Fig. 3: SMAD2/3-dependent TGFβ signalling regulates myoblast fusion in vivo.
From: TGFβ signalling acts as a molecular brake of myoblast fusion
a–d, f–h, i–l Dorsal views of confocal stacks of somites observed at E5.5. To test myoblast fusion, they were electroporated in the DML at E2.5 with the following: a–d electroporation of MLC promoter driving expression of the indicated genes, together with membranal GFP (green) and nuclear mCherry (red) allowing the quantification of nuclei per myocyte. e Column graph for a–d showing the frequency distribution of electroporated myocytes distributed in those that contained one, two to three, and four or more nuclei, relative to its control (in %). f–h Electroporation of the BMP-related SMADs together with membranal GFP (green) and nuclear mCherry (red). i Column graph for f–h showing the frequency distribution of fusion events relative to controls (in %). j–l Electroporation of a CRISPR/Cas9 construct targeting the chicken SMAD3 sequence alone (k) or together with a CRISPR/Cas9 construct targeting the chicken SMAD2 sequence (l) together with membranal GFP (green) and nuclear mCherry (red). m Column graph for j–l showing the frequency distribution of fusion events relative to controls (in %). Arrowheads point to cell nuclei in selected fibres. Statistical analyses: SMAD4: \(\bar x\): 1.72; n = 7; Ctrl: \(\bar x\): 2.29; n = 19; P-value < 0.0001. SMAD3: \(\bar x\): 1.48; n = 15; Ctrl: \(\bar x\): 2.29; n = 19; P-value < 0.0001; SMAD7: \(\bar x\): 7.55; n = 8; Ctrl: \(\bar x\): 2.29; n = 19; P-value < 0.0001; CA SMAD1: \(\bar x\): 2.11; n = 11; Ctrl: \(\bar x\): 1.98; n = 7; P-value: 0.16; CA SMAD5: \(\bar x\): 1.95; n = 10; Ctrl: \(\bar x\): 1.98; n = 7; P-value: 0.97; CRISPR SMAD3: \(\bar x\): 2.86; n = 24; Ctrl: \(\bar x\): 2.57; n = 29; P-value = 0.0002; CRISPR SMAD2/3: \(\bar x\): 2.95; n = 47; Ctrl: \(\bar x\): 2.57; n = 36; P-value < 0.0001; Error bars in e, i, m: SEM. Scale bars: 50 μm. Source data are provided (see ‘Data availability’).
