decrease in Conn-Dens after OVX: 39.7% in the control vs 14.56% in Csf1∆Adipoq mice; an increase in Tb. Sp after OVX: 12.51% in the control vs 1.97% in Csf1∆Adipoq mice. These results support our conclusion that M-CSF deficiency in AdipoQlineage progenitors alleviates estrogen-deficiency induced osteoporosis. These value changes have been included in Fig. 7C and discussed on pg. 7.

   Reviewer #3 (Public Review):

   Macrophage colony-stimulating factor (M-CSF) plays key roles in the differentiation of myeloid-lineage cells, including monocytes, macrophages and osteoclasts. The latter mediate bone resorption, which is important for physiological bone remodelling but, unrestrained, contributes to bone loss in conditions such as in post-menopausal osteoporosis. M-CSF production within the bone marrow is implicated in the maintenance of myeloid and skeletal homeostasis, but the cellular source of bone marrow M-CSF has remained elusive. In this study, Inoue et al address this issue through advanced transcriptomic and gene targeting approaches. They conclude that a population of Adipoq-expressing progenitors within the bone marrow, designated "AdipoQ-lineage progenitors", is the key cellular source of M-CSF. Consistent with this, they find that transgenic deletion of M-CSF from these cells disrupts macrophage and osteoclast development, leading to osteopetrosis and possibly preventing bone loss following ovariectomy. However, they have not adequately addressed the possibility that M-CSF production from other cell types, particularly adipocytes in peripheral adipose tissues, may also be influencing these phenotypes. Specific strengths and weaknesses are as follows:

   Strengths:

   1. The manuscript is written in a clear, succinct manner and the data are generally nicely presented. It is therefore a pleasure to read.

   2. The analysis of single-cell transcriptomic data is clear and convincing, and the skeletal phenotyping has been done to a high standard.

   Weaknesses:

   1. The authors underplay the potential contribution of M-CSF production from other cell types, particularly from adipocytes in peripheral adipose tissues. They show that M-CSF expression from these cells is lower than from the bone marrow progenitors that they focus on; however, based on this they allude to "no expression" of M-CSF from these other adipocytes. This overlooks the findings of other studies showing that peripheral adipocytes produce M-CSF and that this has biological functions. Whether their knockout model alters M-CSF expression in peripheral adipose tissue, whether for whole tissue or for isolated adipocytes, has not been tested.

   We performed western blot to analyze M-CSF protein expression in peripheral adipose. As shown in Fig. 3D, the stromal vascular fraction (SVF) cells in adipose, which contain multiple cell populations including adipogenic progenitors, express M-CSF. On the contrary, M-CSF was nearly undetectable in the peripheral mature adipocytes isolated from adipose (Fig. 3D). These data collectively support that mature adipocytes are not a significant source of M-CSF as evidenced by nearly undetectable M-CSF expression compared to the Adipoq-lineage progenitors. However, we understand that current techniques may have limitation in identification of trace amount of M-CSF. We thus deleted descriptions such as ‘exclusive’ or ‘do not produce/express…’ in the revised manuscript.

   2. The decreases in M-CSF have been assessed at the transcript level, but not for M-CSF protein. Whether their knockout model

   We performed immunofluorescence staining of M-CSF on bone slices, and found a drastic decrease in M-CSF protein in bone marrow AdipoQ+ cells in Csf1∆AdipoQ mice compared to the WT control mice. The results are shown in Fig. 4B, and Fig. 3B-D.

   3. It is also unclear if the Adipoq-lineage progenitors consist exclusively of adipogenic cells, or if osteogenic progenitors are also part of this population.

   We thank the reviewer for the insightful comment on this interesting mystery and complicated question, which is drawing more attention in the field.

   In addition to Adipoq-lineage progenitors, Adipoq Cre also labels other clusters. However, the expression levels of Adipoq and frequency of Adipoq+ cells in other cell populations are relatively low. For example, the integrated scRNAseq dataset we analyzed shows that Adipoq is expressed at a low level (with scaled mean expression at 0.68, (27)) in a small proportion of MSPC-osteo cells (Fig. 1), and small amounts (31, 37) (about 4%) of osteoblasts in 8 or 12-week-old mice are Adipoq-lineage. A recent report found that in 24-week-old mice, about 15-40% of osteoblasts are marked with Adipoq Cre (37). This raises a few important possibilities that will need to be distinguished in future work. One possibility is that the Adipoq-lineage cells (adipo-CAR cells/MALPs) have minor or latent osteogenic potential that may become more evident under specific conditions, such as in older animals. However, balanced against this is the alternative that Adipoq-cre could primarily target a population of solely adipogenic adipo-CAR cells but that its specificity is imperfect, leading to progressive low levels of deletion in a separate population expressing very low levels of Adipoq, such as osteo-CAR cells. An additional possibility is that the Adipoq-lineage cells may themselves actually be further subdivided into multiple component cell types, including a major adipogenic and a separate minor osteogenic subpopulation. Ultimately, at the root of these issues is that Adipoq cre primarily defines one or possibly more lineages of cells rather than a cell type within those lineages. Therefore, application of further markers to fractionate the adipoq-lineage into its component cell types will be needed to resolve these possibilities, focusing on whether any potential osteogenic activity present can be fractionated away from the primary adipogenic activity present.

   Of note, the Adipoq expression level and positive cell proportion are much higher in bone marrow Adipoq lineage progenitors than the levels seen in osteoblast lineage (Fig.1, Fig.2, (22, 27, 31)) or endothelial cells in bone marrow (38, 39). For example, the MSPC-Adipo cluster (Adipoq-lineage progenitors) has 6441 cells with the highest level (scaled mean expression level at 3.01 per (27) at Single Cell Portal) of Adipoq seen among bone marrow cells analyzed. In contrast, the MSPC-osteo cluster consists of 2247 cells with a very low Adipoq expression level (scaled mean expression level at 0.68 per (27) at Single Cell Portal). Taken together with both average expression level and cell numbers in each cluster, the relative overall contribution to Adipoq expression by MSPC-osteo vs the Adipoq-lineage progenitors is 7.8% ((2247 x 0.68)/(6441 x 3.01)). Therefore, the expression of Adipoq in MSPC-osteo cluster is marginal compared to that in the Adipoq-lineage progenitors. These data make Adipoq as an important marker to identify bone marrow Adipoq lineage progenitors. Overall, our work not only validates prior research identifying adipoq-lineage cells, identified as MALPs (22, 31), as a key osteoclast regulatory population, but also further extends the scope of their functions to encompass M-CSF production and regulation of macrophages.

   These points have been added to the Discussion section on pp. 9-10.

   If these weaknesses are addressed then this work has potential to yield firm conclusions and new insights into the regulation of myeloid and skeletal homeostasis, both in normal physiology and in clinically relevant conditions.

   Yes, we have addressed the above 3 major questions.

   Read the original source
6. 

   Version published to 10.7554/elife.82118 on eLife

   Feb 13, 2023
7. 

   Version published to 10.1101/2022.07.28.501837v1 on bioRxiv

   Jul 30, 2022