Peer review process
Not revised: This Reviewed Preprint includes the authors’ original preprint (without revision), an eLife assessment, public reviews, and a provisional response from the authors.
Read more about eLife’s peer review process.Editors
- Reviewing EditorLee RubinHarvard Stem Cell Institute, Harvard University, Cambridge, United States of America
- Senior EditorChristopher HuangUniversity of Cambridge, Cambridge, United Kingdom
Reviewer #1 (Public review):
Summary:
Madigan et al. assembled an interesting study investigating the role of the MuSK-BMP signaling pathway in maintaining adult mouse muscle stem cell (MuSC) quiescence and muscle function before and after trauma. Using a full body and MuSC-specific genetic knockout system, they demonstrate that MuSK is expressed on MuSCs and that eliminating the BMP binding domain from the MuSK gene (i.e., MuSK-IgG KO) in mice at homeostasis leads to reduced PAX7+ cells, increased myonuclear number, and increase myofiber size, which may be due to a deficit in maintaining quiescence. Additionally, after BaCl2 injury, MuSK-IgG KO mice display accelerated repair after 7 days post-injury (dpi) in males only. Finally, RNA profiling using nCounter technology showed that MuSK-IgG KO MuSCs express genes that may be associated with the activated state.
Strengths:
Overall, the biology regulating MuSC quiescence is still relatively unexplored, and thus, this work provides a new mechanism controlling this process. The experiments discussed in the paper are technically sound with great complementary mouse models (full body versus tissue-specific mouse KO) used to validate their hypothesis. Additionally, the paper is well written with all the necessary information in the legends, methods, and figures being reported.
Weaknesses:
While the data largely supports the author's conclusions, I do have a few points to consider when reading this paper.
(1) For Figure 1, while I appreciate the author's confirming MuSK RNA and protein in MuSCs, I do think they should (a) quantify the RNA using qPCR and (b) determine the percentage of MuSCs expressing MuSK protein in their single fiber system in multiple biological replicates. This information will help us understand if MuSK is expressed in 1/10 or 10/10 PAX7-expressing MuSCs. Also, it will help place their phenotypes into the right context, especially when considering how much of the PAX7-pool is expressing MuSK from the beginning.
(2) Throughout the paper the argument is made that MuSK-IgG KO (full body and MuSC-specific KOs) are more activated and/or break quiescence more readily, but there is no attempt to test directly. Therefore, the authors should consider measuring the activation dynamics (i.e., break from quiescence) of MuSCs directly (EdU assays or live-cell imaging) in culture and/or in muscle in vivo (EdU assays) using their various genetic mouse models.
(3) For Figure 2, given that mice are considered adults by 3 months, it is really surprising how just two months later they are starting to see a phenotype (i.e., reduced PAX7-cells, increased number of myonuclei, and increased myofiber size)-which correlates with getting older. Given that aged MuSCs have activation defects (i.e., stuck somewhere in the quiescence cycle), a pending question is whether their phenotype gets stronger in aged mice, like 18-24 months. If yes, the argument that this pathway should be used in a therapeutic sense would be strengthened.
(4) For Figure 4, the same question as in point (2), the increase in fiber sizes by 7dpi in MuSK-IgG KO males is minimal (going from ~23 to 27 by eye) and no difference at a later time point when compared to WT mice. However, if older mice are used (18-24 months old) - which are known to have repair deficits-will the regenerative phenotype in MuSK-IgG KO mice be more substantial and longer lasting?
(5) For Figure 6, this gene set is not glaringly obvious as being markers of MuSC activation (i.e., no MyoD), so it's hard for the readers to know if this gene set is truly an activation signature. Also, the Shcherbina et al. data presented as a column with * being up or down (i.e. differentially expressed) is not helpful, since you don't know whether those mRNAs in that dataset are going up with the activation process. Addressing this point as well as my point (1) will further strengthen the author's conclusions about the MuSK-IgG KO MuSCs not being able to maintain quiescence as effectively.
Reviewer #2 (Public review):
Summary:
The work by Madigan et al. provides evidence that the signaling of BMPs via the Ig3 domain of MuSK plays a role during muscle postnatal development and regeneration, ultimately resulting in enhanced contractile force generation in the absence of the MuSK Ig3 domain. They demonstrate that MuSK is expressed in satellite cells initially post-isolation of muscle single fibers both in WT and whole-body deletion of the BMP binding domain of MuSK (ΔIg3-MuSK). In mice, ΔIg3-MuSK results in increased muscle fiber size, a reduction in Pax7+ cells, and increased muscle contractile force in 5-month-old, but not 3-month-old, mice. These data are complemented by a model in which the kinetics of regeneration appear to be accelerated at early time points. Of note, the authors demonstrate muscle tibialis anterior (TA) weights and fiber feret are increased in a Pax7CreERT2;MuSK-Ig3loxp/loxp model in which satellite cells specifically lack the MuSK BMP binding domain. Finally, using Nanostring transcriptional the authors identified a short list of genes that differ between the WT and ΔIg3-MuSK SCs. These data provide the field with new evidence of signaling pathways that regulate satellite cell activation/quiescence in the context of skeletal muscle development and regeneration.
On the whole, the findings in this paper are well supported, however additional validation of key satellite cell markers and data analysis need to be conducted given the current claims.
(1) The Pax7CreERT2;MuSK-Ig3loxp/loxp model is the appropriate model to conduct studies to assess satellite cell involvement in MuSK/BMP regulation. Validation of changes to muscle force production is currently absent using this model, as is quantification of Pax7+ tdT+ cells in 5-month muscle. Given that MuSK is also expressed on mature myofibers at NMJs, these data would further inform the conclusions proposed in the paper.
(2) All Pax7 quantification in the paper would benefit from high magnification images including staining for laminin demonstrating the cells are under the basal lamina.
(3) The nanostring dataset could be further analyzed and clarified. In Figure 6b, it is not initially apparent what genes are upregulated or downregulated in young and aged SCs and how this compares with your data. Pathway analysis geared toward genes involved in the TGFb superfamily would be informative.
(4) Characterizing MuSK expression on perfusion-fixed EDL fibers would be more conclusive to determine if MuSK is expressed in quiescent SCs. Additional characterization using MyoD, MyoG, and Fos staining of SCs on EDL fibers would help inform on their state of activation/quiescent.
(5) Finally, the treatment of fibers in the presence or absence of recombinant BMP proteins would inform the claims of the paper.
Reviewer #3 (Public review):
Summary:
Understanding the molecular regulation of muscle stem cell quiescence. The authors evaluated the role of the MuSK-BMP pathway in regulating adult SC quiescence by the deletion of the BMP-binding MuSK Ig3 domain ('ΔIg3-MuSK').
Strengths:
A novel mouse model to interrogate muscle stem cell molecular regulators. The authors have developed a nice mouse model to interrogate the role of MuSK signaling in muscle stem cells and myofibers and have unique tools to do this.
Weaknesses:
Only minor technical questions remain and there is a need for additional data to support the conclusions.
(1) The authors claim that dIg3-MuSK satellite cells break quiescence and start fusing, based on the reduction of Pax7+ and increase of nuclei/fiber (Fig 2-3), and maybe the gene expression (Fig6). However, direct evidence is needed to support these findings such as quantifying quiescent (Pax7+Ki67-) or activated (Pax7+Ki67+) satellite cells (and maybe proliferating progenitors Pax7-Ki67+) in the dIg3-MuSK muscle.
(2) It is not clear if the MuSK-BMP pathway is required to maintain satellite cell quiescence, by the end of the regeneration (29dpi), how Pax7+ numbers are comparable to the WT (Fig4d). I would expect to have less Pax7+, as in uninjured muscle. Can the authors evaluate this in more detail?
(2) Figure 4 claims that regeneration is accelerated, but to claim this at a minimum they need to look at MYH3+ fibers, in addition to fiber size.
(3) The Pax7 specific dIg3-MuSK (Fig5) is very exciting. However, it will be important to quantify the Pax7+ number. Could the authors check the reduction of Pax7+ in this model since it would confirm the importance of MuSK in quiescence?
(3) Rescue of the BMP pathway in the model would be further supportive of the authors' findings.
(4) Is the stem cell pool maintained long term in the deleted dIg3-MuSK SCs? Or would they be lost with extended treatment since they are reduced at the 5-month experiments? This is an important point and should be considered/discussed relevant to thinking about these data therapeutically.
(5) Without the Pax7-specific targeting, when you target dIg3-MuSK in the entire muscle, what happens to the neuromuscular nuclei?
(6) Why were differences seen in males and not females? Is XIST downregulation occurring in both sexes? Could the authors explain these findings in more detail?