Extraocular muscle stem cells exhibit distinct cellular properties associated with non-muscle molecular signatures

  1. Daniela Di Girolamo
  2. Maria Benavente-Diaz
  3. Melania Murolo
  4. Alexandre Grimaldi
  5. Priscilla Thomas Lopes
  6. Brendan Evano
  7. Mao Kuriki
  8. Stamatia Gioftsidi
  9. Vincent Laville
  10. Jean-Yves Tinevez
  11. Gaëlle Letort
  12. Sebastian Mella
  13. Shahragim Tajbakhsh
  14. Glenda Comai

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Abstract

Skeletal muscle stem cells (MuSCs) are recognised as functionally heterogeneous. Cranial MuSCs are reported to have greater proliferative and regenerative capacity when compared with those in the limb. A comprehensive understanding of the mechanisms underlying this functional heterogeneity is lacking. Here, we have used clonal analysis, live imaging and single cell transcriptomic analysis to identify crucial features that distinguish extraocular muscle (EOM) from limb muscle stem cell populations. A MyogeninntdTom reporter showed that the increased proliferation capacity of EOM MuSCs correlates with deferred differentiation and lower expression of the myogenic commitment gene Myod. Unexpectedly, EOM MuSCs activated in vitro expressed a large array of extracellular matrix components typical of mesenchymal non-muscle cells. Computational analysis underscored a distinct co-regulatory module, which is absent in limb MuSCs, as driver of these features. The EOM transcription factor network, with Foxc1 as key player, appears to be hardwired to EOM identity as it persists during growth, disease and in vitro after several passages. Our findings shed light on how high-performing MuSCs regulate myogenic commitment by remodelling their local environment and adopting properties not generally associated with myogenic cells.

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  1. Version published to 10.1242/dev.202144
  2. Version published to 10.1101/2023.03.10.532049v2 on bioRxiv
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    Reply to the reviewers

    Reply to Reviewers

    We thank both Reviewers for their comments which we believe could greatly improve the manuscript by adding more functional data. Part of the revision process has been already carried out and part of it is ongoing as detailed in the following sections.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)):

    Comments to the Authors In this manuscript, Girolamo et al., describe the differences in molecular signature and biological features of MuSC populations between extraocular (EOM) and limb (Tibialis anterior, TA) muscles. Comprehensive approaches including scRNA-seq, bioinformatics, and live-cell imaging reveal that EOM MuSCs is more proliferative in vitro and express ECM components at high levels as well as non-myogenic markers such as Foxc1 and Pdgfrb. The transcription factor network described in this study will characterize the identity of EOM MuSCs, providing insights into stem cell-based therapies for muscle diseases.

    Major comments

    1. In Figure 1A, EOM-MuSCs appear to form myotubes as efficiently as TA-MuSCs. If so, why myotube formation is not affected in EOM-MuSCs even with lower Myog expression compared to TA-MuSCs? The authors should discuss about this point.

    We thank the reviewer for raising this point. Actually, EOM myoblasts show a delay in activation of Myog that allows their sustained expansion, however Myog activation per se is not impaired. This is reflected by the presence of the EOM Differentiating cluster in our sc-RNAseq analysis which converges towards the TA Differentiating cluster and shows co-expression of Myog and canonical differentiation markers such as Myh3 and *Troponins *(Figure 2C, D). Accordingly, Stuelsatz et al. (Dev Biol 2015) showed efficient and robust differentiation of EOM myoblasts in vitro, while a higher fraction of self-renewed (Pax7+) cells was observed in long term cultures.

    To formally address this point, we propose to:

    • Isolate EOM and TA MuSCs from Tg:Pax7-nGFP;MyogtdTom mice, plate them at low density and quantify the tdTom+ cell proportions in short vs longer term cultures (D3 vs D5, D8).
    • Re-isolate the tdTom+ mononucleated fraction at D4-5 upon activation to:
    1. plate EOM and TA myoblasts at high density and assess their fusion index. Here, we will evaluated fusion independent of proliferation history and Myog activation.
    2. perform qRT-PCR for Myomixer/Myomaker on the tdTom+ mononucleated fraction to assess the extent of fusion potential.
    1. In Figure 2, activated EOM-MuSCs express Myod1e at lower levels compared to TA-MuSCs. Are MyoD protein levels are also lower in EOM-MuSCs?

    We have performed quantitation of the intensity of fluorescence for Myod at D3.5 in culture. In agreement with the sc-RNAseq in vitro activated dataset, we have noted lower Myod protein levels in EOM MuSCs. This data is now included as part of Suppl Fig 2A.

    In Figure 7E-G: Does the Foxc1 knockdown change the myogenic potential, especially on myotube formation? In addition to FOXC1 and Myog, the mRNA levels of Pax7 and Myod1 would be better to be provided.

    Previously, we used siRNA for short term silencing. As siRNA are diluted upon cell division, we now use lentivirus-mediated KD, which provides more consistent results. To address this point, we propose to use lentivirus carrying 3 different shRNAs for Foxc1 and address expression levels of Pax7, Myod, Myog together with EdU detection and assessment of myotube formation. Given that cell density influences myotube formation, we will pre-amplify the cells, replate them at high density, and silence Foxc1 concomitantly with induction of differentiation. These 3 shRNAs have been already validated in vitro and induce a massive reduction in EOM cell numbers. This data is now included as part of __Figure 7E-L. __

    Please state a reasonable explanation for the physiological role of high amounts of ECM produced by EOM-MuSCs.

    We regret that this explanation did not come across clearly on the manuscript. Previous studies have demonstrated a greater cellular output of cranial MuSCs in clonal assays in vitro (Ono Dev Biol 2010, Stuelsatz et al. Dev Biol 2015; Randolph et al. Stem Cells 2015) and better engraftment capacity in vivo (Stuelsatz et al. Dev Biol 2015). On the other hand, it has been shown that fetal MuSCs, which produce high amounts of extracellular matrix (ECM) cell autonomously, expand more efficiently and contribute more to muscle repair than the adult counterparts (Tierney et al. Cell Reports 2016). Finally, while in vitro expanded MuSCs show a reduced engraftment potential (Montarras et al. Science 2005, Ikemoto et al. Mol Therapy 2007), recapitulation of the endogenous niche ex vivo, allows maintenance of an undifferentiated proliferative state and their capacity to support regeneration in vivo (Ishii et al. Stem Cell Reports 2017). Therefore, we hypothesize that in vitro activated EOM MuSCs secrete high amounts of ECM to self-autonomously maintain stemness when removed from their niche. Alternatively, EOM MuSCs might contribute to connective tissue cells postnatally in vivo as we described previously in the embryo (Grimaldi et al. elife 2022). Secretion of ECM and expression of ECM-related regulons when activated in vivo might recapitulate this process.

    To address these hypotheses, we propose to:

    • Culture EOM and TA MuSCs, generate EOM and TA decellularized ECM (dECM) and test the proliferation/differentiation potentials of MuSCs on the dECM versus that on wells coated with Matrigel or Fibronectin alone. Shall this experiment not give conclusive results, we propose to assess the proliferation/differentiation potential of TA MuSCs on dishes coated with proteins present in the EOM ECM such as Sparc, Bgn, Mgp, Fbn1, Fn1, for which recombinant proteins exist.
    • Assess the expression levels of EOM-specific ECM proteins and TF regulons identified in vitro expressed in EOM MuSCs activated in vivo. To do so we are trying to optimize an EOM injury protocol based on previous observations highlighting the sensitivity of EOMs to anesthetics (Carlson et al. Arch Opthal 1985).
    • Use Pax7CreERT2:R26tdTom:PdgfraH2BGFP reporter mice to determine whether EOM MuSCs can give rise to muscle connective tissue postnatally. Minor comments :

    On p7, the 3rd line: "marker" is redundant.

    This has been corrected.

    On p9: what is meant by "force-directed environment"? Is this the aggregation of regulons and targets based on interaction strength determined by the algorithmic arrangement used by pySCENIC?

    A force-directed graph is a type of visualization technique where nodes are positioned based on the principles of physics that assign forces among the set of edges and the set of nodes. Spring like attractive forces are used to attract pairs of edges towards each other (connected nodes) while repulsive forces, like those of electrically charged particles, are used to separate all pairs of nodes. In the equilibrium state for this system, the edges tend to have uniform length (because of the spring forces), and nodes that are not connected by an edge tend to be drawn further apart (because of the electrical repulsion). This results in a layout that visually represents the relationships between the nodes, where each node (circle) is an active transcription factor and each edge (distance between nodes) is an inferred regulation between 2 transcription factors.

    The text has been changed accordingly.

    On p11: add space between (Sambasivan et al., 20009a)and.

    Corrected

    On p11: 1.7 fold increase → 2.7 fold?

    Corrected

    On p13: please describe the GRN abbreviation since it is the first used here and not clarified beforehand.

    Corrected to gene regulatory network (GRN)

    On p15: add space between (Vallejo et al., 2018)Other.

    Corrected

    On p16: add "a" after "Klf4 is" → Klf4 is a pioneer...

    Corrected

    In Fig1: it would be better to show the quantification of EdU incorporation, especially at D5 for highlighting the difference between EOM and TA

    This has been done and now included Figure 1B.

    In Fig1: MF20 staining seemingly describes larger myotubes in EOM compared with TA at D5. Is this most likely due to the higher number of cells to start with in EOM rather than having more fusion ability?

    Indeed, as discussed in the Main Comments (question 1), the fusogenic ability of EOM an TA MuSCs will be addressed by plating the same number of cells at high density.

    In Fig 2E,F: some font is very faint in color and hard to read in printed format e.g. "TP53 regulates transcription of". May want to change color. In addition, the bottom edge of the Figure is slightly cut off.

    We have enhanced the color of some words so all terms are properly seen. The figure has been adjusted in order for the bottom edge to be seen.

    In Fig 4D: COLVI should be changed to COLIV.

    Corrected

    In Fig 5B: Myod1 is redundant.

    Actually Myod as regulon (Myod1_(+)) in Fig 5B is not redundant. It is written twice as it is a regulon of both EOM Diff and TA Diff.

    In Fig 6D; please specify "Amp". Is Hey1 a myogenic marker?

    Amp stands for Activated/Amplified cells. We have changed this to Act to keep consistency.

    Hey1 is a bHLH transcription factor that is required in a cell-autonomous manner for maintenance of MuSCs (Noguchi et al. Development 2019). This information has now been added to the text.

    In Fig 7F; there are Ctrl and control- please unify them.

    Corrected

    In Fig7C,D: When does Foxc1 start to be expressed in EOM progenitors in the embryo? If the authors tested, please mention it.

    Foxc1 is a DEG and top regulon of EOM progenitors in the early embryo (E11.5, Grimaldi et al. elife 2022). We will formally address this point by immunostaining on tissue sections of E10.5, E12.5 and E14.5 embryos.

    In Supp Fig 7A: graphs are lacking color-coded legends.

    Corrected

    In Supp Fig 7C: Font is very small and illegible in a paper format.

    Corrected. For networks it is sometimes difficult to get bigger font size.

    In Supp Fig 7F: what are the dynamics of PDGFβ+ cell populations in successive passages in culture? If the authors tested please mention it.

    We have been that Pdgfrb expression increases upon passages (Fig 6D). However, this experiment does not tell us whether there is a higher fraction of PDGFRβ+ cells or a similar fraction compared to Day 3 but expressing higher levels of the protein. To distinguish these possibilities, we propose to assess the PDGFRβ+ fraction by FACS upon passages in culture.

    In Supp Fig 8 A: Are the colors reversed for EOM? In Fig 8 E, EOM progenitor and differentiation stream are light blue and yellow respectively but in Supp 8A the colors are flipped and don't seem to match the Map laid out in Fig 8E.

    The scvelo pipeline will be re-run to correct this error on the graphical output.

    Some references are listed as redundant.

    Corrected

    Reviewer #1 (Significance (Required)):

    Skeletal muscle stem cells (MuSCs) play an indispensable role in muscle regeneration in adults. MuSCs are distributed in all muscles throughout the body and their function and molecular properties are diverse among muscles. Extraocular muscles (EOMs) are known to be preferentially spared in muscular dystrophies and during aging. In addition, EOM-derived MuSCs are highly transplantable compared to those of limb muscles. However, intrinsic regulators of EOM MuSCs have not been fully characterized yet. This study by Girolamo et al. shows the differences in molecular signature and biological features of MuSC populations between EOM and limb (Tibialis anterior, TA) muscles. Comprehensive approaches including scRNA-seq, bioinformatics, and live-cell imaging revealed that a subset of the EOM-derived MuSC population is highly proliferative and expresses extracellular matrix components at high levels. The analysis also shows that EOM-derived MuSCs have non-myogenic signatures such as Foxc1 and Pdgfrb. A transcriptional factor Foxc1 is described as a pro-mitogenic factor in the cancer field and is known as a driver of endothelial/smooth muscle fate. In this study, the authors find that Foxc1 is expressed in EOM MuSCs but not TA MuSCs. A siRNA-mediated gene silencing study shows that Foxc1 is important for the population expansion of EOM MuSCs. Furthermore, the authors demonstrated that the EOM MuSCs contain a PDGFRβ+ve cell population that is more proliferative and less myogenic compared to a PDGFRβ-ve cell population. Altogether, this study provides new insights into the regional differences in MuSCs and will contribute to the development of stem cell-based therapies for muscle-wasting diseases including muscular dystrophies and age-related sarcopenia.

    We appreciate the assessment of the Reviewer noting the new insights that our work provides on muscle stem cell heterogeneity.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): Benavente-Diaz et al. address a question of why do progenitors isolated from extra ocular muscles have higher proliferative and regenerative capacity compared to progenitors from limb muscles. They perform transcriptomic profiling of the two progenitor populations and report which genes and functional groups are differentially expressed. They perform multiple bioinformatic analyses aimed at providing insights into which differentially expressed genes may be master regulators of these differences.

    Major comments:

    Many conclusions in this paper are based on bioinformatic analyses with little experimental data provided as a confirmation. Overall, to increase significance of the manuscript I would suggest expanding the experimental validation part of the study. By following up on the findings from the bioinformatic analyses and confirming them in vitro this manuscript could move from beyond preliminary and only potentially interesting.

    The previous version of our manuscript relied on bioinformatics analysis of single cell transcriptomics and some experimental validations to investigate muscle stem cell heterogeneity. Now, we performed loss of function experiments in EOM cells and gain of function experiments in TA cells using lentiviruses to validate those points mechanistically (Figure 7E-L).

    Authors suggest a number of TFs and ECM components as master regulators of progenitor identity. An experiment of a rather limited scope was provided in Figure 7E-F, where effects of silencing TF Foxc1 on EOM progenitor proliferation was assessed. It would be highly beneficial to expand these experiments to include other TFs found in their dataset. Importantly, by overexpressing the proposed master regulators in TA progenitors authors should investigate whether these TFs indeed confer higher proliferative and regenerative capacity. Otherwise, the authors should make it clear that their conclusions about TFs regulating or maintaining EOM progenitors are preliminary and based on bioinformatic analyses.

    Indeed, these experiments are necessary to consolidate the bioinformatic analysis and provide further mechanistic insights on this point. We have obtained lentiviruses and optimized gain of function assays in TA cells. Gain of function experiments with Creb3l1 and Dmrta2, two other top regulon transcription factors have been planned besides the experiments already included with Foxc1.

    Minor comments:

    In Figure 2, authors describe single-cell RNAseq analysis of EOM and TA progenitors. They report a number of markers differentially expressed between these two populations. It would be good to perform higher-resolution subclustering of each these populations to understand whether markers are expressed in all EOM progenitors or whether there is a specific subpopulation that is characterized by Mgp/Bgn/... expression. A paper by Yartseva et al. (Cell Reports 2020) did describe a population of activated satellite cells that express extracellular matrix components. In addition, from the data presented it is not clear whether proposed EOM markers are uniquely expressed or only enriched in EOM progenitors.

    Actually, In Figure 2 we are showing what the reviewer is requesting. The heatmap highlights the presence of subpopulations (Progenitor/Differentiating) in both EOM and TA. The 4-way heatmap in Figure 2D is showing the distinctly upregulated genes of each subcluster. Matrix related genes are indeed expressed by a subpopulation of EOM cells, that we identified as "Progenitors". Two of those, Bgn and Mgp, can be seen in Figure 3H. Moreover, in Figure 4B we show the Average Expression and Percentage of cells expressing certain ECM component.

    Actinomycin D is commonly used in single cell preparations for RNAseq to mitigate stress gene activation due to isolation procedure (for example see Wu YE et al. Neuron 2017). It is possible that EOM progenitors are particularly responsive to the isolation procedure, which would imply that stress genes are not involved in EOM progenitor maintenance, but that their expression is an artifact of isolation. These experiments should be repeated with actinomycin D to exclude potential artifacts.

    While this is a possibility, the likelihood of this is relatively low as activated cells were obtained by quick trypsinization of cells. To formally exclude the problem mentioned by the reviewer we will perform qRT-PCR for EOM markers on activated cells at Day 3 that were fixed in PFA or treated with Actinomycin D prior to trypsinization. Moreover, in Suppl Figure 5G-E, we already used the stress index calculation defined by Machado et al. 2020 and this does not seem to affect our activated dataset.

    It is unclear why authors chose to focus on PdgfrB+ population in Figure 7. Was it chosen as a target of Foxc1 or as a marker of proliferative cells? Any other reason? This should be explained as well as significance of this part in connection to the rest of the article.

    We decided to focus on Pdgfrb for several reasons stated in the text: 1) it is a component of the matrisome we have validated; 2) it stands out in the Reactome pathways/Molecular function analysis; 3) it is a target of Foxc1; 4) it is a marker of cells with mesenchymal characteristics. We also chose this marker, for which antibodies for FACs exist, as proof of principle validation of the EOM mesenchymal phenotype. Similarly, sc-RNAseq analysis of human muscle, used Cav1 antibodies to isolate a functionally different subpopulation of MuSCs (Barruet elife 2020). To make the flow of the manuscript more consistent this data is now compiled on Figure 4.

    Please explain the significance and relevance of the results presented in Figure 8A-C.

    These figures highlight the potential of EOM progenitors to specifically regulate matrisome genes with respect to the TA. The 90 top active regulons of the EOM potentially regulate more matrisome genes than the TA, and the ratio (Number of EOM presumptive regulations/ Number of TA presumptive regulations) peaks when looking at the top 5 regulons (a 3-fold difference), and progressively reduces.

    Prior studies are (not) referenced appropriately

    We have corrected reference duplications.

    Figures contain small fonts that are not legible. In particular, networks such as the one in Fig 5C are difficult to read.

    The font size, font color and/or size of networks has been changed for Fig 2E-F, 5C-D, 8G-J and Suppl Fig 2A, 7C.

    General assessment: Functional differences between EOM and TA progenitors have been previously described, but a deep mechanistic understanding of the underlying molecular pathways is lacking. This manuscript takes a step towards elucidating these molecular pathways.

    We now provide more mechanistic data regarding the differential role that Foxc1 plays in extraocular compared to limb myogenic cells. Our revised plan will consolidate and extend these observations.

    Advance: Tajbakhsh group recently published a paper Evano et al. Plos Genetics 2020, that also explored the differences between EOM and TA stem cells. Among other things this paper showed that EOM stem cells have intrinsic molecular mechanisms/programs that differentiate them from TA stem cells. In that context, the experimental insights that Benavente-Diaz et al. provide are not novel. Benavente-Diaz et al. would extend the knowledge in the field if they experimentally confirmed that the proposed master regulators indeed determine progenitor proliferation and regeneration capacity.

    We respectfully disagree regarding the novelty of our work. In our previous study cited above, we showed that EOM stem cells have in vivo transcriptional differences with those in the limb (bulk RNAseq) and they can adopt the fate of limb cells when transplanted into the limb. Interestingly, full “reprogramming” was not achieved in those experiments pointing to limited plasticity.

    Here, we show by scRNAseq and bioinformatic analysis of regulons that Foxc1 and extracellular matrix signature are features that are unique to EOM stem cells. Also, our overall single cell transcriptome analysis and comparative studies show that EOM muscle stem cells have adopted a signature that overlaps with mesenchymal stem cells – a property that has to date not been reported.

    Muscle is arguably the best system to investigate stem cell heterogeneity, and our study provides mechanistic insights into the extend of this diversity.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary:

    Benaverte-Diaz et al. address a question of why do progenitors isolated from extra ocular muscles have higher proliferative and regenerative capacity compared to progenitors from limb muscles. They perform transcriptomic profiling of the two progenitor populations and report which genes and functional groups are differentially expressed. They perform multiple bioinformatic analyses aimed at providing insights into which differentially expressed genes may be master regulators of these differences.

    Major comments:

    • Many conclusions in this paper are based on bioinformatic analyses with little experimental data provided as a confirmation. Overall, to increase significance of the manuscript I would suggest expanding the experimental validation part of the study. By following up on the findings from the bioinformatic analyses and confirming them in vitro this manuscript could move from beyond preliminary and only potentially interesting.
    • Authors suggest a number of TFs and ECM components as master regulators of progenitor identity. An experiment of a rather limited scope was provided in Figure 7E-F, where effects of silencing TF Foxc1 on EOM progenitor proliferation was assessed. It would be highly beneficial to expand these experiments to include other TFs found in their dataset. Importantly, by overexpressing the proposed master regulators in TA progenitors authors should investigate whether these TFs indeed confer higher proliferative and regenerative capacity. Otherwise, the authors should make it clear that their conclusions about TFs regulating or maintaining EOM progenitors are preliminary and based on bioinformatic analyses.

    Minor comments:

    • In Figure 2, authors describe single-cell RNAseq analysis of EOM and TA progenitors. They report a number of markers differentially expressed between these two populations. It would be good to perform higher-resolution subclustering of each these populations to understand whether markers are expressed in all EOM progenitors or whether there is a specific subpopulation that is characterized by Mgp/Bgn/... expression. A paper by Yartseva et al. (Cell Reports 2020) did describe a population of activated satellite cells that express extracellular matrix components. In addition, from the data presented it is not clear whether proposed EOM markers are uniquely expressed or only enriched in EOM progenitors.
    • Actinomycin D is commonly used in single cell preparations for RNAseq to mitigate stress gene activation due to isolation procedure (for example see Wu YE et al. Neuron 2017). It is possible that EOM progenitors are particularly responsive to the isolation procedure, which would imply that stress genes are not involved in EOM progenitor maintenance, but that their expression is an artifact of isolation. These experiments should be repeated with actinomycin D to exclude potential artifacts.
    • It is unclear why authors chose to focus on PdgfrB+ population in Figure 7. Was it chosen as a target of Foxc1 or as a marker of proliferative cells? Any other reason? This should be explained as well as significance of this part in connection to the rest of the article.
    • Please explain the significance and relevance of the results presented in Figure 8A-C.
    • Prior studies are referenced appropriately
    • Figures contain small fonts that are not legible. In particular, networks such as the one in Fig 5C are difficult to read.

    Significance

    • General assessment: Functional differences between EOM and TA progenitors have been previously described, but a deep mechanistic understanding of the underlying molecular pathways is lacking. This manuscript takes a step towards elucidating these molecular pathways.
    • Advance: Tajbakhsh group recently published a paper Evano et al. Plos Genetics 2020, that also explored the differences between EOM and TA stem cells. Among other things this paper showed that EOM stem cells have intrinsic molecular mechanisms/programs that differentiate them from TA stem cells. In that context, the experimental insights that Benaverte-Diaz et al. provide are not novel. Benaverte-Diaz et al. would extend the knowledge in the field if they experimentally confirmed that the proposed master regulators indeed determine progenitor proliferation and regeneration capacity.
    • Audience: basic research, translational. This research would be of interest to regenerative therapy field.

    My field of expertise: molecular and cellular biology, muscle regeneration. I am not a bioinformatician and I am not able to technically evaluate in silico approaches used in this study.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript, Girolamo et al., describe the differences in molecular signature and biological features of MuSC populations between extraocular (EOM) and limb (Tibialis anterior, TA) muscles. Comprehensive approaches including scRNA-seq, bioinformatics, and live-cell imaging reveal that EOM MuSCs is more proliferative in vitro and express ECM components at high levels as well as non-myogenic markers such as Foxc1 and Pdgfrb. The transcription factor network described in this study will characterize the identity of EOM MuSCs, providing insights into stem cell-based therapies for muscle diseases.

    Major comments

    1. In Figure 1A, EOM-MuSCs appear to form myotubes as efficiently as TA-MuSCs. If so, why myotube formation is not affected in EOM-MuSCs even with lower Myog expression compared to TA-MuSCs? The authors should discuss about this point.
    2. In Figure 2, activated EOM-MuSCs express Myod1e at lower levels compared to TA-MuSCs. Are MyoD protein levels are also lower in EOM-MuSCs?
    3. In Figure 7E-G: Does the Foxc1 knockdown change the myogenic potential, especially on myotube formation? In addition to FOXC1 and Myog, the mRNA levels of Pax7 and Myod1 would be better to be provided.
    4. The authors mention that Foxc1 maintains EOM MuSCs in a progenitor-like state through matrix remodeling and cooperation with other TFs. However, such functional analysis does not seem to be provided sufficiently. For example: does Foxc1 siRNA lower matrisome and Pdgfrb genes in EOM-MuSCs; does forced expression of Foxc1in TA-MuSCs acquire the EOM-like state? Although the authors can predict the role and function of Foxc1 in the MuSC population based on the pySCENIC and previous studies, the experimental approach by the authors would be more convincing. The reviewer would like to emphasize that these experiments are not entirely necessary in this manuscript.
    5. Please state a reasonable explanation for the physiological role of high amounts of ECM produced by EOM-MuSCs.

    Minor comments

    On p7, the 3rd line: "marker" is redundant.

    On p9: what is meant by "force-directed environment"? Is this the aggregation of regulons and targets based on interaction strength determined by the algorithmic arrangement used by pySCENIC?

    On p11: add space between (Sambasivan et al., 20009a)and.

    On p11: 1.7 fold increase → 2.7 fold?

    On p13: please describe the GRN abbreviation since it is the first used here and not clarified beforehand.

    On p15: add space between (Vallejo et al., 2018)Other.

    On p16: add "a" after "Klf4 is" → Klf4 is a pioneer...

    In Fig1: it would be better to show the quantification of EdU incorporation, especially at D5 for highlighting the difference between EOM and TA.

    In Fig1: MF20 staining seemingly describes larger myotubes in EOM compared with TA at D5. Is this most likely due to the higher number of cells to start with in EOM rather than having more fusion ability?

    In Fig 2E,F: some font is very faint in color and hard to read in printed format e.g. "TP53 regulates transcription of". May want to change color. In addition, the bottom edge of the Figure is slightly cut off.

    In Fig 4D: COLVI should be changed to COLIV.

    In Fig 5B: Myod1 is redundant.

    In Fig 6D; please specify "Amp". Is Hey1 a myogenic marker?

    In Fig 7F; there are Ctrl and control- please unify them.

    In Fig7C,D: When does Foxc1 start to be expressed in EOM progenitors in the embryo? If the authors tested, please mention it.

    In Supp Fig 7A: graphs are lacking color-coded legends.

    In Supp Fig 7C: Font is very small and illegible in a paper format.

    In Supp Fig 7F: what are the dynamics of PDGFβ+ cell populations in successive passages in culture? If the authors tested please mention it.

    In Supp Fig 8 A: Are the colors reversed for EOM? In Fig 8 E, EOM progenitor and differentiation stream are light blue and yellow respectively but in Supp 8A the colors are flipped and don't seem to match the Map laid out in Fig 8E.

    Some references are listed as redundant.

    Significance

    Skeletal muscle stem cells (MuSCs) play an indispensable role in muscle regeneration in adults. MuSCs are distributed in all muscles throughout the body and their function and molecular properties are diverse among muscles. Extraocular muscles (EOMs) are known to be preferentially spared in muscular dystrophies and during aging. In addition, EOM-derived MuSCs are highly transplantable compared to those of limb muscles. However, intrinsic regulators of EOM MuSCs have not been fully characterized yet. This study by Girolamo et al. shows the differences in molecular signature and biological features of MuSC populations between EOM and limb (Tibialis anterior, TA) muscles. Comprehensive approaches including scRNA-seq, bioinformatics, and live-cell imaging revealed that a subset of the EOM-derived MuSC population is highly proliferative and expresses extracellular matrix components at high levels. The analysis also shows that EOM-derived MuSCs have non-myogenic signatures such as Foxc1 and Pdgfrb. A transcriptional factor Foxc1 is described as a pro-mitogenic factor in the cancer field and is known as a driver of endothelial/smooth muscle fate. In this study, the authors find that Foxc1 is expressed in EOM MuSCs but not TA MuSCs. A siRNA-mediated gene silencing study shows that Foxc1 is important for the population expansion of EOM MuSCs. Furthermore, the authors demonstrated that the EOM MuSCs contain a PDGFRβ+ve cell population that is more proliferative and less myogenic compared to a PDGFRβ-ve cell population. Altogether, this study provides new insights into the regional differences in MuSCs and will contribute to the development of stem cell-based therapies for muscle-wasting diseases including muscular dystrophies and age-related sarcopenia.

  6. Version published to 10.1101/2023.03.10.532049v1 on bioRxiv