Fig. 2

Ezh2-deficient neural progenitor cells (NPCs) show reduced proliferation and precocious cell cycle exit. (a–c) Confocal images of the inferior tectal midbrain at E12.5 with respective quantification. Cartoon insert indicates area of analysis for a–c. (a) Immunostaining against the thymidine analogue EdU after 1-h pulse labeling reveals reduced proliferation of NPCs in Ezh2-deficient cells. n ≥3 in each group, ***P ≤0.001, Student’s t-test. (b) After a 24-h BrdU pulse, staining against BrdU and the proliferation marker Ki67 distinguishes cells that have exited the cell cycle as BrdU-positive and Ki67-negative. Quantification of BrdU⁺Ki67⁻/BrdU⁺ cells demonstrates increased cell cycle exit of mutant NPCs. n ≥3 in each group, ***P ≤0.001, Student’s t-test. (c) Ezh2-deficient NPCs differentiate precociously as the quantification of Sox2-positive NPCs and Dcx-positive neurons show. n ≥3 in each group, *P ≤0.05, Student’s t-test. (d) Immunostaining for cleaved Caspase3 on sagittal midbrain sections reveals increased apoptosis in the dorsal midbrain of the mutant as compared to the control. In contrast, higher magnification confocal micrographs of the inferior tectal midbrain (indicated by the white asterisk) display no apoptosis. DAPI staining serves as nuclear marker for all images. Scale bars: a–c, 40 μm, d, 200 μm (left panel), 40 μm (right panel); Error bars indicate SD; ctrl, Control