Doherty JT et al. (2011), Skeletal muscle differentiation and fusion are ...

XB-ART-43295

J Biol Chem 2011 Jul 22;28629:25903-21. doi: 10.1074/jbc.M111.243030.

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Skeletal muscle differentiation and fusion are regulated by the BAR-containing Rho-GTPase-activating protein (Rho-GAP), GRAF1.

Doherty JT , Lenhart KC , Cameron MV , Mack CP , Conlon FL , Taylor JM .

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Although RhoA activity is necessary for promoting myogenic mesenchymal stem cell fates, recent studies in cultured cells suggest that down-regulation of RhoA activity in specified myoblasts is required for subsequent differentiation and myotube formation. However, whether this phenomenon occurs in vivo and which Rho modifiers control these later events remain unclear. We found that expression of the Rho-GTPase-activating protein, GRAF1, was transiently up-regulated during myogenesis, and studies in C2C12 cells revealed that GRAF1 is necessary and sufficient for mediating RhoA down-regulation and inducing muscle differentiation. Moreover, forced expression of GRAF1 in pre-differentiated myoblasts drives robust muscle fusion by a process that requires GTPase-activating protein-dependent actin remodeling and BAR-dependent membrane binding or sculpting. Moreover, morpholino-based knockdown studies in Xenopus laevis determined that GRAF1 expression is critical for muscle development. GRAF1-depleted embryos exhibited elevated RhoA activity and defective myofibrillogenesis that resulted in progressive muscle degeneration, defective motility, and embryonic lethality. Our results are the first to identify a GTPase-activating protein that regulates muscle maturation and to highlight the functional importance of BAR domains in myotube formation.

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Species referenced: Xenopus laevis
Genes referenced: actl6a arhgap26 dag1 drg1 mapk1 myod1 pxn rho rho.2 rhoa tnnt2 tpm1 tsc1
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	FIGURE 9. GRAF1 is highly expressed during somitogenesis. A, RT-PCR analysis for x.GRAF1 and histone H4 (H4; loading control) was performed using RNA isolated from embryos at the indicated stages. B and C, whole-mount in situ hybridization of stage 29 Xenopus embryo using an antisense probe specific for x.GRAF1: h, heart; b, brain; s, somites; nt, neural tube; drg, dorsal root ganglia. B, lateral view. C, top, transverse; bottom; cross-section through mid-somite region (somite borders indicated with brackets). D, Western blot analysis of GRAF1 and ERK (loading control) expression at the indicated developmental stages. E, laser scanning confocal images of whole-mount GRAF1 (red), tropomyosin (green), and ToPro3 (blue nuclei) stained somites from wild type stage 37 embryo. IHC, immunohistochemistry.
	FIGURE 10. GRAF1 is essential for maintaining somite architecture and myoseptal boundaries. A, Western blot analysis for GRAF1 in Con MO- and GRAF1 Mo-injected embryos at the indicated stages of development. IB, immunoblot; St., stain. B, stage 39 GRAF1 Mo-injected embryos exhibit edema (panel b) and anteroposterior axis defects (panel c) relative to control embryo (panel a). C, laser scanning confocal images of whole-mount stage 30 –37 Con Mo- and GRAF1 Mo-injected somites reveal progressive degeneration. Embryos were stained with Tm or laminin (LAM) to visualize cells or myosepta, respectively.
	FIGURE 11. GRAF1 depletion leads to fiber rupture. A, laser scanning confocal microscopy of mildly affected stage 34 somites with Tm (green) and laminin (LAM) (red) reveal mid-somite tears (denoted by arrows). Laminin association with fiber tip is denoted by an asterisk. Scale bar, 100 m. B, TEM ( 2500 magnification) of somite-matched stage 37 Con Mo- and GRAF1 Mo-injected embryos. The myoseptum is denoted by a thick white arrow in the Con Mo panel. The sarcolemma remained attached to the intersomitic boundary in GRAF1 morphants, but mid-myofiber tears were prevalent (black arrows).
	FIGURE 12. GRAF1 morphants exhibit elevated RhoA activity and impaired skeletal muscle differentiation. A, RNA from Con Mo (C)- and GRAF1 Mo (G)-injected embryos (n 10) was isolated and utilized for semi-quantitative RT-PCR analysis of indicated marker gene. B, lysates from Con Mo- and GRAF1 Mo-injected embryos at the indicated stages were analyzed by Western analysis. Note that the lysates shown in the left panel are identical to those shown in Fig. 2A. Lysates used for right panel were collected from a separate experiment. IB, immunoblot. C, top, laser scanning confocal microscopy of whole-mount 12–101 (red) and ToPro3 (blue)-stained stage 25 Con Mo- and GRAF1 Mo-injected embryos (scale bar, 500 m). Note appropriate alignment of nuclei but reduced skeletal muscle differentiation (also see Fig. S3, A and B and Table 1). Bottom, TEM ( 2500 magnification) from somite-matched stage 25 Con Mo- and GRAF1 Mo-injected embryo. Note reduced myofiber content in GRAF1 morphants relative to controls. D, ELISA-based RhoA activity assays were performed on lysates isolated from stage 22 and 25 Con Mo- and GRAF1 Mo-injected embryos. Ten embryos were processed in batch for each stage and treatment.
	Supplemental Figure 4. Characterization of GRAF-specific antibody. A. COS cells were transfected with constructs containing flag-tagged human or Xenopus GRAF cDNAs. Lysates were immunoblotted using either anti-Flag or the GRAF-specific Ab. Both antibodies recognize a single protein species at approximately 110kDa. B. Extracts obtained from stage 32 Xenopus embryo or adult skeletal muscle (sk.m.) or heart were immunoblotted with GRAF-specific Ab. Lysate from COS cells transfected with Xenopus F-GRAF (XFGRAF) is shown as a positive control.
	Supplemental Figure 5. GRAF expression in Xenopus laevis. A. Western analysis of GRAF expression levels in Stage 37 and 52 embryos. Erk is shown as a loading control. B. Immunohistochemical staining for GRAF (red) in Stage 25 somites. Nuclei were detected with TOPRO3 (blue).
	Supplemental Figure 6. Characterization of GRAF-specific morphilinos. A,B) Flag-tagged human or Xenopus GRAF cDNA constructs were subjected to an in vitro transcription/translation assay containing 35Smethionine in the presence or absence of GRAF-specific anti-sense morpholinos (denoted mo1 and mo2) and exposed to autoradiography (panel A, panel B top) or immunoblotted using GRAF-specific antibody (panel B, bottom). Mo1 and mo2 each significantly reduced flag-tagged Xenopus GRAF1 expression in this in vitro transcription/translation assay but had no effect on translation of a control plasmid encoding human GRAF1 (panel A). When injected individually, these morphilinos each led to lethality by stage 42 and all embryos exhibited a similar range of phenotypes as observed when mixed together (i.e. fully penetrent motility defects, cardiac edema, and a small percentage with shortened AP axis). Since, the mixture of the two morpholinos (hereafter termed GRAF1 Mo) led to the most significant depletion of GRAF1 protein, (panel B) we used this combination for the remaining studies limiting the potential for off-target effects. C. Whole-mount GRAF antibody staining and laser scanning confocal microscopic analysis of Con Mo- (top) and GRAF Mo- (bottom) injected embryos at stage 37 reveals that GRAF Ab recognizes GRAF protein by IHC. The specificity of this staining is confirmed by markedly reduced immunoreactivity in GRAF morphant embryos. Dorsal is to the top, anterior to the left
	Supplemental Figure 7. Early development of Co- and GRAF Mo-injected Xenopus embryos. A. Gross morphological assessment of Con Mo- and GRAF Mo- injected embryos at the indicated stages. Stage 10 embryos are visualized with vegetal layer to the top. Stage 22 embryos are oriented with dorsal to the top, anterior to the left. Early stage GRAF MO-injected embryos were identified by FITC fluorescence. No apparent defects in gross morphology were observed at these stages. B. GRAF depletion does not affect the spatial temporal expression of MyoD. Embryos were injected with GRAF Mo or control Mo allowed to develop to the indicated stages and in situ hybridization analysis for Myo-D was performed as described in Materials and Methods.
	Supplemental Figure 8. GRAF depletion does not affect somite rotation or myoseptal alignment but leads to progressive loss of segmental integrity. A,B) Laser scanning confocal microscopy and digital deconvolution of whole-mount stage 25 Con Mo- and GRAF Mo-injected embryos stained with designated antibodies. Note appropriate alignment of nuclei at the mid-point of organized myoseptal boundaries (demarcated by LAM, -dystroglycan, and paxillin). Panel B shows example of mild phenotype observed in 3/25 Stage 25 GRAF morphants. C. Stage 37 Con Mo- and GRAF Mo-injected embryos co-stained with - dystroglycan (green) and TOPRO (nuclei, red) reveals progressive disruption of myoseptal boundaries in late stage GRAF morphants.

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