TGFβ signalling acts as a molecular brake of myoblast fusion

Fusion of nascent myoblasts to pre-existing myofibres is critical for skeletal muscle growth and repair. The vast majority of molecules known to regulate myoblast fusion are necessary in this process. Here, we uncover, through high-throughput in vitro assays and in vivo studies in the chicken embryo, that TGFβ (SMAD2/3-dependent) signalling acts specifically and uniquely as a molecular brake on muscle fusion. While constitutive activation of the pathway arrests fusion, its inhibition leads to a striking over-fusion phenotype. This dynamic control of TGFβ signalling in the embryonic muscle relies on a receptor complementation mechanism, prompted by the merging of myoblasts with myofibres, each carrying one component of the heterodimer receptor complex. The competence of myofibres to fuse is likely restored through endocytic degradation of activated receptors. Altogether, this study shows that muscle fusion relies on TGFβ signalling to regulate its pace.

Supplementary Fig. 3 Diagram depicting the somitic regions taking part in the formation of the myotome and the fusion of myocytes.Selected epithelial cells originating from the medial border of the dermomyotome (DML) undergo an epithelial to mesenchyme transition (EMT) that allows their translocation in a region located beneath the dermomyotome, the transition zone (TZ), where they orient in the antero-posterior axis of the embryo [23][24][25] .The EMT triggers the entry of cells derived from the DML into the myogenic program.Terminal myogenic differentiation (e.g.MyHC expression) is observed when cells attach to the anterior and posterior borders of somites, at which time they are named myocytes (green elongated fibres).Fusion of myocytes is observed about 24 hours after myotome formation was initiated.Progenitors from the anterior (AL) and posterior (PL) border of the dermomyotome translocate in the myotome where they fuse to existing myocytes 9 .nt: neural tube.Supplementary Fig. 4 Electroporation protocol used throughout this study.Chicken embryos at 2.5 days of incubation (a) were electroporated in the medial border of newly formed somites (b,c).d, shows the epithelial progenitors (PR) expressing a GFP reporter driven by a CAGGS promoter in as short as 3 hours.e, Dorsal view of a somite, showing the myocytes that were generated during the two-day incubation time.NT, neural tube.Supplementary Fig. 5 Myosin Light Chain promoter is expressed in terminally differentiated myocytes.a, Overlay confocal stacks of a single somite electroporated at HH15 (E2.5), and imaged at HH25 (E4.5).The white solid line delineates the somite.The dotted lines delineate the transition zone (TZ).b, Electroporation control, a CAGGS ubiquitous promoter driving nuclear H2B-RFP.Expression is observed in the dorsomedial lip (DML), the TZ and the myotome.c, Expression of GFP driven by a myosin light chain (MLC) promoter.No expression of the reporter is seen in the DML.Cells within the DML initiate MYF5 and MYOD expression 25    Supplementary Table 1 Scores (as normalized to 100 to the effect of negative controls, RLuc and FLuc), accompanied with their respective P-values, obtained after esiRNA transfection of the indicated genes into the mouse myogenic cell line C2C12.Effects on fusion (nuclei per fibre, in red), proliferation/cell survival (nuclei count, in blue) and myogenic differentiation (myogenin expression, in green) after loss of function of those molecules are indicated.The list encompasses the gene names (Entrez gene IDs) of the esiRNAs that obtained the highest score in the fusion assay.The corresponding genes are therefore predicted to act as strongest inhibitors of C2C12 fusion.Supplementary Table 2 Scores (as normalized to 100 to the effect of negative controls, RLuc and FLuc), accompanied with their respective P-values, obtained after esiRNA transfection of the indicated genes into the mouse myogenic cell line C2C12.Effects on fusion (nuclei per fibre, in red), proliferation/cell survival (nuclei count, in blue) and myogenic differentiation (myogenin expression, in green) after loss of function of those molecules are indicated.The list encompasses the Entrez gene IDs of the esiRNAs that obtained the lowest score in the fusion assay.The corresponding genes are therefore predicted to act as most necessary for C2C12 fusion.

Supplementary Table 3
List of genes known from the literature to modulate fusion (with references) and that were tested or not in the esiRNA screen in the present study.

7 8 Supplementary Figure 9
Fig. 14 TGF signalling is activated upon fusion of PL-derived progenitors to DML-derived myofibres.Scatter plots showing the proportion of electroporated cells activating the TGF b reporter 6 hours, one day or 3 days after electroporation.Shown are means and standard deviations.Supplementary Figure 14 Melendez et al.
upon RAB11 function.Scatter plots showing the number of nuclei per fibre in each condition.Shown are means and standard deviations; ***P<0.001.Supplementary Figure 15 Melendez et al. dependent upon RAB7 function.Scatter plots showing the number of nuclei per fibre in each condition.Shown are means and standard deviations; ***P<0.001;**P<0.01.Supplementary Figure 16 Melendez et al. mediated inhibition of fusion by CA RAB7.Scatter plots showing the number of nuclei per fibre in each condition.Shown are means and standard deviations; ***P<0.001.Supplementary Figure 17 Melendez et al.

Table 3 . Role of TGFb-associated genes on fusion
TGFB1,2,3 bind TGFBR1 or ACVRL1 (type I receptors) and only bind TGFBR2 (type II receptors) MST binds TGFBR1 or ACVR1B (type I receptors) and only binds ACVR2B (type II receptor)