Fig 2: Expression of Chd8 in the developing mouse brain. a Chd8 mRNA expression in the developing mouse brain (n = 5 at each time point) by real-time RT-PCR. The level was normalized to the E16 mice. Error bars represent SEM. b Western blot analysis of CHD8 protein using whole brain lysates at the different developmental stages of mouse brains. c Quantification of CHD8 protein levels in developing mouse brain (n = 6 at each time point) by western blot analysis that is normalized to E16 level. d Chd8 RNA in situ hybridization of sagittal and coronal sections from mouse brains at the stages indicated. Abbreviations: cortex (Ctx), lateral ventricle (LV), hippocampus (Hip), thalamus (Tha), olfactory bulb (Ob), cerebellum (Cb). Scale bar, 600 μm. e RNA in situ hybridization shows the Chd8 expression pattern in coronal sections of the cerebral cortex at the stages indicated. Scale bar, 500 μm. High levels of Chd8 mRNA are detected in the cortex at perinatal stages. Abbreviations: cortical plate (CP), ventricular zone (VZ), subventricular zone (SVZ), corpus callosum (CC). Roman numerals (I–VI) indicate layers of the cerebral cortex (scale bar, 100 μm)

Fig 3: Cell type-specific expression pattern of CHD8 in mouse brains. a Endogenous mouse CHD8 is selectively enriched in the nucleus of cultured DIV3 cortical neurons. Cells were immunolabeled with CHD8 (green), the somatodendritic marker MAP2 (red), and with counterstaining of nuclear marker Hoechst (blue). Scale bar, 2 μm. b Double-immunofluorescent staining shows that CHD8 is highly expressed in NeuN-positive neurons. Scale bar, 5 μm. The open arrows indicate both CHD8 and NeuN-positive neurons, while the filled arrows indicate neither CHD8 nor NeuN-positive non-neuronal cells. c Double-immunofluorescent staining shows that CHD8 is weakly expressed in GFAP-positive astrocytes. The open arrows indicate GFAP-positive astrocytes with weak CHD8 expression. Scale bar, 25 μm. d Representative western blot shows that CHD8 is predominately expressed in neurons but also in cultured glia cells

Fig 4: More glial cells after CHD8 targeting in brain slice culture system.a Cortical brain region near the LV and SVZ in the newborn monkeys was isolated for brain slice culture. OB olfactory bulb, Hippo hippocampus, Cere cerebellum. b After infection with lentivirus for 10 days, the brain slices showed strong GFP signaling, reflecting lentiviral transduction. Scale bar: 100 μm. c Immunostaining of the infected brain slices showing that GFP-positive cells contain NeuN positive and negative cells. Scale bar: 25 μm. d Double immunostaining showing that lentiviral CHD8 gRNA/Cas9 expression, reflected by GFP expression, reduced CHD8 expression and occurred in astrocytes and oligodendrocytes. Scale bar: 10 μm. e Double immunostaining of the brain slice region near the LV showing that the density of olig2-labeled oligodendrocytes and SOX9-labeled astrocytes was increased in the lentiviral CHD8 gRNA/Cas9 infected region as compared with lentiviral control gRNA/Cas9 infection. Scale bar: 25 μm. f Quantitation of the relative numbers of oligodendrocytes and astrocytes. Nine brain slices from three newborn monkeys were used for statistical analysis. The data are presented as means ± SEM. **P < 0.01; ***P < 0.001.

Fig 5: Expression of CHD8 in human cerebral cortex. a Immunohistochemical staining revealed the expression pattern of CHD8 in human cerebral cortex from Brodmann 19 region. b Human CHD8 is expressed in neurons. The sections were immunostained with CHD8 antibody and counterstained with NeuroTrace 500/525 green fluorescent Nissl (Thermo Fisher Scientific). c CHD8 is expressed in GFAP+ astrocytes (open arrows: astrocytes). d CHD8 is expressed in MAP2-positive neurons (open arrows for MAP2 positive neurons). e CHD8 is expressed in pavalbumin (Pvalb)-positive interneurons (open arrows for Pvab-positive interneuron). f CHD8 is expressed in DCX-positive neurons (open arrows for DCX+ neuron) (scale bar, 20 μm)