Fig. 3

Neural progenitor cell proliferation is controlled by Ezh2-mediated repression of cell cycle and Wnt/β-catenin signaling inhibitors. (a) Microarray analysis of three dissected E10.5 control and mutant midbrains identified 126 differentially expressed genes (≥1.75×, P ≤0.01), the majority of which (114) are upregulated upon Ezh2 ablation. Genes further analyzed are indicated. (b) qRT-PCR for Ezh2, cell cycle regulators Cdkn2a and Cdkn2c, and Wnt signaling inhibitors Wif1 and Dkk2 on control and mutant E11.5 midbrains confirms microarray data. n ≥3 in each group, ***P ≤0.001, **P ≤0.01, *P ≤0.05, Student’s t-test. (c) Chromatin immunoprecipitation confirms the presence of H3K27me3 at the transcription start site (±500 bp) of Cdkn2a, Cdkn2c, Wif1, and Dkk2. Intergenic region Int1 serves as unmethylated negative control. n ≥3 in each group, ***P ≤0.001, **P ≤0.01, Student’s t-test. (d–e) In situ hybridization for Cdkn2a (d) and Wif1 (e) mRNA illustrates increased gene expression in Ezh2 mutants. (f) Immunostaining for β-galactosidase + cells on the BAT-gal Wnt/β-catenin signaling reporter line demonstrates diminished signaling in Ezh2-deficient midbrains. n ≥3 in each group, **P ≤0.01, Student’s t-test. Cartoon insert indicates area of analysis for f and g. (g) Immunostaining against CyclinD1 and qRT-PCR. (h) Ccnd1 and Lef1 Wnt signaling downstream targets show decreased expression upon Ezh2 ablation. n ≥3 in each group, ***P ≤0.001, **P ≤0.01, Student’s t-test. (i) H&E staining of E12.5 sagittal midbrain sections of controls and Wnt/β-catenin signaling-ablated embryos. Mutant embryos exhibit reduced neuroepithelium thickness indicated with grey brackets in the magnifications. DAPI staining serves as nuclear marker: f, g; Scale bars: d, e, 100 μm; f, g, 40 μm; i, 400 μm; Error bars indicate SD; ctrl, Control; dMB, Dorsal midbrain; vMB, Ventral midbrain