Functional substitutions of amino acids that differ between GDF11 and GDF8 impact skeletal development and skeletal muscle

Replacement of amino acids unique to GDF11 and GDF8 into the native locus of the other ligand yields measurable, differential skeletal and muscle phenotypes, revealing distinct features between the ligands and a requirement for GDF11 in early-stage skeletal development.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authors We encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript. If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information. These files will be linked online as supplementary "Source Data" files. ***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available. Failure to provide original images upon request will result in unavoidable delays in publication. Please ensure that you have access to all original microscopy and blot data images before submitting your revision.*** This was a very rigorous paper that dove deeply into the differences between GDF8 versus GDF11, and more specifically at the level type I receptor binding interface and the amino acids that differ between these proteins. The results are interesting and show alterations in axial skeletal development suggesting how these 2 regions differ in vivo in how each protein functions. Overall the study is very well done and it represents a great deal of work. The study is also very straightforward and I do not have any significant technical concerns or problems with data presentation or interpretation. My only comment is that the work is highly focused and reductionary, and it might only be of interest to experts in the field. Despite this concern, the study is meritorious and describes mice and a resulting functional phenotype that better distinguishes GDF8 from GDF11, again supporting the concept that these 2 related family members do not overlap in function Reviewer #2 (Comments to the Authors (Required)): Lian and Walker et al. Work focuses in two highly homologous TGFB proteins, GDF11 and GDF8 (also known as myostatin) and their role and biological actions in musculoskeletal development and muscle regeneration. Remarkably, the authors challenge previous -and recent findings-about the role of GDF11 and GFD8 activities in vivo using CRISPR/CAS transgenic and chimeric mice in which they substituted key structural amino acids (signalling domains) that differ between these two GDF proteins. Here, the authors show differential in in vivo and unique roles of GDF11 and 8, which cannot be merely explained by differential ligand concentration and patterns of expression. First, they show decrease GDF11 potency when replacing these key amino acids with those of GDF8, and therefore affected function, cause defects in axial musculoskeletal patterning, craniofacial bone, and limb development, in which in some the defects seem to recapitulate previous findings using KO models. However, no over phenotype was found in mice lacking GDF8, except for minor effects in muscle mass (atrophy? Development?). Overall, this study reveals distinct roles of GDF11 and 8 mature domains in vivo based on two key signalling domain-associated amino acids. Note, I am pleased to see the authors put their work in context with a recent (and quite similar) manuscript just published, well done.
Comments and revisions: 1. Please clarify what potency refers in the introduction, paragraph 4. 2. The authors indicate that previous studies have shown that administration of rGDF11 to aged mice reduces cardiac hypetrophy, but then they evaluated some heart parameters (weight, cardiomyocyte CSA, and others) in young mice using their transgenic lines. Why is that? I think there is some miscommunication to what has been demonstrated and their goals in evaluating a cardiac function of GDF11 or GDF8. Please, clarify or explain better the argumentative rationale behind. 3. How could the serum concentrations correlate with the tissue concentration? I ask this because I assume that the extracellular binding capabilities of both molecules are not affected, or are they? 4. Figure 3. Could the authors show pictures from above the cranium? 5. Figures 4, 5 and 6 are hard to read, and therefore, interpret. Please, increase the font size for enhancing eligibility. 6. "This was a very rigorous paper that dove deeply into the differences between GDF8 versus GDF11, and more specifically at the level type I receptor binding interface and the amino acids that differ between these proteins. The results are interesting and show alterations in axial skeletal development suggesting how these 2 regions differ in vivo in how each protein functions. Overall, the study is very well done, and it represents a great deal of work. The study is also very straightforward, and I do not have any significant technical concerns or problems with data presentation or interpretation. My only comment is that the work is highly focused and reductionary, and it might only be of interest to experts in the field. Despite this concern, the study is meritorious and describes mice and a resulting functional phenotype that better distinguishes GDF8 from GDF11, again supporting the concept that these 2 related family members do not overlap in function." Author Response: Thank you for your positive feedback. We understand that our study is highly focused, but we suggest that there is broad interest in these ligands. It is particularly significant that many recent clinical trials on inhibiting GDF8 function have failed in patients. Therefore, we believe that the molecular differences between the molecules in vivo are an important topic, and our study represents a step forward worthy of publication.

Reviewer #2: "Lian and Walker et al. Work focuses in two highly homologous TGFB proteins, GDF11 and GDF8 (also known as myostatin) and their role and biological actions in musculoskeletal development and muscle regeneration. Remarkably, the authors challenge previous -and recent findings-about the role of GDF11 and GFD8 activities in vivo using CRISPR/CAS transgenic and chimeric mice in which they
substituted key structural amino acids (signaling domains) that differ between these two GDF proteins.
Here, the authors show differential in in vivo and unique roles of GDF11 and 8, which cannot be merely explained by differential ligand concentration and patterns of expression. First, they show decrease GDF11 potency when replacing these key amino acids with those of GDF8, and therefore affected function, cause defects in axial musculoskeletal patterning, craniofacial bone, and limb development, in which in some the defects seem to recapitulate previous findings using KO models. However, no over phenotype was found in mice lacking GDF8, except for minor effects in muscle mass (atrophy?

Development?). Overall, this study reveals distinct roles of GDF11 and 8 mature domains in vivo based
on two key signaling domain-associated amino acids. Note, I am pleased to see the authors put their work in context with a recent (and quite similar) manuscript just published, well done." "Comments and revisions:

Please clarify what potency refers in the introduction, paragraph 4."
Author Response: Thank you for your helpful advice and detailed suggestions to help make our manuscript easier to comprehend. We have added a definition for "potency" as it relates to our study and clarified in paragraph 4 of the Introduction that "potency" refers to "signaling potency" differences between GDF11 and GDF8 ligands with amino acid substitutions, previously shown in Walker et al., 2017.
"In a prior study, we demonstrated that GDF11 and GDF8 differ in their signaling properties in multiple cell lines and cultured primary myoblasts, with GDF11 signaling at lower concentrations than GDF8 and more efficiently utilizing the type I receptors ALK4, ALK5, and ALK7 (Walker et al., 2017). We define this ability to activate downstream pathways at lower concentrations as having greater potency." Introduction, Paragraph 4. "These sequence alterations in GDF11, which previously were shown to diminish signaling potency of the resulting protein (Walker et al., 2017), caused a perturbation of the axial skeletal structure of mutant mice during development that persists into adulthood. In contrast, the sequence alterations introduced into GDF8, which previously were shown to increase the signaling potency of the resulting ligand (Walker et al., 2017), did not produce observable developmental phenotypes." "2. The authors indicate that previous studies have shown that administration of rGDF11 to aged mice reduces cardiac hypertrophy, but then they evaluated some heart parameters (weight, cardiomyocyte show that GDF11 is a more potent ligand than GDF8, there was potential for the genetically engineered mice to have changes in cardiac parameters. We agree that aging the mice will be an interesting analysis, and we have clarified our rationale for characterizing heart parameters, as follows: "Previous studies have shown that administration of exogenous recombinant GDF11 to aged mice reduces cardiac hypertrophy (Loffredof et al., 2013), and fetal cardiac GDF8 has also been implicated in early-stage heart development (Sharma et al., 1999). Since our in vitro experiments showed that GDF11 is a more potent signaling ligand than GDF8, there is potential for the genetically engineered mutants to have changes in cardiac parameters." "3. How could the serum concentrations correlate with the tissue concentration? I ask this because I assume that the extracellular binding capabilities of both molecules are not affected, or are they?"

CSA, and others) in young mice using their transgenic lines. Why is that? I think there is
Author Response: We have not measured tissue concentrations, and the mass spectrometry assay has been validated only for serum. Based on the biochemistry of the ligands, we do not anticipate that the matrix binding of the ligands would be affected, but we have not studied this rigorously. We have modified the manuscript as follows:

Revised manuscript:
Results, Circulating GDF11 concentration increases 50-fold in Gdf8 Gdf11MD mutants, while GDF11 and GDF8 levels in Gdf11 Gdf8aa and Gdf8 Gdf11aa mutants remain unchanged, Paragraph 1. "Our study further expands this analysis of the impact of mature domain sequence on ligand expression by measuring endogenous GDF11 and GDF8 in serum from Gdf11 Gdf8aa and Gdf8 Gdf11aa mutants as well, showing that serum GDF11 and GDF8 protein concentrations are not significantly altered in either monoallelic or bi-allelic Gdf11 Gdf8aa or Gdf8 Gdf11aa mutants, compared to wild-type mice ( Fig. 2A, B, C). Based on the biochemistry of the ligands, we do not anticipate that the matrix binding of GDF11 and GDF8 would be affected, though we have not studied this."     Author Response: We did not observe a specific timepoint during development at which muscle formation abruptly stops or fails, rather the development of the chimeric mice appeared gradual and normal. There were no distinct measurable defects or patterning malformations in other muscle groups, such as craniofacial muscles or diaphragm, either. For measuring muscle regeneration capacity of the mutant mice, we damaged the tibialis anterior (TA) muscle via cryoinjury in order to activate quiescent satellite cells in the basal lamina of myofibers, then harvested the muscle at 7 and 14 days post-injury. However, we observed normal muscle regeneration capability similar to wild-type. We have modified the manuscript as below:

Revised manuscript:
Results, Gdf8 Gdf11MD mutants exhibit decreased skeletal muscle mass, while the muscles of Gdf11 Gdf8aa and Gdf8 Gdf11aa mutants are not significantly altered, Paragraph 2.
"Overall, we did not observe any distinct defects or malformations in muscle development or patterning at any point during development, and the chimeric mice appeared similar to wild-type into early adulthood. In all cases, separation by males and females resulted in shifts in the mean muscle mass between the sexes." Along with points mentioned below, please tend to the following: -please add ORCID ID for secondary corresponding author-they should have received instructions on how to do so -please add the Twitter handle of your host institute/organization as well as your own or/and one of the authors in our system -please add a figure legend section to your main manuscript, including your main and supplementary figures and your table legends -please upload your table files as editable doc or excel files, or make sure they're included in the doc file of your main manuscript -we encourage you to introduce the panels in your figure legends in alphabetical order -please add a callout for Figure S3A and Figure S1D, E, F, I to your main manuscript text -please mention panel H in the Figure 3 legend If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
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