Lack of Mecp2 does not affect the commitment of NPCs but delays their differentiation A: Schematic representation of the differentiation protocol and the cell types composing cultures differentiated from NPCs (adapted from Carlessi et al, 2013).Figure 1A: Schematic representation of the differentiation protocol and the cell types composing cultures differentiated from NPCs (adapted from Carlessi et al, 2013). B-J: Representative immunofluorescences of the cell types composing the WT and Mecp2 null cultures differentiated from NPCs. Antibodies against Tuj1, Gfap and Olig2 were used to recognize neurons, astrocytes and oligodendrocytes. In D, G and J the percentage of cells positive (mean ± SEM) for each marker was counted over the total of DAPI positive cells (100%). No difference between genotypes was highlighted (Two-way ANOVA); the variation through time resulted significant for the levels of Tuj1 and Gfap (Two-way ANOVA: F(1,16)=9.542, p-value<0.05 and F(1,10)=68.95, p-value<0.0001 respectively). Sample size: n ≥3 wells obtained from two independent preparations; for each well 10 random fields were acquired. Scale bars: 30 µm. K: Representative immunostaining of Neun (green) and Tuj1 (red) positive neurons at DIV14. Scale bars: 15 µm. L,M: Graphs show the percentage of Neun and Tuj1 positive cells and the average Neun intensity in wt and null samples (L: mean ± SEM; M: central band shows the median, boxes show the lower and upper quartiles, whiskers show max and min values). The percentage of cells positive for each marker was counted over the total of DAPI positive cells. Student's t-test highlighted a significant decrease of Neun intensity in null samples. **: p-value<0.01. Sample size: n≥10 wells obtained from two independent preparations. N,n': The binned distribution of Neun intensity is plotted as both frequency distribution graphs, in panel N, and cumulative probability plot in panel n'. The null neurons distribution is significantly shifted towards low levels of Neun intensity, Kolmogorov-Smirnov test **: p-value<0.01. Each value in panel N is represented as mean ± SEM obtained by measuring 900 cells from n ≥10 wells derived from two different preparations. O: Neun expression levels assessed at DIV0, DIV3 and DIV14 are represented as 2-∆ct. Box plots show the median (central band), the lower and upper quartiles (boxes) and the max and min values (whiskers) from at least 5 samples obtained from two independent preparations. Two-way ANOVA indicated both a significant genotype effect (F(2,28)=19,60; p<0.0001) and time effect (F(2,28)=93,70; p<0.0001). Bonferroni post-hoc test revealed significant differences between genotypes at DIV 14 but not at DIV0 and DIV 3. ****: p-value<0.0001. P: Scatter plots describing the transcriptional levels of selected differentially expressed genes in WT and Mecp2 null cultures differentiated from NPCs and analysed at DIV3 and DIV14. For each gene Two-way ANOVA and Šidák multiple comparison test were used to assess difference between genotypes at the two selected time points (*p<0.05, **p<0.01, ***p<0.001, ****p<0.0001). Values are represented as mean ± SEM. Sample size: ≥5 samples obtained from two independent preparations.