Fig. 7: Downregulating Mef2c counteracts the enhanced cortical neurogenesis induced by MeCP2 duplication.

A MEF2C protein level in the embryonic cortices of WT and MeCP2^Tg1 male mice were detected by western blot. Vinculin served as the loading control in the Western blot. (N = 6 males from 4 dams). B, C MEF2C⁺ cell distribution in the embryonic cortices of WT and MeCP2^Tg1 male mice were detected by immunofluorescence staining. (N = 6 males from 6 dams). MEF2C⁺ cell distribution across 10 cortical bins is shown in (B), and total MEF2C⁺ cell counts per 100 μm bin are quantified in (C). Scale bars, 50 μm. D, E EYFP and either shCont or Mef2c shRNA were introduced into the cerebral cortex of WT or MeCP2^Tg1 embryos via IUE at E14.5 (D). Tissues were collected at E18.5 for immunostaining. (E) Distribution of EYFP⁺ cells across cortical zones was analyzed (N = 6 male offspring from 4 dams). Scale bars, 50 μm. Orange and blue labels denote comparisons between WT vs MeCP2^Tg1 (shCont) and MeCP2^Tg1 (shCont) vs MeCP2^Tg1 (shMef2c), respectively. F–I Primary NPCs from WT or MeCP2^Tg1 embryos were infected with shCont or shMef2c lentivirus. Protein levels of MAP2, GFAP, Nestin, and MEF2C were assessed by western blot (N = 4 independent cultures), with Vinculin as the loading control. Representative blots are shown in (F), and quantification in (G–I). J–M Immunofluorescence staining for MAP2, GFAP, and Nestin in NPCs infected with EGFP-labeled shCont or shMef2c lentivirus. Marker-positive cells within the EGFP⁺ population were quantified (N = 4 independent cultures). Representative images are shown in (J); quantification is shown in (K–M). Scale bar, 50 μm. All data are expressed as the means ± SEM. Box plots show the median (center line), 25th–75th percentiles (box limits), and whiskers indicate the minimum and maximum values. Exact P-values are indicated. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Statistical analyses were performed using two-tailed unpaired Student’s t test (A, C), two-way ANOVA with Sidak’s post hoc test (B, E), or one-way ANOVA with Tukey’s post hoc test (G–I, K–M).