Deletion of Fibroblast growth factor 9 globally and in skeletal muscle results in enlarged tuberosities at sites of deltoid tendon attachments

  1. Connor C. Leek
  2. Jaclyn M. Soulas
  3. Iman Bhattacharya
  4. Elahe Ganji
  5. Ryan C. Locke
  6. Megan C. Smith
  7. Jaysheel D. Bhavsar
  8. Shawn W. Polson
  9. David M. Ornitz
  10. Megan L. Killian

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Abstract

The growth of most bony tuberosities, like the deltoid tuberosity (DT), rely on the transmission of muscle forces at the tendon-bone attachment during skeletal growth. Tuberosities distribute muscle forces and provide mechanical leverage at attachment sites for joint stability and mobility. The genetic factors that regulate tuberosity growth remain largely unknown. In mouse embryos with global deletion of fibroblast growth factor 9 ( Fgf9 ), the DT size is notably enlarged. In this study, we explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9 . We showed that cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9 . Loss of Fgf9 during embryonic growth led to increased chondrocyte hypertrophy and reduced cell proliferation at the DT attachment site. This endured hypertrophy and limited proliferation may explain the abnormal mineralization patterns and locally dysregulated expression of markers of endochondral development in Fgf9 null attachments. We then showed that targeted deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. Taken together, we discovered that Fgf9 may play an influential role in muscle-bone crosstalk during embryonic and postnatal development.

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    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    We thank the reviewers for their constructive and critical feedback on our original manuscript.

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): In this study, the authors explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. They found cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. However, the innovation of this paper is not enough, the phenotypes of global deletion of Fgf9 were previously reported, most of the data in this paper are mainly descriptive analysis of the phenotypes, and internal cellular and molecular mechanisms were not well investigated.

    Here are the major issues:

    1.The data showed that fewer osteoclasts were present at both E16.5 and P0 in Figure 2R, V. Whether FGF9 affects both osteogenesis and osteoclast formation?

    • __Authors’ response to Reviewer: __*Thank you for your feedback. We revised this manuscript to reflect the concerns of Reviewer 1 related to the lack of cellular and molecular mechanisms as described below. **Based on this question from the Reviewer, we have revised our discussion to clarify our findings as follows: * “From our EdU proliferation assays, we observed a decline in cell proliferation in Fgf9null attachments, suggesting an accelerated chondrocyte maturation. Though we saw similar levels of Pthlh expression (a chondrocyte hypertrophy suppressor) in both WT and Fgf9null attachments, we also saw increased expression of Gli1 (a marker of chondrocyte hypertrophy) localized to the attachment in Fgf9null embryos compared to WT embryos. This decrease in proliferation was in parallel with increased hypertrophy of chondrocytes adjacent to the attachment cells within the Fgf9null DT, which may have led to a rapid expansion of matrix in the DT. Even though the DT was enlarged in Fgf9null mutants, we found fewer Sost+ cell clusters in their DTs compared to WT mice. Mature osteocytes express Sost (Winkler et al., 2003), and fewer Sost+ cells may indicate an impaired ability of Fgf9null osteoblasts to embed and mature into osteocytes. Overexpression of FGF9 in the perichondrium has been previously shown to suppress chondrocyte proliferation and limit bone growth in the limb (Karuppaiah et al., 2016); in our study, we found that loss of Fgf9 globally leads to an accelerated enlargement of chondrocytes in the tuberosity. This accelerated enlargement may limit the ability of these cells to deposit matrix and mineral and therefore limit osteocyte differentiation. We also found fewer osteoclasts in the Fgf9null DT which mirrors previous reports using the same mutation to study the length and vascularity of developing limb (Hung et al., 2007). Because the DT is enlarged and resides on the surface of a shortened bone, this phenotype may elucidate a divergent role of FGF9 in patterning of an arrested (e.g., attachment) growth plate compared to a regular (e.g., long bone) growth plate. This includes unexplored roles of FGF9 in vascularity of the tendon attachment and formation of bone ridges that overlap with or deviate from its role in growth plate development that are beyond the scope of the current study.”
    1. RNA-sequencing analysis showed the decreased expression of mitochondria/ energy and lipid associated genes in Fgf9 null muscle compared to WT muscle, how does this relate to the enlargement of the DT? What are the detailed molecular mechanisms?
    • __Authors’ response to Reviewer: __
    • Based on this question from the Reviewer, we have revised our discussion to reflect the potential molecular mechanisms related to muscle mitochondria, fiber type, and metabolism as follows:

    “Fgf9 is expressed in muscle during embryonic stages, which we and others have observed using ISH (Colvin et al., 1999; Garofalo et al., 1999; Hung et al., 2007; Yang and Kozin, 2009). Previous work has established a connection between Fgf9 and muscle, as treatment of muscle and muscle progenitor cells with FGF9 slows maturation, enhances proliferation, and decreases expression of various myogenic genes (Huang et al., 2019). This study found supporting evidence that Fgf9 expression in muscle may be a limiting factor in tuberosity growth. However, it remains unknown how other FGFs and their receptors, FGFRs, regulate superstructure and attachment formation. In this study, we identified potential mediators of skeletal muscle metabolism in Fgf9null muscle, including downregulated mitochondrial-related genes associated with oxidative respiration and proton transport (i.e., Slc36a2 and Ucp1, amongst others). In cultured myoblasts, FGF9 can inhibit myogenic differentiation potentially via increased production of Myostatin (Huang et al., 2019), a well-established mediator of fast glycolytic muscle fibers (Girgenrath et al., 2005; Hennebry et al., 2009). While the role of FGF9 in myoblast fusion has been investigated in vitro, its effect on muscle fiber type and fiber metabolism (i.e., oxidative vs. glycolytic) has not yet been explored. Our findings from bulk RNA-seq of Fgf9null muscle point to potential mechanisms in muscle metabolism that may contribute to the enlarged phenotype that is mimetic of that found in Myostatin deficient mice and other animals (Elkasrawy and Hamrick, 2010; Hamrick et al., 2002). Additionally, further investigations are needed to investigate the potential role of Fgf9 in mitochondrial function and lipid metabolism. Recent work by Huang et al. also identified FGF9 as a potent regulator of calcium signaling and homeostasis in myoblast culture in vitro, and calcium release from the sarcoplasmic reticulum in muscle plays a critical role during embryonic skeletal myogenesis via ryanodine receptor 1 (RYR1). Although Ryr1 was not significantly different in between Fgf9null and WT muscle in the present study, we did find that calmodulin-associated genes (e.g., Calm4, Calml3, Camsap3, Calm5) were all significantly upregulated in Fgf9null muscle compared to WT muscle. Calmodulin interacts with RYR1 and its activation is required for intracellular binding of calcium (Newman et al., 2014, 1). Calmodulin is a crucial component of the calcium signal transduction pathway and also plays an important role in lipid and glucose metabolism (Nishizawa et al., 1988). Taken together, our findings along with recent work by Huang et al. support more mechanistic studies to investigate the metabolic effects of loss and gain of function of Fgf9 on skeletal muscle as well as the muscle secretome.”

    Reviewer #1 (Significance (Required)):

    R1 The authors compared the phenotypes between globally and muscle-specifically deletion of Fgf9 in mice, and found that Fgf9 secreted by muscle may induced the enlargement of the DT. However, the detailed molecular mechanisms were not well investigated.

    **Referees cross-commenting**

    R2 I do not disagree with Rev 1, but I do not think such a task is so trial reason why I don't suggest; it could take years to determine molecular mechanisms of anything. The authors could expand the discussion, offer some possibilities. If they had some RNAseq data they maybe could suggest some of the key signaling pathways involved.

    **Referees cross-commenting**

    R1 We still suggested that the internal cellular and molecular mechanisms should be well investigated in this papaer.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)):

    • This paper deals with an important topic which is exact molecular mechanisms regulating the growth of bony tuberosities; because this region is essential for force transmission and movement.
    • Based on the previous information they had that in the global KO of the gene FGF9 the deltoid muscle is enlarged; and this muscle is in a very important tuberosity; they decided to look at FGF9 as a potential genetic regulator.
    • The manuscript is clear, objective, concise. Very clear. Authors used both the global and targeted deletions, very high reproducibility. Reviewer #2 (Significance (Required)):
    • This manuscript advances several areas since we know little about the mechanisms controlling local mechanisms of tuberosities. It also advances our knowledge of FGF9. There were several studies before mostly in vitro showing that FGF9 when added to muscle cells could arrest myogenesis, but the types of experiments in vivo had not been performed yet. The authors used an array of methods; the studies are unbiased and very rigorous and also they always show all experimental points, which is excellent. The conclusions are supported by the data.
    • The main suggestion for authors: They essentially do not discuss the nature of the potential muscle to bone signaling occurring when they target the deletion of FGF9 in skeletal muscles and muscles enlarge and there is a series of adaptions in the tuberosity. Do the authors believe this to be all the genetic changes or potentially through secreted myokines? In the paper of Huang et al, 2019 the authors document an effect of FGF9 in intracellular calcium homeostasis/signaling; could this be part of the mechanism? Perhaps the authors could propose a model?

    __Authors’ response to Reviewer: __

    • *Future studies could investigate the secretome of muscle in Fgf9null or muscle-specific knockouts, as well as assess calcium signaling homeostasis in Fgf9 mutant muscles. We did find calcium- and ion-associated genes in the RNAseq and revised the discussion to include this information. *
    • Based on this question from the Reviewer, we have revised our discussion to reflect the potential molecular mechanisms related to muscle mitochondria, fiber type, and metabolism as follows: “Fgf9 is expressed in muscle during embryonic stages, which we and others have observed using ISH (Colvin et al., 1999; Garofalo et al., 1999; Hung et al., 2007; Yang and Kozin, 2009). Previous work has established a connection between Fgf9 and muscle, as treatment of muscle and muscle progenitor cells with FGF9 slows maturation, enhances proliferation, and decreases expression of various myogenic genes (Huang et al., 2019). This study found supporting evidence that Fgf9 expression in muscle may be a limiting factor in tuberosity growth. However, it remains unknown how other FGFs and their receptors, FGFRs, regulate superstructure and attachment formation. In this study, we identified potential mediators of skeletal muscle metabolism in Fgf9null muscle, including downregulated mitochondrial-related genes associated with oxidative respiration and proton transport (i.e., Slc36a2 and Ucp1, amongst others). In cultured myoblasts, FGF9 can inhibit myogenic differentiation potentially via increased production of Myostatin (Huang et al., 2019), a well-established mediator of fast glycolytic muscle fibers (Girgenrath et al., 2005; Hennebry et al., 2009). While the role of FGF9 in myoblast fusion has been investigated in vitro, its effect on muscle fiber type and fiber metabolism (i.e., oxidative vs. glycolytic) has not yet been explored. Our findings from bulk RNA-seq of Fgf9null muscle point to potential mechanisms in muscle metabolism that may contribute to the enlarged phenotype that is mimetic of that found in Myostatin deficient mice and other animals (Elkasrawy and Hamrick, 2010; Hamrick et al., 2002). Additionally, further investigations are needed to investigate the potential role of Fgf9 in mitochondrial function and lipid metabolism. Recent work by Huang et al. also identified FGF9 as a potent regulator of calcium signaling and homeostasis in myoblast culture in vitro, and calcium release from the sarcoplasmic reticulum in muscle plays a critical role during embryonic skeletal myogenesis via ryanodine receptor 1 (RYR1). Although Ryr1 was not significantly different in between Fgf9null and WT muscle in the present study, we did find that calmodulin-associated genes (e.g., Calm4, Calml3, Camsap3, Calm5) were all significantly upregulated in Fgf9null muscle compared to WT muscle. Calmodulin interacts with RYR1 and its activation is required for intracellular binding of calcium (Newman et al., 2014, 1). Calmodulin is a crucial component of the calcium signal transduction pathway and also plays an important role in lipid and glucose metabolism (Nishizawa et al., 1988). Taken together, our findings along with recent work by Huang et al. support more mechanistic studies to investigate the metabolic effects of loss and gain of function of Fgf9 on skeletal muscle as well as the muscle secretome.

    In conclusion, this work established a new role of skeletal muscle derived Fgf9 during skeletal development and tuberosity growth. Additionally, our unbiased transcriptomic approaches and rigorous analyses identified new potential mechanisms associated with muscle development, mitochondrial bioenergetics, and muscle metabolism that warrant further investigation into the role of FGF9 in muscle-bone crosstalk.”

  2. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #2

    Evidence, reproducibility and clarity

    This paper deals with an important topic which is exact molecular mechanisms regulating the growth of bony tuberosities; because this region is essential for force transmission and movement. Based on the previous information they had that in the global KO of the gene FGF9 the deltoid muscle is enlarged; and this muscle is in a very important tuberosity; they decided to look at FGF9 as a potential genetic regulator.

    The manuscript is clear, objective, concise. Very clear. Authors used both the global and targeted deletions, very high reproducibility.

    Significance

    This manuscript advances several areas since we know little about the mechanisms controlling local mechanisms of tuberosities. It also advances our knowledge of FGF9. There were several studies before mostly in vitro showing that FGF9 when added to muscle cells could arrest myogenesis, but the types of experiments in vivo had not been performed yet. The authors used an array of methods; the studies are unbiased and very rigorous and also they always show all experimental points, which is excellent. The conclusions are supported by the data.

    The main suggestion for authors: They essentially do not discuss the nature of the potential muscle to bone signaling occurring when they target the deletion of FGF9 in skeletal muscles and muscles enlarge and there is a series of adaptions in the tuberosity. Do the authors believe this to be all the genetic changes or potentially through secreted myokines? In the paper of Huang et al, 2019 the authors document an effect of FGF9 in intracellular calcium homeostasis/signaling; could this be part of the mechanism? Perhaps the authors could propose a model?

    Referees cross-commenting

    I do not disagree with Rev 1, but I do not think such a task is so trial reason why I don't suggest; it could take years to determine molecular mechanisms of anything. The authors could expand the discussion, offer some possibilities. If they had some RNAseq data they maybe could suggest some of the key signaling pathways involved.

  3. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

    Learn more at Review Commons

    Referee #1

    Evidence, reproducibility and clarity

    In this study, the authors explored the tissue-specific regulation of DT size using both global and targeted deletion of Fgf9. They found cell hypertrophy and mineralization dynamics of the DT, as well as transcriptional signatures from skeletal muscle but not bone, were influenced by the global loss of Fgf9. Deletion of Fgf9 in skeletal muscle leads to postnatal enlargement of the DT. However, the innovation of this paper is not enough, the phenotypes of global deletion of Fgf9 were previously reported, most of the data in this paper are mainly descriptive analysis of the phenotypes, and internal cellular and molecular mechanisms were not well investigated.

    Here are the major issues:

    1.The data showed that fewer osteoclasts were present at both E16.5 and P0 in Figure 2R, V. Whether FGF9 affects both osteogenesis and osteoclast formation?

    2.RNA-sequencing analysis showed the decreased expression of mitochondria/ energy and lipid associated genes in Fgf9 null muscle compared to WT muscle, how does this relate to the enlargement of the DT? What are the detailed molecular mechanisms?

    Significance

    The authors compared the phenotypes between globally and muscle-specifically deletion of Fgf9 in mice, and found that Fgf9 secreted by muscle may induced the enlargement of the DT. However, the detailed molecular mechanisms were not well investigated.

    Referees cross-commenting

    We still suggested that the internal cellular and molecular mechanisms should be well investigated in this papaer.

  4. Version published to 10.1101/2021.02.23.432498 on bioRxiv