Figure 5

Tamoxifen regulated CreERT2 recombinase activity in F0 somatic targeted embryos. (a) nacre; ubi:Switch embryos injected with Cas9mRNA, sgRNAs and the neurod1-2A-CreERT2 targeting vector. At 6 hpf embryos were mock treated or treated with 5um 4-hydroxytamoxifen and imaged at 3 dpf. (b–b′′) Injected embryos without treatment or (c–c′′) mock treated show a low level of expression in the brain. (d–d′′) 4-hydroxytamoxifen treatment resulted in switching from EGFP to mCherry expression in cells in the midbrain, hindbrain and neural tube. Arrows point to off-target expression. (e–e′′) Dorsal imaging of the brain in the larvae shown in d–d′′ revealed mCherry expression was detected in cells throughout the midbrain and hindbrain parenchymal tissue. 4-OHT 4-hydroxytamoxifen, c cerebellum, EGFP enhanced green fluorescent protein, hb hindbrain, mb midbrain, nt neural tube, t optic tectum, v ventricle. Scale bars (b′′,c′′,d′′) 200 μm; (e′′) 50 μm.