Figure 4

Figure 4

(A) Schematic of single myofibre culture and analysis of derived MPCs after 4 days from plating in growth medium (left). Total number of cells (= GFP^+ve + GFP^-ve) is increased in myog^-/- (centre). n = 3 fish/genotype, n = 8 myofibre/fish. Unpaired t-test on average number of cells/fish/genotype. Representative immunostaining of Desmin (red) and nuclei (white) confirming myogenic identity of both sib and myog^-/- cells from single-plated myofibres at 4 days (right). (B) Schematic of 90–100 myofibres culture in growth medium (GM) and analysis of derived MPCs (left). Representative immunostaining of Desmin (red), GFP (green) and nuclei (white) confirming myogenic identity of both sib and myog^-/- cells after 2 days of culture (centre). Myog^-/- myofibres yielded higher number of Desmin⁺ MPCs per field of view (FOV). Symbol shapes denote paired sib and myog^-/- samples, n = 3 fish/genotype, paired t-test (right). (C) 90–100 myofibres cultured from each genotype yielded over 90% of Desmin⁺ cells, confirming high purity of the MPC population, n = 3 fish/genotype, unpaired t-test. (D) qPCR analysis shows expression dynamics of cdkn1a (p21) and cdkn1ca (p57) mRNAs in sib and myog^-/- MPCs cultured for 2 (n = 4) or 3 days (n = 3), unpaired t-test. Statistical significance within (coloured p) or between (black p) genotypes is indicated. (E) Schematic of 90–100 myofibres culture and analysis of derived MPCs cultured for 5 days in differentiation medium (DM), following expansion for 4 days in growth medium (GM) (left). Representative immunostaining of MyHC (red), nuclei (white) confirming that fusion into multinucleated myotubes occurred only in sib MCPs, whereas differentiated myog^-/- cells remained mostly mononucleated, as reported previously (Ganassi et al., 2018) (right). Dashed cyan squares highlight 3x-magnified cells, coloured arrowheads indicate nuclei of each separate differentiated cell. All graphs report mean ± SEM. Scale bars = 100 µm.