Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This manuscript describes the limb phenotype of mice treated with CSF1R inhibitor PLX5622. The drug treatment leads to partial loss of Csf1r+ cells and postnatal shortening of the limbs. This work complements earlier work on this specific mouse model that described defects in other organ systems but not limbs.

 

• Both Csf1 and Csf1r KO mice and rats exist and limb phenotypes have been described. The authors need to do a better job discussing why it is relevant to report limb phenotype in this drug-treated model, and what new insight we can gain on top of what are already known from the KO models.

2. Throughout the results section, comparisons should be made between the current model and what are known in KO models. Are these differences due to partial blocking of the Csf1r activity by the drug, or does the drug has off-target activities?
3. The authors should be praised for including the sexes of the samples throughout the work. It is better to indicate whether there is any difference between the sexes in various measurements besides simply showing the sexes though.
4. It should be indicated in the figure legends that the blue and red colors indicate sexes of the samples. It took me a while to realize this is the case.
5. The immunofluorescence images in various figures need enhancement to better show the red and green color (or just make the images larger). I cannot see any without zooming in significantly.
6. Page 17, line 506 The authors used the word "delay", which should be used only when the authors have evidence that the structure catches up later in development. Change it if it is not the case.

 

Author Response

Reviewer #1 stated that the “authors should be praised for including the sexes of the samples throughout the work”, but did include some suggestions to improve the clarity of the manuscript, which we have responded to below:

 

Comments:

1. “Both Csf1 and Csf1r KO mice and rats exist and limb phenotypes have been described. The authors need to do a better job discussing why it is relevant to report limb phenotype in this drug-treated model, and what new insight we can gain on top of what are already known from the KO models.”

 

We would like to thank Reviewer #1 for this comment and have included additional information in the Introduction of the manuscript to address this point (please see lines 79-87).

 

2. “Throughout the results section, comparisons should be made between the current model and what are known in KO models. Are these differences due to partial blocking of the Csf1r activity by the drug, or does the drug has off-target activities?”

 

We would like to thank Reviewer #1 for this comment and have included additional information in the Results (please see lines 432-436) and Discussion (please see lines 526-531). Moreover, we have now included a section in the limitations describing off target impacts of PLX5622 (please see lines 576-588). As described in the text, our findings are in line with what has been observed in Csf1 and Csf1r KO rodent models (i.e., reduced limb lengths and altered mineralization); therefore, we would argue that our findings are indicative of partial blocking of CSF1R activity by the drug and not off target impacts.

 

3. “The authors should be praised for including the sexes of the samples throughout the work. It is better to indicate whether there is any difference between the sexes in various measurements besides simply showing the sexes though.”

 

We would like to thank Reviewer #1 for this comment. We have now included a description in the methodology section explaining that we have assessed sex differences for each analyses using a two-way ANOVA and results for those assessments which showed differences have been included in Table 2 in the methods section. Considering that these differences were minimal and did not show consistent changes (i.e., TRAP staining only showed a mild sex difference for the ulna and no other structures examined), we explain that all the data was then pooled, colour-coded by sex for graphical representation, and then analyzed using a t-test (please see lines 241-246 & Table 2).

 

4. “It should be indicated in the figure legends that the blue and red colors indicate sexes of the samples. It took me a while to realize this is the case.”

 

We apologize for this oversight and have now included this information both in a

legend beside the first graph in the Figures themselves, and also in the legends for each of the Figures.

 

5. “The immunofluorescence images in various figures need enhancement to better show the red and green color (or just make the images larger). I cannot see any without zooming in significantly.”

 

We apologize for not providing clearer immunofluorescent images and have addressed this problem by including higher magnification insets (please see Figure 1).

 

6. “Page 17, line 506 The authors used the word "delay", which should be used only when the authors have evidence that the structure catches up later in development. Change it if it is not the case.”

 

We apologize for this oversight and have modified our language to remove the word delay.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

The Authors describe the effect of the lack of CSFR1+ during the limb bone development.  The work is well conceptualized. The experimental procedure consisted in the pharmacological treatment with PLX5622  on the pregnant miThe introduction is well-structured and documented, the materials and methods are well-described, the results are important and relevant, and the discussion is thorough. The figures are very beautiful and useful. The extended and updated bibliographic citations.The entire manuscript is well-edited and precise. Although I searched for critical points, errors, or limitations, I was unable to identify any. This is the first time, after 30 years of peer review, that I have concluded that the work deserves to be published without revisions. 

Author Response

Reviewer #2 stated that “The work is well conceptualized” and “The introduction is well-structured and documented, the materials and methods are well-described, the results are important and relevant, and the discussion is thorough. The figures are very beautiful and useful.” and “The entire manuscript is well-edited and precise. Although I searched for critical points, errors, or limitations, I was unable to identify any. This is the first time, after 30 years of peer review, that I have concluded that the work deserves to be published without revisions.”

 

We would like to thank Reviewer #2 for their review and support of our manuscript.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

This study aims to investigate the effect of CSF1R inhibition via PLX5622 on limb bone development during embryogenesis. The authors administered PLX5622 to pregnant mice and examined the resulting embryos using whole-mount staining and histological analyses. Overall, this is a well-written manuscript that is easy to follow. I only have a few comments and suggestions, which are outlined below.

Major comments

1. Positive staining in Fig. 1 immunofluorescent images is difficult to see. Please provide higher magnification images or insets such as shown in Fig 2.
2. It would be beneficial to include quantification in Figs 4 and 5 (e.g. length and width measurements) to support stated claims. The representative images in Fig.5M-P appear to show differences in the size of the muscle groups and staining intensity between control and treated groups so quantification would be valuable.
3. Sp7 does not distinguish between immature and mature osteoblasts. Therefore, the possibility of differences in osteoblast maturation cannot be ruled out. Have the authors tried staining for additional osteoblast maturation markers, such as collagen type I or osteocalcin?
4. In the Fig.2 results description, the authors state that they examined TRAP staining as a measure of osteoclast function, which is arguable. TRAP is widely used to identify osteoclasts, but it does not necessarily reflect active bone resorption. Cathepsin K, which the authors also examined, is a better indicator of osteoclast resorptive activity, as it is secreted into the resorption lacuna and helps degrade the organic component of bone matrix. The function of TRAP in osteoclasts remains incompletely understood, but recent work suggests it may be dispensable for resorption in vivo (PMID: 39021958). In models targeting osteoclasts, such as bisphosphonate/denosumab treatment, TRAP positive multinucleated cells can still be present despite inhibited resorption. Therefore, I suggest that the authors dial back their interpretation of TRAP staining alone as evidence of osteoclast function in both the Results and Discussion sections.

Minor comments

1. Some figure panels don’t have scale bars. Are these panels the same magnification as the adjacent panels? Please clarify and include scale bars where appropriate.
2. I am not fully convinced that transverse sections would necessarily be more informative than sagittal sections for assessing embryonic bone density. As the authors acknowledge, micro-CT would be the more appropriate method for directly assessing bone density. The authors may wish to consider collagen type I staining, which could provide useful information on bone matrix deposition regardless of the sectioning plane.

Author Response

Reviewer #3 stated that “Overall, this is a well-written manuscript that is easy to follow” and that they “only have a few comments and suggestions”, which we have responded to below:

 

Comments:

1. “Positive staining in Fig. 1 immunofluorescent images is difficult to see. Please provide higher magnification images or insets such as shown in Fig 2.”

 

We apologize for not providing clearer immunofluorescent images in Figure 1 and have addressed this problem by including higher magnification insets (please see Figure 1).

 

2. “It would be beneficial to include quantification in Figs 4 and 5 (e.g. length and width measurements) to support stated claims. The representative images in Fig.5M-P appear to how differences in the size of the muscle groups and staining intensity between control and treated groups so quantification would be valuable.”

 

We would like to thank Reviewer #3 for this comment. We have now included nerve measurements for the E11.5 radial nerve, ulnar nerve, ventral axon, and dorsal axon (please see Figure 4E-H), the E12.5 radial nerve, ulnar nerve, peroneal nerve, and tibial nerve (please see Figure 4M-P), and the E13.5 radial nerve, ulnar nerve, peroneal nerve, and tibial nerve (please see Figure 4U-X). We have also now included muscle length and width measurements for the E13.5 extensor carpi radialis longus, extensor carpi radialis brevis, extensor carpi ulnaris, extensor digitorum, triceps, gastrocnemius, quadriceps, and tibialis anterior (please see Figure 5M-T’). Lastly, we have also included quantification of staining intensity for the E15.5 triceps and gastrocnemius (please see Figure 5Y-Z).

 

3. “Sp7 does not distinguish between immature and mature osteoblasts. Therefore, the possibility of differences in osteoblast maturation cannot be ruled out. Have the authors tried staining for additional osteoblast maturation markers, such as collagen type I or osteocalcin?”

 

We would like to thank Reviewer #3 for this comment. We have now included staining for collagen type I (please see Figure 6G-J’).

 

4. “In the Fig.2 results description, the authors state that they examined TRAP staining as a measure of osteoclast function, which is arguable. TRAP is widely used to identify osteoclasts, but it does not necessarily reflect active bone resorption. Cathepsin K, which the authors also examined, is a better indicator of osteoclast resorptive activity, as it is secreted into the resorption lacuna and helps degrade the organic component of bone matrix. The function of TRAP in osteoclasts remains incompletely understood, but recent work suggests it may be dispensable for resorption in vivo (PMID: 39021958). In models targeting osteoclasts, such as bisphosphonate/denosumab treatment, TRAP positive multinucleated cells can still be present despite inhibited resorption. Therefore, I suggest that the authors dial back their interpretation of TRAP staining alone as evidence of osteoclast function in both the Results and Discussion sections.”

 

We apologize for this oversight and have modified our language in the Result and Discussion. We would like to thank Reviewer #3 for sharing this literature with us.

 

5. “Some figure panels don’t have scale bars. Are these panels the same magnification as the adjacent panels? Please clarify and include scale bars where appropriate.”

 

We apologize for this oversight. Yes, all panels show the same magnification as adjacent panels with scale bars. We have corrected this in all Figures by placing scale bars on all images.

 

6. “I am not fully convinced that transverse sections would necessarily be more informative than sagittal sections for assessing embryonic bone density. As the authors acknowledge, micro-CT would be the more appropriate method for directly assessing bone density. The authors may wish to consider collagen type I staining, which could provide useful information on bone matrix deposition regardless of the sectioning plane.”

 

We would like to thank Reviewer #3 for this comment and have now removed the discussion of the limitations that might be related to sectioning plane. Moreover, we have performed collagen type I staining (please see Figure 6).

Author Response File: Author Response.pdf