Fig. 3: The RHEB p.Y35L mutation induces neuronal migration defects, cytomegalic neurons, and mTOR pathway activation in vivo.

E14.5 mouse embryos were electroporated with control vehicle, wild-type (WT) or p.Y35L RHEB plasmids coexpressing GFP through an IRES sequence. a At E18.5, embryos were collected and brain coronal sections were stained with DAPI and imaged under a confocal microscope. WT and p.Y35L sample images were aligned in the same orientation and roughly defined into two bins, with bin 1 representing the regions below the cortical plate and bin 2 representing the cortical plate. GFP-positive cells are indicated in green, and nuclei are indicated in blue. Scale bars: 100 μm. b The percentage of GFP + neurons in each bin was quantified according to the fluorescence intensity using ImageJ. Data are presented as the mean ± s.e.m, n = 4, *P < 0.05 (Student’s t-test). c At E18.5, embryo brain coronal sections were immunostained using an anti-phosphorylated S6 (pS6) antibody and a corresponding secondary antibody. Images were acquired by confocal microscopy. GFP (green) and the pS6 immunoreactivities (red) were visualized as indicated. Scale bars: 20 μm. Soma sizes of GFP-positive cells (d) and colocalization of GFP and pS6 (e) as indicated by yellow convergent signals in c were quantified using ImageJ for comparison. Data are presented as the mean ± s.e.m, n = 4, *P < 0.05, **P < 0.01 (Student’s t-test). f At P30, electroporated brain samples were immunostained using an anti-NeuN antibody and a corresponding secondary antibody, and images were acquired by confocal microscopy. GFP (green) and NeuN immunoreactivity (red) were visualized as indicated. Scale bars: 20 μm. g Soma sizes of GFP- and NeuN-double positive cells in f were quantified using ImageJ. Data are presented as the mean ± s.e.m. ***P < 0.001 (Vehicle: n = 3, WT: n = 4, p.Y35L: n = 3, one-way ANOVA with Tukey’s post test)