Adult fish were bred according to standard methods. Embryos were raised at 28.5°C in E3 embryo medium with 0.003% phenylthiourea to inhibit pigment formation and staged by time and morphology [46 (link)]. For in situ staining, embryos were fixed in 4% paraformaldehyde (PFA) (in PBS) for 3 h at room temperature (RT) or overnight (O/N) at 4°C, washed briefly in PBS, dehydrated, and stored in 100% MeOH at -20°C until use.
Transgenic fish lines and alleles used were as follows: Df(LG01)x8 [29 (link)]; Tg(hsp70l:Δtcf-GFP)w26 [30 (link)]; Tg(foxP2-enhancerA:EGFP)zc42; Tg(foxP2-enhancerA.1:EGFP)zc44; Tg(foxP2-enhancerA.2:EGFP)zc46; Tg(foxP2-enhancerB:EGFP)zc41; Tg(foxP2-enhancerD:EGFP)zc47; Tg(pax2a:GFP)e1 [36 (link)]; Tg(elavl3:EGFP)zf8 [35 (link)]. Df(LG01)x8 homozygotes were identified by their smaller forebrain and flattened hindbrain phenotype (J.E.L. and R.I.D., unpublished). Tg(hsp70l:Δtcf-GFP)w26 embryos were identified by GFP expression after heat shock.
Heat shock was performed by incubation of 32 hpf embryos for 1 hour at 37°C, then collecting 3 hours after the end of the heat shock.
Transgenic fish lines and alleles used were as follows: Df(LG01)x8 [29 (link)]; Tg(hsp70l:Δtcf-GFP)w26 [30 (link)]; Tg(foxP2-enhancerA:EGFP)zc42; Tg(foxP2-enhancerA.1:EGFP)zc44; Tg(foxP2-enhancerA.2:EGFP)zc46; Tg(foxP2-enhancerB:EGFP)zc41; Tg(foxP2-enhancerD:EGFP)zc47; Tg(pax2a:GFP)e1 [36 (link)]; Tg(elavl3:EGFP)zf8 [35 (link)]. Df(LG01)x8 homozygotes were identified by their smaller forebrain and flattened hindbrain phenotype (J.E.L. and R.I.D., unpublished). Tg(hsp70l:Δtcf-GFP)w26 embryos were identified by GFP expression after heat shock.
Heat shock was performed by incubation of 32 hpf embryos for 1 hour at 37°C, then collecting 3 hours after the end of the heat shock.
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