Zebrafish strain including AB and Tg(cmlc2:GFP) transgenic lines were raised and maintained at 28.5°C in system water and staged as previously described (40). This study was approved by the Ethical Review Committee of Institute of Zoology, Chinese Academy of Sciences, China. Two antisense morpholino oligonucleotides (MOs) were used: ccm2 MO (GAAGCTGAGTAATACCTTAACTTCC) and mekk2 MO (GGCTGAAAAATGCTGCTCACCTTGC).

Morpholinos were injected at the one-cell stage. Tg(cmlc2:GFP) embryos at 3 days after fertilization were mounted in 1% low–melting point agarose. Images were acquired with an Andor dragonfly spinning disk confocal microscope (Dragonfly 505).

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