Fig. 5

a Timeline. Application of 4-Hydroxytamoxifen (4OHT) induces Cre activity in otx2b:CreER^T2-positive animals at 6 hpf eliciting recombination (blue box) in cells of the developing anterior neural plate. At 12 hpf, a heat treatment triggers expression of Cas9-GFP and the subsequent mutagenesis of the tyr target site (green box). Analysis of GFP expression and pigmentation was conducted at 22 and 50 hpf, respectively. b Expression of GFP is detected in cells of the developing fore-, midbrain and eyes at 22 hpf. c, d In comparison to controls (ctrl), GFP-positive embryos (GFP+) display no pigmentation loss in cells along the body, but significant pigmentation defects in the developing eyes. Examples shown are representatives across ten experiments showing the same result. A total of >200 GFP-positive individuals and their respective non-GFP-positive siblings were analyzed. Scale bars: 150 µm in b and d; 500 µm in c. e Graph showing quantification of RPE pigmentation determined using the mean of the histogram from images of control embryos expressing no fluorescent protein (Non-FP), DsRed-positive (DsRed+), and GFP-positive (GFP+) siblings as well as tyrosinase mutants (tyr^−/−) at 50 hpf. n (Non-FP, DsRed+, tyr^−/−) = 12; n (GFP+) = 18. Data were analyzed using one-way ANOVA, followed by Tukey’s post hoc test. Values are presented as mean ± SEM; ****p ≤ 0.0001, ns (non-significant) = >0.9999.