EYA1/EYA2 and EYA3/EYA4 act as stage-specific SIX cofactors in embryonic and adult regenerative skeletal myogenesis

Abstract

Eya3 and Eya4 are two Eya genes expressed in adult myogenic stem cells, where they may act as SIX cofactors. We analyzed muscle regeneration in single and compound Eya3 and satellite cell-specific Eya4 mutant mice. A kinetic analysis of muscle regeneration after Notexin injury of the Tibialis Anterior revealed no major phenotype at 4, 14, and 30 days after injury in terms of PAX7+ cell number and myofiber cross-sectional area in Eya3 mutants, while all parameters were decreased in Eya4 mutants and further worsened in Eya3/Eya4 double mutants, in which we also observed a modification of the myofiber phenotype at 30 days after injury. Satellite cells were cultured ex vivo and Eya4 deletion was induced by Ad-Cre-mediated recombination. While single Eya3 mutant cells showed normal proliferation and differentiation, double mutant cells exhibited normal proliferation but failed to fuse. Analysis of their transcriptome revealed that the expression of Myomixer, Follistatin, and Noggin was severely downregulated specifically in double mutant cells, explaining their fusion deficiency. To gain a better understanding of the involvement of Eya genes during embryonic development and the genesis of PAX7+ myogenic stem cells, we analyzed Eya1 / ;Eya2 / , Eya3 / , Eya4 / , and Eya3 / ;Eya4 / E18.5 mutant fetuses at the limb and craniofacial levels. In Eya1 / ;Eya2 / fetuses, we confirmed the absence of distal limb muscles and observed reduced craniofacial muscles. In Eya3 / ;Eya4 / fetuses, craniofacial myogenesis appeared preserved and PAX7+ myogenic stem cells were present. BackgroundThe Eyes absent (Eya) genes encode transcriptional co-activators and phosphatases that function within the PAX-SIX-EYA-DACH (PSED) regulatory network. In skeletal muscle, EYA proteins cooperate with SIX homeoproteins to control myogenic gene expression during both embryonic development and adult regeneration. While Eya1 and Eya2 are predominantly expressed in embryonic myogenic progenitors and Eya3 and Eya4 are the dominant paralogs in adult satellite cells (SC), the specific and redundant contributions of individual family members to myogenesis remain poorly characterized. MethodsWe analyzed compound Eya mutant mice during adult Tibialis anterior muscle regeneration and during embryogenesis. We complemented this analysis by performing ex vivo myogenic stem cell cultures from compound Eya mutants and examining their fusion capacity. ResultsAnalysis of muscle regeneration following Notexin injury revealed that Eya2 and Eya3 single mutants display no major regenerative deficit. In contrast, satellite cell-specific deletion of Eya4 (Eya4sc/sc) caused a transient impairment of early regeneration, with reduced numbers of smaller regenerating MYH3+ (embryonic myosin heavy chain) myofibers and a transient decrease in SC number at 4 days post-injury (dpi). Compound Eya3-/-;Eya4sc/scdouble mutants showed a more severe and persistent phenotype, with decreased myofiber cross-sectional area, reduced myonuclear accretion, accumulation of PAX7+ cells associated with regenerated myofibers, and altered fiber-type composition at 14 and 30 dpi. Ex vivo analysis of double mutant SCs revealed a specific and complete blockade of myogenic fusion without defects in proliferation or MYOD expression. Transcriptomic analysis identified severe downregulation of Myomixer, Noggin, and Follistatin in differentiating Eya3-/-;Eya4-/- SCs. Open-access SIX1 and SIX4 ChIP-seq publicly available data confirmed direct binding at the Myomixer, Noggin, and Follistatin loci, supporting a direct SIX-EYA transcriptional mechanism. In parallel, embryonic analysis demonstrated that Eya1-/-;Eya2-/-E18.5 fetuses lack distal limb musculature and display severe craniofacial muscle hypoplasia, while in Eya3-/-;Eya4-/-fetuses limb and craniofacial musculature developed with no detectable defects. ConclusionsThese results reveal distinct temporal requirements for EYA proteins in skeletal muscle: EYA1 and EYA2 are essential SIX cofactors for embryonic myogenic fate acquisition in hypaxial and craniofacial progenitors, while EYA3 and EYA4 act redundantly in adult satellite cells to enable myogenic fusion by maintaining BMP antagonist expression and Myomixer activation downstream of the SIX-EYA transcriptional complex.