Zebrafish

Zebrafish (Danio rerio) were raised on a 14/10 h light/dark cycle and a water temperature of 28.5°C. Embryos at early larval stages were used before sex is apparent. Thus, both presumptive male and female embryos were used for all experiments. Embryos were incubated at 28.5°C in E3 buffer (0.3 × g/l Instant Ocean salt, 0.1% methylene blue). For imaging purposes, pigment formation was blocked by treating embryos with phenylthiourea (PTU; 1 × PTU, 0.2 mm) at 22–24 h post-fertilization (hpf). Embryos were then manually dechorionated using forceps. All mutant and transgenic lines were created using AB wild-type fish (ZFIN: ZDB-GENO-960809–7). Experimental procedures were approved by the Chancellor's Animal Research Care Committee at University of California, Los Angeles and the Pennsylvania State Institutional Animal Care and Use Committee.