Ptu e3

2 nL of a 10-μL injection mix, comprised of 100 ng krt4:EGFP-CAAX, 150 ng krt4:EGFP-dt-KRas^V12, krt4:EGFP-T2A-KRas^V12, or UAS:EGFP-KRas^{V12 17 (link)}, or 200 ng krt4:mCherry-T2A-cMyc + 200 ng transposase mRNA + 1 μL phenol red (Sigma) in nuclease-free dH₂O (Ambion), was microinjected into one-cell embryos. Some experiments included 0.2 pmol p53 morpholino (Gene Tools, 5′-GCGCCATTGCTTTGCAAGAATTG-3′) or 25 ng α-bungarotoxin mRNA^{35 (link)}. Embryos were sorted for expression of transgenes at 1 dpf using a fluorescence dissection microscope, dechorionated with forceps at 1 or 2 dpf or with 1 mg/mL pronase, incubated in E3 with 0.003% N-phenylthiourea (PTU-E3, Merck), and prepared for live imaging or fixed and immunostained. Our studies were not blinded as injected embryos are very easy to visually distinguish, owing to the development of epidermal cell masses in EGFP-KRas^V12 embryos (both “dt” and “T2A” versions), absent in the majority of EGFP-CAAX embryos (see Results, Fig. 1A, and Supplementary Fig. 3A). All zebrafish embryos and adults were treated ethically in compliance with our UK Project Licence P946C972B.

All animal procedures were performed according to the UK Animal (Scientific Procedures) Act 1986 and carried out under Home Office Project Licence number PPL P946C972B, which was subject to local AWERB Committee review and Home Office approval. The following zebrafish lines were used: Ekkwill, AB/Tuebingen, Tuepfel long fin, Tg(actb1:mCherry–utrCH) (Krens et al., 2017 (link)), Tg(bAct:hRas-eGFP) (Cooper et al., 2005 (link)), and lamc1^sa379 mutant (sleepy) (Kettleborough et al., 2013 (link)). Embryos were obtained by natural spawning and raised in E3 medium at 28.5°C. Embryos used for imaging were transferred to E3 with 0.003% N-phenylthiourea (PTU-E3, Merck) at 24 hpf to inhibit pigmentation. Embryos were used at 2 or 5 dpf, as indicated in each experiment. Sex cannot be determined before 5 dpf in zebrafish, so was not factored here.