Eya1 and Eya2 proteins are required for hypaxial somitic myogenesis in the mouse embryo

Abstract

In mammals, Pax3, Six4, Six1 and Six5 genes are co-expressed with Eya1, Eya2 and Eya4 genes during mouse somitogenesis. To unravel the functions of Eya genes during muscle development, we analyzed myogenesis in Eya2-/- and in Eya1-/- embryos. A delay in limb myogenesis was observed between E10 and E13 in Eya1-/- embryos only, that is later compensated. Compound E18 Eya1-/-Eya2-/+ fetuses present a muscle phenotype comparable with that of Six1-/- fetuses; lacking a diaphragm and with a specific absence of limb muscles, suggesting either genetic epistasis between Six and Eya genes, or biochemical interactions between Six and Eya proteins. We tested these two non-exclusive possibilities. First, we show that Six proteins recruit Eya proteins to drive transcription during embryogenesis in the dermomyotomal epaxial and hypaxial lips of the somites by binding MEF3 DNA sites. Second, we show that Pax3 expression is lost in the ventrolateral (hypaxial) dermomyotomes of the somite in both Eya1-/-Eya2-/- embryos and in Six1-/-Six4-/- embryos, precluding hypaxial lip formation. This structure, from which myogenic cells delaminate to invade the limb does not form in these double mutant embryos, leading to limb buds without myogenic progenitor cells. Eya1 and Eya2, however, are still expressed in the somites of Six1Six4 double mutant and in splotch embryos, and Six1 is expressed in the somites of Eya1Eya2 double mutant embryos and in splotch embryos. Altogether these results show that Six and Eya genes lie genetically upstream of Pax3 gene in the formation of ventrolateral dermomyotome hypaxial lips. No genetic links have been characterized between Six and Eya genes, but corresponding proteins activate key muscle determination genes (Myod, Myogenin and Mrf4). These results establish a new hierarchy of genes controlling early steps of hypaxial myogenic commitment in the mouse embryo.

MyoD Directly Up-regulates Premyogenic Mesoderm Factors during Induction of Skeletal Myogenesis in Stem Cells

“…Eya2 acts genetically upstream of Pax3 in the formation of the hypaxial lip of the dermomyotome, and it is a cofactor of Six for the activation of Pax3 expression and migration (42). Pax3 and Meox1 regulate their own expression, although Pax3 can also regulate the activity of Meox1 and Six1 but not Gli2 (36,37).…”

Molecular pathogenesis of congenital diaphragmatic hernia revealed by exome sequencing, developmental data, and bioinformatics

“…We identified a de novo mutation in the EYA1 gene in one CDH patient. Mice lacking two alleles of Eya1 and one allele of Eya2 show diaphragm defects (21). Mutations in EYA1, responsible for 40% of BOR syndrome patients, have never been reported in CDH patients (22).…”

Genome-wide discovery of Pax7 target genes during development

“…Likewise, Pax7 and Eya4 share significantly similar developmental expression patterns; both play critical roles in neurogenesis and myogenesis and are highly expressed in developing brain, craniofacial mesenchyme, dermomyotome and limb during embryogenesis (6,25,49), and both Eya4 and Pax7 are robustly expressed in brain and skeletal muscle in adult mouse tissues (65,70,82). Eya family members function as transcriptional coactivators controlling cell fate specification, cell survival and apoptosis, proliferation, differentiation, and morphogenesis (34,49,57).…”

“…Conservation of this pathway by paralogous genes has been observed in murine myogenesis, where Pax3 and Eya2 function upstream of myogenic regulatory factors to induce myogenesis (60). Eya1 and Eya2 are functionally redundant during myogenesis and act upstream of Pax3 during dermomyotome development (25). Retroviral misexpression of Eya2 (with either six1 or Dach2) initiates Pax3 expression in somite explant cultures; conversely, retroviral misexpression of Pax3 upregulates Eya2 expression (26).…”