Fig. 1. H₂O₂ levels shape the Engrailed 2 distribution in the tectum.

a Immunodetection of Eng2a and Eng2b (green) and DAPI staining (red) in zebrafish embryos (24 hpf) revealed different nuclear/extranuclear distributions along the anteroposterior axis (a’–a”‘: insets of sections at higher magnification; MHB midbrain hindbrain boundary, HB hindbrain). Eng2 staining alone is shown in Supplementary Fig. 1. b Quantification of total Eng2 levels (inferred from Eng immunostaining) along the anteroposterior axis of the tectum at 24, 26, and 28 hpf. All values were normalized to the maximum value for 26 hpf. c Ratio of Eng2 nuclear over extranuclear signals at 24, 26, and 28 hpf. d H₂O₂ levels in the tecta of zebrafish embryos from 24 to 28 hpf. H₂O₂ levels were inferred from the YFP₅₀₀/YFP₄₂₀ excitation ratio of HyPer7 in time-lapse recordings. Arrowhead: MHB position. The quantification is presented in e. f H₂O₂ levels in the tecta of control (Ctrl) and two hours Nox-i (100 nM) treated zebrafish 26 hpf embryos. g Quantification of immunodetected Eng2 along the anteroposterior axis of the tectum in control (Ctrl) and Nox-i-treated embryos (26 hpf). h ratio of Eng2 nuclear/extranuclear signals in control (Ctrl) and Nox-i-treated embryos (26 hpf). Double arrows in g and h indicate the domain where nuclear to extranuclear ratios are statistically different (confidence interval varying from 0.0001 to 0.05 from the MHB to more anterior positions). Scale bars, 50 μm. *p-value ≤ 0.05.