Fig 1: Reconstruction of the myogenic differentiation trajectory in a pseudotime manner. A Pseudotime analysis of myogenic cells (including Pax3+ progenitors, myogenic progenitors, myoblasts, and myocytes in Fig. 1E) was performed by Monocle 2 and revealed seven different cell states (states 1~7). The distributions of cell states were presented along with pseudotime flows. Each dot is a cell. B Visualization of myogenic differentiation trajectory by cell origins (left) and developmental stages (right). C Visualization of myogenic differentiation trajectory by cell identity. D Violin plots showing feature gene expression in each cell cluster. MYMK, FNDC5, MEF2C, and TNNI1 are muscle development-related genes. MYL9 is a cardiomyocyte-specific marker. MYOD1 and MYOG are skeletal muscle cell-specific markers. E Gene Ontology (GO) analysis of the differentially expressed genes with high levels in myocytes (state 2) and myocytes (state 7). F Visualization of myogenic differentiation trajectory by cell state, with cardiac cells distinguished from differentiating skeletal muscle cells. G Bar plot showing the percentage of cells within each sample assigned to the annotated myogenic cell types. H Immunofluorescence staining for Pax7 and MyoD on somite cross sections of ZZ and DZ at E21 and E28. Scale bar = 100μm
Fig 2: Functional analysis of EGR1 and RHOB in myogenesis. A To explore the connection network between TFs and targets in skeletal muscle development, 172 genes associated with muscle development (http://wiki.geneontology.org/index.php/Muscle biology) were extracted as target genes. Then, 215 high-confidence annotation TF-target pairs were selected from regulon activity network to construct the regulatory network. B Target genes counts of TFs. C The mRNA levels of EGR1, RHOB, and myogenic markers during the differentiation of pig primary myogenic cells (PPMCs) at several indicated time points. When the cells were cultured in growth medium (GM) at sub-confluent density, it was defined as day 0 (day 0); when the cells reached 100% confluence, GM was changed to differentiation medium (DM). D The mRNA levels of EGR1, Myf5, and MyoD in proliferating C2C12 cells at 36 h after transfection with the pcDNA3.1-EGR1 vector. E Immunofluorescence staining for MyHC in PPMCs after transfection with the pcDNA3.1-EGR1 vector and differentiation induction for 5 days. The fusion index (the percentage of nuclei in fused myotubes out of the total nuclei) was calculated. F The mRNA levels of myogenic differentiation markers in C2C12 cells after transfection with the pcDNA3.1-EGR1 vector and induction of differentiation for 3 days. G The mRNA levels of RHOB, Myf5, and MyoD1 in proliferating C2C12 cells at 36 h after transfection with the pcDNA3.1-RHOB vector. H Immunofluorescence co-staining for Pax7 and MyoD in C2C12 cells transfected with the pcDNA3.1-EGR1 vector and cultured in growth medium for 36 h. I Statistical analysis was performed to quantify the percentages of the three myogenic cell populations. J Immunofluorescence staining for MyHC in PPMCs after transfection with the pcDNA3.1-RHOB vector and induced differentiation for 5 days. The fusion index was calculated. *P < 0.05, **P < 0.01, ***P < 0.001
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