Fgfr4 Is Required for Effective Muscle Regeneration in Vivo

Abstract

Fgfr4 has been shown to be important for appropriate muscle development in chick limb buds; however, Fgfr4 null mice show no phenotype. Here, we show that staged induction of muscle regeneration in Fgfr4 null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed. By 7 days of regeneration, differentiation of myotubes became poorly coordinated and delayed by both histology and embryonic myosin heavy chain staining. By 14 days much of the muscle was replaced by fat and calcifications. To begin to dissect the molecular pathways involving Fgfr4, we queried the promoter sequences for transcriptional factor binding sites and tested candidate regulators in a 27-time point regeneration series. The Fgfr4 promoter region contained a Tead protein binding site (M-CAT 5-CATTCCT-3), and Tead2 showed induction during regeneration commensurate with Fgfr4 regulation. Co-transfection of Tead2 and Fgfr4 promoter reporter constructs into C2C12 myotubes showed Tead2 to activate Fgfr4, and mutation of the M-CAT motif in the Fgfr4 promoter abolished these effects. Immunostaining for Tead2 showed timed expression in myotube nuclei consistent with the mRNA data. Query of the expression timing and genomic sequences of Tead2 suggested direct regulation by MyoD, and consistent with this, MyoD directly bound to two strong E-boxes in the first intron of Tead2 by chromatin immunoprecipitation assay. Moreover, co-transfection of MyoD and Tead2 intron reporter constructs into 10T1/2 cells activated reporter activity in a dose-dependent manner. This activation was greatly reduced when the two E-boxes were mutated. Our data suggest a novel MyoDTead2-Fgfr4 pathway important for effective muscle regeneration. Fibroblast growth factors (FGFs)2 and their receptors (FGFRs) are critical for the development of most cell types. There are at least 22 distinct FGF ligands and 4 receptors (FGFR1-4), and different ligand/ receptor pairs regulate cell growth in either a positive or negative manner depending on the cell type and stage of development (1, 2). Gainof-function mutations of FGFR1, FGFR2, and FGFR3 cause a series of important human disorders of bone development, most notably achondroplasia (dwarfism) (FGFR3) and different types of craniosynostosis (FGFR1, FGFR2, FGFR3) (3-7).We and others have shown that the major receptor expressed in muscle is FGFR4, whereas the major ligand is FGF6 (8 -17). Consistent with an important role for FGFR4 in muscle development, previous studies have shown that inhibition of FGFR4 leads to the arrest of muscle progenitor differentiation in chick embryo, with reduced expression of Myf5, MyoD, and embryonic myosin heavy chain and dramatic loss of limb muscles (18). This effect is independent of myoblast proliferation (18). Sp1 is shown as one of the factors to control the transcription of FGFR4 in skeletal muscle cells (19,20).Despite the well documented importance of Fgfr4 in muscle development in the chick, Fgfr4 null mice develop normally, with no evident muscle defects (21). The only phenoty...

Synergistic effects of TGFβ2, WNT9a, and FGFR4 signals attenuate satellite cell differentiation during skeletal muscle development

“…The overexpression of TGFβ2 and TGFβ3 can decrease the myogenic differentiation of myoblasts (de Mello, Streit, Sabin & Gabillard, 2015; Schabort, van der Merwe & Niesler, 2011). Moreover, the knockout of FGFR4 attenuated the skeletal muscle regeneration (Zhao et al., 2006). Therefore, TGFβ2, WNT9a, and FGFR4 could regulate the skeletal muscle development by adjusting the activation status of satellite cells.…”

Myogenic progenitor cells and skeletal myogenesis in vertebrates

“…Expression of MyoD depends on Pax3 and Pax7, whereas, in this postnatal context, expression of Myf5, already transcribed in many quiescent satellite cells, is Pax-independent and can promote skeletal muscle differentiation in the absence of MyoD [55 ]. Microarray analysis has begun to reveal other potential Pax targets in satellite cells, such as Fgfr4, which is required for effective muscle regeneration [62].…”

Mandible and Tongue Development

“…Fgf6 −/− mice have a severe regeneration defect with fibrosis and myotube degeneration, consistent with a function during tongue myogenic differentiation (Armand, Laziz, & Chanoine, 2006). Although Fgfr4 −/− mice show no phenotype at birth, muscle regeneration in Fgfr4 -null mice becomes highly abnormal at the time point when Fgfr4 is normally expressed (Zhao et al, 2006). Previous studies suggest that TGFβ2 may function upstream of Smad4 during myogenesis.…”