Myogenin is an Essential Regulator of Adult Myofibre Growth and Muscle Stem Cell Homeostasis

  1. Massimo Ganassi
  2. Sara Badodi
  3. Kees Wanders
  4. Peter S. Zammit
  5. Simon M. Hughes

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Abstract

Growth and maintenance of skeletal muscle fibres depend on coordinated activation and return to quiescence of resident muscle stem-cells (MuSCs). The transcription factor Myogenin (Myog) regulates myocyte fusion during development, but its role in adult myogenesis remains unclear. In contrast to mice, myog −/− zebrafish are viable, but have hypotrophic muscles. By isolating adult myofibres with associated MuSCs we found that myog −/− myofibres have severely reduced nuclear number, but increased myonuclear domain size. Expression of fusogenic genes is decreased, pax7 upregulated, MuSCs are fivefold more numerous and mis-positioned throughout the length of myog −/− myofibers instead of localising at myofibre ends as in wild-type. Loss of Myog dysregulates mTORC1 signalling, resulting in an ‘alerted’ state of MuSCs, which display precocious activation and faster cell cycle entry ex vivo, concomitant with myod upregulation. Thus, beyond controlling myocyte fusion, Myog influences the MuSC:niche relationship, demonstrating a multi-level contribution to muscle homeostasis throughout life.

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  1. eLife

    ###This manuscript is in revision at eLife

    The decision letter after peer review, sent to the authors on August 7 2020, follows.

    Summary

    This manuscript uncovers an unexpected role for myogenin in muscle stem/progenitor cells in adult zebrafish. Further analysis of a previously characterised mutant makes novel contributions to the field of muscle growth. The authors show that Myog helps keep MuSCs quiescent and provide mechanistic insights into how Myog controls MuSC activation. Intriguingly, their work suggests that Myog mutants have increased differentiation markers compared to wild-type siblings. They also offer new models for MuSC positioning within a fiber.

    Essential Revisions

    1. In their previous publication (Ganassi 2018), the authors showed that the differentiation index of cultured adult myoblasts does not differ between WT and myog-/-, but in the current study myog mutant cells show a much higher degree of differentiation compared to their wild-type siblings. Please clarify why these findings differ.

    2. The authors claim that the pax7a:GFP+VE cells represent bona fide MuSCs, which can only be determined by co-label of GFP and an anti Pax7 (or Anti Pax3+7) antibody. Although the authors do provide this co-label in one panel, they only show one cell per genotype. Please provide quantification of how often the GFP+VE cells are also positive for the anti-Pax3/7 antibody. Unless this co-label is extremely common in both genotypes, or confirmed in each experiment, the authors should soften their language abut the GFP expressing cells being verified MuSCs.

    3. Related to the previous point: The authors show that MPCs behave differently in culture and although they show increased pax7a and pax7b expression they also express higher levels of differentiation markers and enter terminal differentiation. This is puzzling and inconsistent with the reduced number of myonuclei and smaller myofibres that are seen in the mutant fish. Furthermore, it is not clear how the increased number of MuSCs per fibre is reached. A plausible explanation for both observations (fewer myonuclei/smaller fibres & more MPCs), is that these cells are myocytes that do not fuse efficiently. The authors raise this possibility in the discussion, however, this should be better assessed and either excluded or supported.

    4. The surface area domain size measurement in Figure 1 is a strange proxy for myonuclear domain. which is best thought of as a volume, as shown in Figure 2. However, figure 2 omits all pax7:GFP+VE cells, some of which may have fused recently enough to retain their GFP label. Please replace the SADS calculation in Figure 1 with a volumetric calculation. This will be important for interpreting and comparing the two findings.

    5. Culture of mononucleated MPCs from plated fibres was used to investigate whether lack of Myog enhanced MPC proliferation. The relative proliferation rates were not significantly different, however, EdU pulse experiments suggest that mutant MPC are more readily entering S-phase. Overall the authors suggest that lack of myog accelerates MuSC transition into the proliferation phase. At present this is not supported convincingly. Indeed, the data shows reduced proliferation and AUC (4E) in mutants. An additional EdU pulse at an earlier time after plating (day 1) should be included to potentially strengthen this idea. Alternatively the statement should be modified and toned down.

    6. The authors want to assess whether there is an earlier onset of differentiation of MPCs in culture. However, they only show expression of mef2d and mylpfa at day 3 (Fig 4H) and day 2 should be included here as well. Overall the differentiation index of MPCs is increased, can they comment on whether the cells remain mononucleated.

    7. In the discussion the paragraph (292 ff) regarding the niche is very speculative and should be toned down/amended. In particular, (Line 318) the conclusion that that Myog is required for assembly of the MTJ MuSC:niche complex is not well supported, there are no MTJ markers shown.

  2. Version published to 10.1101/2020.08.10.244590 on bioRxiv