Fig. 1

Wnt1-Cre-mediated Ezh2/H3K27me3 ablation affects midbrain expansion. (a) Left panel: scheme of the recombination area of the Wnt1-Cre line (indicated in blue) in the murine midbrain at E12.5. Wnt1-Cre ⁺ /Ezh2 ^[SET]lox/wt and Wnt1-Cre ⁻ /Ezh2 ^[SET]lox/lox mice are used as control while Wnt1-Cre ⁺ /Ezh2 ^[SET]lox/lox animals are referred to as Ezh2 conditional knock-out (cko). Right panel: Wnt1-Cre-driven recombination of the R26R reporter allele has been visualized by immunostaining against β-galactosidase, confirming recombination of the midbrain. Note that neural crest cells giving rise to craniofacial structures (white arrow) are also Wnt1-Cre recombined. (b) Immunostaining for Ezh2 reveals complete and partial ablation of Ezh2 protein in the caudal and rostral dorsal midbrain, respectively. (c) β-galactosidase immunostaining confirms full recombination of the caudal midbrain resulting in the absence of Ezh2 protein (upper panel) and the H3K27me3 repressive mark in the mutant (lower panel). (d) Hematoxylin and eosin staining (H&E) on sagittal midbrain sections at E12.5 (upper panel) and E14.5 (lower panel). Ezh2-deficient midbrains show reduced horizontal expansion in the inferior tectal region from E12.5 onwards (white arrowheads). n ≥3 in each group, ***P ≤0.001, Student’s t-test. Also, mutant neuroepithelium at E14.5 is thinner than the control highlighted by the grey brackets in high magnification pictures (asterisk indicates basal). Note that at E14.5 recombined neural crest-derived mesenchyme between neural epithelium and surface ectoderm is expanded in the mutant. DAPI staining serves as nuclear marker: a (right panel), b, c (lower panel); Scale bars: a, b, 500 μm; c, 40 μm; d, 200 μm; Error bars indicate SD; FB, Forebrain; MB, Midbrain; ctrl, Control