Fig. 1

(A) Phylogenetic tree of PIH proteins in yeast, Chlamydomonas, and vertebrates. Amino acid sequences were aligned by MAFFT program with FFT-NS-i option (Katoh and Standley, 2013) and evolutionary distances were calculated using neighbor-joining (Saitou and Nei, 1987). yeast, Saccharomyces cerevisiae; Chlamy, Chlamydomonas reinhardtii; z, zebrafish (Danio rerio); o, medaka (Oryzias latipes); m, Mus musculus; h, Homo sapiens. (B) Genomic organization of PIH genes in zebrafish. Black: exons. Gray: untranslated regions. Red asterisks indicate the target sites of our genome-editing. (C) Immunoblot of PIH proteins. None of the PIH proteins were detected in the sperm flagella fraction (asterisks). α-tubulin: control. (D) Whole-mount in situ hybridization of PIH genes. Arrows in lateral views indicate expression of PIH genes in ciliated organs (Kupffer’s vesicle, floor plate, otic vesicle, and pronephric duct). Flat mount preparations show dorsal views of the posterior regions of 12 hpf embryos. pih1d1 was ubiquitously expressed in 12 hpf embryos (black asterisk) containing Kupffer’s vesicle and floor plate (red arrowheads). ktu was also expressed in brain rudiments at 32 hpf (black arrowhead). White asterisks: non-specific staining of yolk. Scale bars: 200 μm. (E and F) Diagrams of zebrafish embryos at (E) 12 hpf and (F) 32 hpf, showing typical ciliated organs. (G) Left: preparation procedure of flat mount. Right: diagram of Kupffer’s vesicle and floor plate in flat-mounted embryos.