All animal procedures were performed according to the UK Animal (Scientific Procedures) Act 1986 and carried out under Home Office Project Licence number PPL P70880F4C, 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(vsx1:GFP) (Kimura et al., 2008 (link)), Tg(deltaD:Gal4;UAS:GFP) (Scheer et al., 2001 (link)), Tg(TP1:VenusPEST) (Ninov et al., 2012 (link)), and lamc1sa379 mutant (sleepy; Kettleborough et al., 2013 (link)). Tg(vsx1:GFP) and lamc1sa379 lines were crossed to establish a Tg(vsx1:GFP);lamc1sa379 line. Adults were maintained under standard conditions as previously described (Westerfield, 2000 ), in a 14/10 hour light/dark cycle.
Embryos were obtained by natural spawning and raised in water or E2 medium at 28.5°C. If necessary, they were transferred to 0.003% 1-phenyl-3-(2-thiazolyl)-2-thiourea (Sigma-Aldrich) at 24 hpf to inhibit pigmentation.
Injections were performed at 16-64-cell stage. Embryos positive for mRNA expression, transgenic GFP expression and/or lamc1sa379-/- phenotype were selected for imaging. Live imaging was performed at 18-42 hpf. In situ hybridisation was performed at 22 hpf and immunohistochemistry at 22-28 hpf. Sex is not yet determined at these stages in zebrafish so was not taken into account.
Embryos were obtained by natural spawning and raised in water or E2 medium at 28.5°C. If necessary, they were transferred to 0.003% 1-phenyl-3-(2-thiazolyl)-2-thiourea (Sigma-Aldrich) at 24 hpf to inhibit pigmentation.
Injections were performed at 16-64-cell stage. Embryos positive for mRNA expression, transgenic GFP expression and/or lamc1sa379-/- phenotype were selected for imaging. Live imaging was performed at 18-42 hpf. In situ hybridisation was performed at 22 hpf and immunohistochemistry at 22-28 hpf. Sex is not yet determined at these stages in zebrafish so was not taken into account.
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