Fig 2: Single cell transcriptomic analysis of P0 WT and Maf cDKO neocortex.(A) Experimental design for 10X genomics analysis of the P0 WT and Maf cDKO neocortex. Neocortical tissues were dissociated for single cell gel bead-in-emulsion (GEMs) particle preparation, followed by cDNA library preparation, sequencing and data analysis. (B) UMAP plot of WT and cDKO cells, color coded with cluster identities (see Figure 1—figure supplement 2). (C) UMAP plot of WT and cDKO cells, color coded with genotypes; cluster one are MGE INs. Note the genotype separation of the cells in cluster 1 (left bottom), but not other clusters, suggesting the lack of non-cell autonomous effects of the cDKO. (D) Heatmap showing cluster identities with corresponding marker genes. (E) Heatmap of cluster 1 cells from WT and cDKO showing average expression of selected MGE IN markers and pan-IN markers. Note the nearly full depletion of Mafb and Maf and increased expression of Sst, and the decreased expression of Mef2c, while other MGE-derived IN markers such as Dlx1/2/5, Gad2 and Lhx6 were not changed. This suggests that the deletion of Mafs in the MGE-lineage does not lead to a gross change in MGE-derived IN fate. (F–G) Enlarged feature plots showing the expression of Mef2c (F) and Sst (G) from WT and cDKO from cluster 1. (H–I) FISH in Sst together with EdU labeling showing the amount of E15.5 born MGE-derived CINs that became Sst+ in WTs and cDKOs. Arrows point to the cells that are EdU+;Sst+. (J) Quantification of Sst+ CINs at P2 WT and cDKO neocortex. (K) Quantification of EdU+;Sst+ CINs at P2 WT and cDKO neocortex. (L–M) Immunofluorescent staining of MEF2C colocalized with Nkx2.1-cre-driven tdTomato reporter. P2 Maf cDKOs have decreased MEF2C expression (M) compared to WTs (L). (N) Quantification of the proportion of tdTomato+ CINs that were MEF2C+ by bins. N = 3–4 animals per group and multiple brain sections were used for quantification. Scale bar in (I) = 100 um and in (M) = 50 um. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.Figure 1—source data 1.Cluster marker gene list Here we provide a list of genes that were used to define each cluster’s identity.

Fig 3: Protein expression of METTL3 is regulated by MEF2C via direct DNA-binding. (A) Overexpression of MEF2C promotes the expression of METTL3 mRNA on the 2nd and 4th days after differentiation. (B,C) METTL3 protein levels after MEF2C knockdown and overexpression on the 4th day after differentiation. *p < 0.05, **p < 0.01, siNC vs. siMEF2C samples; ##p < 0.01, empty Vector vs. MEF2C-WT samples; Student's t-test. (D) Knockdown of MEF2C expression decreased the m6A level of total RNA in bovine skeletal myoblasts after siRNA transfection for 3 days (scale bar: 100 µm). (E) Using Celleste Image Analysis Software (Invitrogen, USA), the average optical density was calculated as the cumulative optical density of m6A red fluorescence divided by the number of nuclei, n = 3. (F,G) Dot blot and LC-MS/MS assays showed the level of m6A after siMEF2C for 3 days. (H,I) The consensus MEF2C-binding site is indicated, and a schematic diagram illustrates two potential MEF2C-binding sites in the METTL3 promoter and the corresponding mutant sequences. (J,K) Luciferase assay with the wild-type or mutant sequences of METTL3 binding sites verified the activity of sites 1 and 2 in MEF2C knockdown or overexpression myoblasts. (L) ChIP and PCR analyses of myoblasts revealed the binding of MEF2C to two sites in the METTL3 promoter. (M) ChIP-qPCR detection of the binding of MEF2C to the 2 sites in the METTL3 promoter in myoblasts transfected with the empty vector and MEF2C-WT. (A,E,G,J,K,M) The results are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, using Student's t-test.

Fig 4: MEF2C promotes bovine skeletal myoblast differentiation, and its expression and function in myoblast differentiation are regulated by m6A modification. (A) Relative mRNA expression of MEF2C during bovine skeletal myoblast differentiation. (B–F) After myoblasts were transfected with siRNA for 24 h, myogenic differentiation was induced. (B) Myotube formation was observed on the 4th day of differentiation (scale bar: 200 µm). (C) The fusion index was calculated as the percentage of nuclei in fused myotubes out of the total nuclei. (D) Relative mRNA expression levels of MYOD1, MYOG, MYH3, and MYMK were measured on the fourth day of differentiation. (E) The protein expression of MEF2C, MYH3, MYOG, and GAPDH on the 4th day after myogenic induction. (F) Semi-quantitative analysis of the protein expression. (G) Synonymous mutations at the m6A motif in the MEF2C CDS. (H–L) After transfection of the empty control, MEF2C-WT and MEF2C-MUT plasmids for 24 h, myoblasts were induced to differentiate. (H) Myotubes with MYHC expression on the 4th day after differentiation (scale bar: 200 µm). (I) The fusion index was counted. (J) Relative mRNA expression levels of MYOD1, MYOG, MYH3, and MYMK were measured. (K,L) The protein expression of MEF2C, MYH3, MYOG, GFP, and GAPDH after myogenic induction for 4 days. (D,F,J,L) The results were normalized to GAPDH levels and are presented as the means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance.

Fig 5: METTL3 increases MEF2C protein expression in an m6A-YTHDF1-dependent manner. (A,B) Relative dual-luciferase reporter activity of WT (MEF2C-WT) or mutated (MEF2C-MUT) reporters in METTL3 knockdown or overexpression 293A cells. (C) Representative mRNA profile of MEF2C in MEF2C-WT and MEF2C-MUT myoblasts at indicated time points after actinomycin D treatment. (D,E) MEF2C protein levels after YTHDF1 knockdown and overexpression on the 4th day after differentiation. *p < 0.05, **p < 0.01, siNC vs. siYTHDF1 samples; #p < 0.05, empty Vector vs. OE-YTHDF1 samples, using Student's t-test. (F,G) MEF2C, YTHDF1, MYH3, GFP, and GAPDH protein levels in MEF2C-WT or MEF2C-MUT with YTHDF1 expression knocked down after myogenic induction for 4 days. (H) Myotube formation in MEF2C-WT or MEF2C-MUT with YTHDF1 expression knocked down after myogenic induction for 4 days (scale bar: 200 µm). (I) The fusion index was counted. (J) Relative dual-luciferase reporter activity of MEF2C-WT or MEF2C-MUT reporters in YTHDF1 overexpression 293A cells. (K) RIP-qPCR detection of the binding of YTHDF1 to the transcript of MEF2C in myoblasts transfected with the empty vector and MEF2C-WT. The results are presented as the means ± SD. ***p < 0.001, using Student's t-test. (A–C,G,I,J) *p < 0.05, **p < 0.01, ***p < 0.001, ns, no significance, using one-way ANOVA with Tukey's correction for multiple comparisons.