Fig. 4

Establishment of forebrain identity in Ezh2-ablated midbrain cells. (a) Upper panel: antibody staining for forebrain-specific transcription factor Foxg1 on sagittal brain sections at E12.5 demonstrates forebrain-restricted Foxg1 expression in the control and ectopic Foxg1 expression in the dorsal mutant midbrain. The cartoon indicates regular Foxg1 expression. Lower panel: high-magnification confocal images of immunostaining for Ezh2 and Foxg1 show ectopic Foxg1 expression in Ezh2-ablated cells (asterisk indicates basal). (b) Upper panel: antibody staining for Pax6 reveals ectopic Pax6 expression in the Ezh2 mutant midbrain. The cartoon inset illustrates regular Pax6 distribution at E12.5 rostral of the di-mesencephalic boundary indicated by a white dotted line. Lower panel: immunostaining for Ezh2 and Pax6 reveals ectopic mosaic-like upregulation of Pax6 in Cre-recombined cells without Ezh2 (asterisk indicates basal). (c) ChIP confirms the presence of H3K27me3 repressive mark at the transcription start site of Pax6 and Foxg1 (±500 bp). The intergenic region Int1 is unmethylated and serves as negative control. n ≥3 in each group, ***P ≤0.001, Student’s t-test. (d–e) In situ hybridization for Dlx2 mRNA and qRT-PCR for Foxg1, Pax6, Dlx2, and Emx1 demonstrate elevated expression of the forebrain transcription factors in Ezh2-deficient midbrains. n ≥3 in each group, ***P ≤0.001, **P ≤0.01, *P ≤0.05, Student’s t-test. Ectopic Dlx2 expression is indicated by a black arrow (d). Downstream targets of Pax6 – Dmrta1 and Neurog2 – are induced in mutant midbrains as shown by in situ hybridization for Neurog2 mRNA (f) and qRT-PCR for Dmrta1 and Neurog2 (g). n ≥3 in each group, ***P ≤0.001, **P ≤0.01, Student’s t-test. DAPI staining serves as nuclear marker: a, b; Scale bars: a, b, 500 μm (upper panel); a, b, 40 μm (lower panel); d, f, 100 μm; Error bars indicate SD; ctrl, Control; FB, Forebrain; MB, Midbrain; dMB, Dorsal midbrain; vMB, Ventral midbrain