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A conserved molecular pathway mediates myoblast fusion in insects and vertebrates

Abstract

Skeletal muscles arise by fusion of precursor cells, myoblasts, into multinucleated fibers. In vertebrates, mechanisms controlling this essential step in myogenesis remain poorly understood1,2. Here we provide evidence that Kirrel, a homolog of receptor proteins that organize myoblast fusion in Drosophila melanogaster3,4, is necessary for muscle precursor fusion in zebrafish. Within developing somites, Kirrel expression localized to membranes of fusion-competent myoblasts of the fast-twitch lineage. Unlike wild-type myoblasts that form spatially arrayed syncytial (multinucleated) fast myofibers, those deficient in Kirrel showed a significant reduction in fusion capacity. Inhibition of Rac, a GTPase and the most downstream intracellular transducer of the fusion signal in D. melanogaster1,5,6, also compromised fast-muscle precursor fusion in zebrafish. However, unlike in D. melanogaster6, constitutive Rac activation in zebrafish led to hyperfused giant syncytia, highlighting an entirely new function for this protein in zebrafish for gating the number and polarity of fusion events. These findings uncover a substantial degree of evolutionary conservation in the genetic regulation of myoblast fusion.

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Figure 1: Expression of kirrel mRNA and protein in fast-muscle precursors.
Figure 2: Structural organization of D. melanogaster Kirre, Rst and zebrafish Kirrel proteins as analyzed by the SMART domain annotation tool.
Figure 3: Kirrel is essential for fast-muscle precursor fusion.
Figure 4: Fusion behavior of kirrel morphant fast-muscle precursors in genetic mosaics.
Figure 5: Alteration of Rac activity affects fast-muscle precursor fusion.

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Acknowledgements

We thank A. Mahadevan for technical assistance; K. Sampath (Temasek Life Sciences Laboratory) for the 393.RN3-EGFP-plasmid and S.D. Menon, P.W. Ingham, K. Sampath and members of our laboratory for discussion and constructive criticism. This work was funded by the Institute of Molecular and Cell Biology and the Agency for Science, Technology and Research of Singapore. S.R. is an adjunct faculty member in the Department of Biological Sciences, National University of Singapore.

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Contributions

B.P.S. and S.R. designed the study; B.P.S., S.R., J.W. and W.Y.L. performed all the experiments and S.R. wrote the paper with constructive input from B.P.S.

Corresponding author

Correspondence to Sudipto Roy.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Dynamic profile of kirrel expression in non-muscle tissues. (PDF 86 kb)

Supplementary Fig. 2

Sequence comparison and phylogenetic analysis of Kirrel protein family members. (PDF 378 kb)

Supplementary Fig. 3

Quantitative analysis of nuclear numbers in fast muscle fibers of wild-type, kirrel morphant, rac1 morphant and caRac-expressing embryos and donor-host fusion index of fast myocytes in transplantation experiments. (PDF 23 kb)

Supplementary Fig. 4

Specific activity of the anti-kirrel morpholinos in zebrafish embroys. (PDF 97 kb)

Supplementary Fig. 5

Analysis of Kirrel and Rac1 protein levels in wild-type and morphant embryos using anti-Kirrel and anti-Rac1 antibodies by protein blot. (PDF 84 kb)

Supplementary Fig. 6

Sequence alignment of the full-length human and zebrafish Rac1 proteins, expression of the zebrafish rac1 gene and loss of Rac1 protein expression in embryos injected with anti-rac1 morpholinos. (PDF 108 kb)

Supplementary Table 1

PCR primers and morpholinos and their sequences. (PDF 15 kb)

Supplementary Methods (PDF 67 kb)

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Srinivas, B., Woo, J., Leong, W. et al. A conserved molecular pathway mediates myoblast fusion in insects and vertebrates. Nat Genet 39, 781–786 (2007). https://doi.org/10.1038/ng2055

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