Journal: Cell Discovery

Article Title: CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

doi: 10.1038/s41421-023-00525-3

Figure Lengend Snippet: a Immunostaining of the prefrontal cortex gray matter (GM) and white matter (WM) of control and CHD8 mutant monkeys (M1, M2) with anti-CHD8 antibody. CHD8 shows a higher expression in E125–E130 than newborn (E163 ± 5). Scale bar: 50 μm. b Western blotting of the prefrontal cortex of the newborn monkeys (control-2 and M2). The blots were probed with antibodies to neuronal proteins (NeuN, DcX, PV, and Hom) and glial proteins (GFAP, MBP). Tubulin served as a loading control. c Quantification of the ratios of neuronal and glial proteins to the loading control on western blots. The data were obtained from four western blotting experiments and are presented as means ± SEM. * P < 0.05; ** P < 0.01. d Immunostaining of the white matter of control and CHD8 mutant monkeys with an antibody to olig2, a marker protein for oligodendrocytes. The nucleus was stained with hematoxylin. Scale bar: 50 μm. e Density of oligodendrocytes in the white matter of control and CHD8 mutant monkeys. The data were obtained by counting 12 images and presented as means ± SEM per mm 2 . *** P < 0.001.

Article Snippet: Olig2 , Rabbit AB9610 , , WB 1:1000 Merck Millipore , .

Techniques: Immunostaining, Control, Mutagenesis, Expressing, Western Blot, Marker, Staining

Journal: Cell Discovery

Article Title: CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

doi: 10.1038/s41421-023-00525-3

Figure Lengend Snippet: a The timeline for embryonic neurogenesis and gliogenesis in the macaque monkey. b The coronal section of the control-1 monkey (E130) showing the cortex, caudate, corpus callosum and LV. Rectangle indicates the location of brain slices that were used to examine the staining and numbers of neuronal and glial cells. Scale bar: 1 mm. c The brain slice used for examination consists of different lays including the inner subventricular zone (iSVZ), outer subventricular zone (oSVZ), intermediate zone (IZ), and subplate (SP). d Immunostaining of progenitor cells with antibodies to EGFR and Pax6. The nucleus was stained with hematoxylin. Scale bar: 100 μm. e Immunostaining of the white matter near the LV with antibodies to olig2 and GFAP, respectively. Scale bar: 100 μm. f Quantification of the relative numbers of EGFR- and Pax6-positive cells in iSVZ and the numbers of oligodendrocytes and astrocytes in the brain slices. The data were obtained by counting 4–7 brain slices per group and presented as means ± SEM. ** P < 0.01; *** P < 0.001.

Article Snippet: Olig2 , Rabbit AB9610 , , WB 1:1000 Merck Millipore , .

Techniques: Control, Staining, Slice Preparation, Immunostaining

Journal: Cell Discovery

Article Title: CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

doi: 10.1038/s41421-023-00525-3

Figure Lengend Snippet: a In utero delivery of lentiviral CHD8 gRNA/Cas9 into the LV of fetal monkey brain at ~G55. b Ultrasound image of injection of lentivirus into the LV of the fetal monkey brain at G55. Arrows indicate a needle. c Widespread expression of GFP, which reflects lentiviral CHD8 gRNA/Cas9 transduction in the brain of newborn monkey. Scale bar: 1 mm. d Neuronal expression of GFP in the newborn monkey brain cortex. Scale bar: 10 μm. e GFAP labeling of the white matter of the brain cortical region in control and CHD8-targeted newborn monkeys. Scale bar: 1 mm. f Western blotting of the lentiviral-infected brain regions showing that CHD8 gRNA/Cas9 expression reduced CHD8 expression and increased levels of glial proteins (Sox9 and Olig2). g Double immunostaining of lentiviral-infected brain regions showing that CHD8 gRNA/Cas9 expression increased Sox9-labeled astrocytes and Olig2-labeled oligodendrocytes when compared with control gRNA/Cas9 expression. Scale bar: 20 μm. h Quantitation of the relative number of oligodendrocytes and astrocytes. The data were obtained from one newborn monkey targeted by control gRNA/Cas9 and two newborn monkeys targeted by CHD8 gRNA/Cas9. Brain sections (1.3 mm apart from each other) from the brain cortical region in the control gRNA/Cas9 (six sections) and CHD8 gRNA/Cas9 (eight sections) targeted monkeys were used for quantitative analysis. At least four images (×20) from each brain section were taken to obtain the average numbers of glial cells per mm 2 . The data are presented as means ± SEM ( n = 24 images for control gRNA/Cas9 and n = 32 images for CHD8 gRNA/Cas9). *** P < 0.001.

Article Snippet: Olig2 , Rabbit AB9610 , , WB 1:1000 Merck Millipore , .

Techniques: In Utero, Injection, Expressing, Transduction, Labeling, Control, Western Blot, Infection, Double Immunostaining, Quantitation Assay

Journal: Cell Discovery

Article Title: CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

doi: 10.1038/s41421-023-00525-3

Figure Lengend Snippet: 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.

Article Snippet: Olig2 , Rabbit AB9610 , , WB 1:1000 Merck Millipore , .

Techniques: Isolation, Slice Preparation, Infection, Transduction, Immunostaining, Double Immunostaining, Expressing, Labeling, Control, Quantitation Assay

Journal: Cell Discovery

Article Title: CHD8 mutations increase gliogenesis to enlarge brain size in the nonhuman primate

doi: 10.1038/s41421-023-00525-3

Figure Lengend Snippet:

Article Snippet: Olig2 , Rabbit AB9610 , , WB 1:1000 Merck Millipore , .

Techniques: