Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice

Abstract

In vertebrates, skeletal muscle is a model for the acquisition of cell fate from stem cells1. Two determination factors of the basic helix–loop–helix myogenic regulatory factor (MRF) family, Myf5 and Myod, are thought to direct this transition because double-mutant mice totally lack skeletal muscle fibres and myoblasts2,3,4. In the absence of these factors, progenitor cells remain multipotent and can change their fate5,6. Gene targeting studies have revealed hierarchical relationships between these and the other MRF genes, Mrf4 and myogenin, where the latter are regarded as differentiation genes7. Here we show, using an allelic series of three Myf5 mutants that differentially affect the expression of the genetically linked Mrf4 gene, that skeletal muscle is present in the new Myf5:Myod double-null mice only when Mrf4 expression is not compromised. This finding contradicts the widely held view that myogenic identity is conferred solely by Myf5 and Myod, and identifies Mrf4 as a determination gene. We revise the epistatic relationship of the MRFs, in which both Myf5 and Mrf4 act upstream of Myod to direct embryonic multipotent cells into the myogenic lineage.

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Figure 1: Allelic series of Myf5 mutants.
Figure 2: Myod expression depends on Mrf4 in Myf5 mutants.
Figure 3: Myotome formation and desmin staining in Myf5-null and Mrf4:Myf5 double-mutant embyros.
Figure 4: Myf5:Myod double mutants make muscle.
Figure 5: Genetic hierarchy governing the acquisition of skeletal muscle identity in the mouse.

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Acknowledgements

We thank S. Shorte, E. Perret and P. Roux at the Pasteur Dynamic Imaging Center, and C. Cimper for assistance. The laboratories of S.T. and M.B. were funded by the Pasteur Institute, CNRS, AFM, ACI Integrative Biology programme of the French Ministry. M.B. was funded by the European Union. S.T. was also funded by grants from the HFSPO, Association pour la Recherche sur le Cancer. B.G.-M. received a fellowship from the ARC, and V.S. a grant from the Fondation pour la Recherche Médicale and Pasteur Institute. L.K.-D. was funded by the HFSPO, the EU and Pasteur Institute.

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Correspondence to Shahragim Tajbakhsh.

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The authors declare that they have no competing financial interests.

Supplementary information

Supplementary Data

Null status of Myf5 allelic mutants. (DOC 25 kb)

Supplementary Figure 1

Characterisation of Myf5nlacZ, Myf5GFP-P and Myf5loxP alleles. (JPG 72 kb)

Supplementary Figure 1 legend

Characterisation of Myf5nlacZ, Myf5GFP-P and Myf5loxP alleles. (DOC 21 kb)

Supplementary Figure 2

Myogenic conversion assay of Myf5 allelic constructs by transient transfections. (JPG 37 kb)

Supplementary Figure 2 legend

Myogenic conversion assay of Myf5 allelic constructs by transient transfections. (DOC 20 kb)

Supplementary Figure 3

Ex vivo analysis of Myf5:Myod mutants ; no myogenesis in embryos lacking Mrf4, Myf5 and Myod. (JPG 198 kb)

Supplementary Figure 3 legend

Ex vivo analysis of Myf5:Myod mutants ; no myogenesis in embryos lacking Mrf4, Myf5 and Myod. (DOC 21 kb)

Supplementary Methods

Explant cultures and immunofluorescence. (DOC 42 kb)

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Kassar-Duchossoy, L., Gayraud-Morel, B., Gomès, D. et al. Mrf4 determines skeletal muscle identity in Myf5:Myod double-mutant mice. Nature 431, 466–471 (2004). https://doi.org/10.1038/nature02876

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