Abstract
Skeletal muscle arises from the fusion of precursor myoblasts into multinucleated myofibres1,2. Although conserved transcription factors and signalling proteins involved in myogenesis have been identified, upstream regulators are less well understood. Here we report an unexpected discovery that the membrane protein BAI1, previously linked to recognition of apoptotic cells by phagocytes3, promotes myoblast fusion. Endogenous BAI1 expression increased during myoblast fusion, and BAI1 overexpression enhanced myoblast fusion by means of signalling through ELMO/Dock180/Rac1 proteins4. During myoblast fusion, a fraction of myoblasts within the population underwent apoptosis and exposed phosphatidylserine, an established ligand for BAI1 (ref. 3). Blocking apoptosis potently impaired myoblast fusion, and adding back apoptotic myoblasts restored fusion. Furthermore, primary human myoblasts could be induced to form myotubes by adding apoptotic myoblasts, even under normal growth conditions. Mechanistically, apoptotic cells did not directly fuse with the healthy myoblasts, rather the apoptotic cells induced a contact-dependent signalling with neighbours to promote fusion among the healthy myoblasts. In vivo, myofibres from Bai1−/− mice are smaller than those from wild-type littermates. Muscle regeneration after injury was also impaired in Bai1−/− mice, highlighting a role for BAI1 in mammalian myogenesis. Collectively, these data identify apoptotic cells as a new type of cue that induces signalling via the phosphatidylserine receptor BAI1 to promote fusion of healthy myoblasts, with important implications for muscle development and repair.
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Acknowledgements
We thank members of the Ravichandran laboratory for their valuable suggestions at many stages of this work. We also thank L. Haney, A. Bruce and A. Dutta for technical suggestions and assistance and M. Hufford and A. Fond for help with statistical analysis. We thank members of the University of Virginia Flow Cytometry Core, Research Histology Core, and Gene Targeting and Transgenic Facility for cell sorting, histological services and transgenic mouse generation. This work was supported by a grant from the National Institute of General Medical Sciences/National Institutes of Health and the Center for Cell Clearance at the University of Virginia.
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A.E.H.-H. designed, performed and analysed most of the experiments in this study with input from K.S.R. C.S.L. generated and supplied the Bai1−/− mice, and provided GST-tagged BAI1 TSR. J.M.K. assisted with time-lapse and shRNA studies, and myofibre cross-sectional area analyses. S.A. helped with the in vivo muscle regeneration and ex vivo primary myoblast cultures. J.D.S. processed, stained and analysed the mouse embryonic tissues. A.L.K. provided phosphatidylserine liposomes for these studies. J.A.C. and Z.Y. assisted with the cardiotoxin injury model. J.W.M. provided intellectual input on the in vivo BAI1 studies. A.E.H.-H. and K.S.R. wrote the manuscript with comments from co-authors.
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This file contains Supplementary Figures 1-7. (PDF 39261 kb)
A population of myoblasts undergo cell death during fusion and remain in close contact with nascent myotubes
This video shows C2C12 fusing myoblasts labeled with TO-PRO-3 (red) to identify apoptotic cells. The time-lapse covers a period of 72 hours during which time a number of myoblasts die but do not appear to be phagocytosed. These TO-PRO-3 positive cells are passed along the newly forming myotubes culture maintain contact with the myotubes for the duration of the video. (MOV 28781 kb)
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Hochreiter-Hufford, A., Lee, C., Kinchen, J. et al. Phosphatidylserine receptor BAI1 and apoptotic cells as new promoters of myoblast fusion. Nature 497, 263–267 (2013). https://doi.org/10.1038/nature12135
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DOI: https://doi.org/10.1038/nature12135
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