Zebrafish lines

All zebrafish work was approved by Gyeongsang National University Institutional Animal Care and Use Committee. Zebrafish were raised and maintained by the Animal Protection Act (2017), Korea. Tg(∼3.4her5:EGFP) (Tallafuss and Bally-Cuif 2003) and Tg(sox:EGFP) (Carney et al. 2006) lines used in this study were published. To generate egfl6 mutant lines with CRISPR/Cas9 system, 150 pg of in vitro-synthesized gRNA and 900 pg in vitro-transcribed mRNA encoding a nuclear-localized Cas9 were injected into one-cell stage wild-type Tübingen (TU) embryos. To identify carriers with germline transmission deletions in the egfl6 gene, embryos were raised to adulthood and outbred to wild-type TU zebrafish. The carriers for egfl6 mutant alleles were in-crossed, and the resulting embryos were used for in situ hybridization, immunochemistry, and alcian blue staining. For genotyping of egfl6 mutant alleles, PCR amplicons produced by primers egfl6_F (5′-CAGCCATGCATACACAAA-3′) and egfl6_R (5′-CTGTCAGTATGGGCTGCT-3′) were digested with TaqI; while a wild-type allele had 208 and 252 bp, egfl6 mutant alleles generated 452 bp (egfl6^GNU12), 455bp (egfl6^GNU13), and 468 bp (egfl6^GNU14). egfl6-morpholino (MO) and egfl7-MO published previously (Parker et al. 2004; Wang et al. 2015) were obtained from Genetools, and 1 nl of a 300-μM solution was injected at the one-cell-stage.