Generation of the gpx4b mutant using the CRISPR/Cas9 system, and the gross phenotypes of maternal and maternal-zygotic loss of gpx4b in zebrafish embryos. (A) Schematic representation of the gpx4b locus. Exons are shown as boxes (filled box, protein-coding region; open box, UTR). Introns are shown as lines. The location of the Sec codon, TGA, is indicated with a red line. The green line indicates the location of the Cas9 target site. Black arrows show the position of genotyping primers. (B) The sequence of Cas9 target site and predicted protein sequences of wild type (WT) and mutant gpx4b alleles (allele1 and allele2). Upper panel: the sequence of the Cas9 target site is indicated with a black line and the two gpx4b-null mutant alleles are shown. The black dashed line indicates a deletion. Red line, restriction enzyme BciT1301 recognition sequence. Lower panel: the protein sequences of wild type and mutants are shown. (C) Genotyping result using restriction enzyme BciT1301 digestion assay. (D) Representative views of wild type as well as maternal (M) and maternal-zygotic (MZ) mutants of gpx4b at the indicated stages. The arrow shows the reduced ventral tail fin. The frequency of embryos with the indicated patterns is shown in the bottom right corner of each panel. n=4 females for allele1, n=3 females for allele2. At least two clutches of embryos from each female were analyzed. Lateral views with anterior towards the left. Scale bar: 200 µm.