Abstract
Migration of skeletal muscle precursor cells is a key step during limb muscle development and depends on the activity of PAX3 and MET. Here, we demonstrate that BRAF serves a crucial function in formation of limb skeletal muscles during mouse embryogenesis downstream of MET and acts as a potent inducer of myoblast cell migration. We found that a fraction of BRAF accumulates in the nucleus after activation and endosomal transport to a perinuclear position. Mass spectrometry based screening for potential interaction partners revealed that BRAF interacts and phosphorylates PAX3. Mutation of BRAF dependent phosphorylation sites in PAX3 impaired the ability of PAX3 to promote migration of C2C12 myoblasts indicating that BRAF directly activates PAX3. Since PAX3 stimulates transcription of the Met gene we propose that MET signaling via BRAF fuels a positive feedback loop, which maintains high levels of PAX3 and MET activity required for limb muscle precursor cell migration.
Highlights
In vertebrates, skeletal muscles of the trunk and limbs originate from condensations of the paraxial mesoderm, the somites (Braun and Gautel, 2011; Buckingham and Relaix, 2007)
We found that RCAS virus-mediated expression of BRAF and PAX3 but not human alkaline phosphatase (AP) increased cellular migration out of somitic explants isolated from chicken embryos and cultivated in matrigel further corroborating the results obtained with C2C12 cells (Figure 1—figure supplement 1)
We demonstrate that BRAF is critical for migration of limb muscle precursor cells, which serves an essential role for normal limb muscle development
Summary
Skeletal muscles of the trunk and limbs originate from condensations of the paraxial mesoderm, the somites (Braun and Gautel, 2011; Buckingham and Relaix, 2007). Several genes including Pax (Bober et al, 1994), Met (Bladt et al, 1995; Dietrich et al, 1999), Cxcr (Vasyutina et al, 2005), Gab (Sachs et al, 2000), Six1;Eya (Heanue et al, 1999) and Lbx (Brohmann et al, 2000; Gross et al, 2000; Schafer and Braun, 1999) have been identified to control somite maturation and compartmentalization, delamination of muscle precursor cells from the dermomyotomal epithelium as well as muscle precursor cell migration, proliferation and differentiation. It is known that PAX3 controls expression of Met in the ventro-lateral dermomyotome (Relaix et al, 2005; Yang et al, 1996) by direct binding to the Met gene promoter (Epstein et al, 1996), thereby enabling delamination and migration of limb muscle precursor cells
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