Skip to main content
. Author manuscript; available in PMC: 2017 Apr 11.
Published in final edited form as: Nat Neurosci. 2016 Oct 3;19(11):1477–1488. doi: 10.1038/nn.4400

Figure 1. Chd8 regulates neural progenitor proliferation in developing cortex.

Figure 1

(a) Images of E16 mouse cortices electroporated at E13 with nontargeting (Control), Chd8-directed small hairpin RNA (Chd8 shRNA), full-length human CHD8 construct (hCHD8), combination of Chd8 shRNA with hCHD8 (Chd8 shRNA + hCHD8) and GFP expression plasmid. Single-pulse BrdU was injected 24 h before brain dissection. Images were stained for GFP (green), BrdU (red) and Ki67 (cyan). Dashed lines represent the ventricular (bottom) and basal (top) surfaces. Scale bars: 50 µm. (b) Distribution of GFP+ cell in different cortical zones 72 h after transfection at E16 shows reduced percentage of cell in VZ/SVZ with similar increase in cortical plate (CP) following Chd8 knockdown (one-way ANOVA followed by Dunnett’s multiple comparison test; DF = 5, F = 19.45, P < 0.0001 for VZ/SVZ; DF = 5, F = 0.2843, P < 0.9159 for IZ; DF = 5, F = 13.00, P < 0.0001 for CP; Control, n = 8; Chd8 sh1, n = 3; Chd8 sh2, n = 3; hCHD8, n = 5; Chd8 sh1 + hCHD8, n = 3; Chd8 sh2 + hCHD8, n = 3). (c) Chd8 knockdown resulted in decreased BrdU incorporation, which could be rescued by overexpression of hCHD8 (one-way ANOVA followed by Dunnett’s multiple comparison test; DF = 5, F = 28.62, P < 0.0001; Control, n = 12; Chd8 sh1, n = 4; Chd8 sh2, n = 6; hCHD8, n = 4; Chd8 sh1 + hCHD8, n = 3; Chd8 sh2 + hCHD8, n = 3). (d) Chd8 knockdown resulted in increased cell cycle exit, which could be rescued by overexpression of hCHD8 (one-way ANOVA followed by Dunnett’s multiple comparison test; DF = 5, F = 14.12, P < 0.0001; Control, n = 9; Chd8 sh1, n = 4; Chd8 sh2, n = 3; hCHD8, n = 4; Chd8 sh1 + hCHD8, n = 3; Chd8 sh2 + hCHD8, n = 3). (e) Images of E16 mouse cortices electroporated at E13. Images were stained for GFP (green), Tuj1 (red) and pHH3 (cyan). Arrows indicate GFP–pHH3 double-positive cells. Dashed lines represent the ventricular (bottom) and basal (top) surfaces. Scale bars: 50 µm. (f) Chd8 knockdown reduced mitotic index measured by pHH3 staining (one-way ANOVA followed by Dunnett’s multiple comparison test; DF = 5, F = 11.95, P < 0.0001; Control, n = 8; Chd8 sh1, n = 3; Chd8 sh2, n = 3; hCHD8, n = 5; Chd8 sh1 + hCHD8, n = 3; Chd8 sh2 + hCHD8, n = 3). (g) Fraction of GFP+ cell overlapping with Tuj1 staining is increased after Chd8 knockdown (one-way ANOVA followed by Dunnett’s multiple comparison test; DF = 5, F = 16.74, P < 0.0001; Control, n = 8; Chd8 sh1, n = 3; Chd8 sh2, n = 3; hCHD8, n = 5; Chd8 sh1 + hCHD8, n = 3; Chd8 sh2 + hCHD8, n = 3). *P < 0.05; **P < 0.01; ***P < 0.001; ns., nonsignificant. n, number of different brain samples analyzed. Results are presented as mean ± s.e.m.