Fig. 5

Mutation of zebrafish efcab1 causes abnormal motility of Kupffer’s vesicle cilia. a Genomic organization of zebrafish efcab1. Black boxes: exons. Gray boxes: untranslated regions. Red asterisk indicates the genome-editing target site. b CRISPR/Cas9 target sequence. c Sanger sequencing of efcab1^+/+ and efcab1^−/− fish around the genome-editing target site. The 11 bp-insertion in efcab1^−/− includes a stop codon (red asterisk). d Immunoblot of testis lysate. The induced mutation deleted Efcab1 (asterisk). α-tubulin: loading control. e Kupffer’s vesicle cilia were visualized by immunofluorescence staining with acetylated-tubulin antibodies. f Measurement of the number of Kupffer’s vesicle cilia showed no significant differences between efcab1^+/+ and efcab1^−/− fish. N (embryo) = 26 (efcab1^+/+) and 27 (efcab1^−/−). Values indicate mean ± SD. g Typical kymographs of Kupffer’s vesicle cilia in efcab1^+/+, efcab1^−/−, and mRNA-rescued efcab1^−/− fish. Kymograph patterns were categorized into two classes: rotating (blue) and irregular (red). h Ratios of each motility class. N (cilia) = 55 (efcab1^+/+), 72 (efcab1^−/−) and 65 (mRNA-rescued efcab1^−/−). i Rotational frequencies of Kupffer’s vesicle cilia. Boxes correspond to the first and third quartiles, and whiskers extend to the full range of the data. N (cilia) = 55 (efcab1^+/+), 25 (efcab1^−/−) and 63 (mRNA-rescued efcab1^−/−). ***p < 0.001 vs. efcab1^+/+ (Student’s t-test). j Ventral views of 48 hpf embryos. Heart looping was visualized by whole-mount in situ hybridization of cmlc2. L, left; R, right; V, ventricle; A, atrium. k Directions of heart looping. N (embryo) = 108 (efcab1^+/+), 114 (efcab1^−/−), and 51 (mRNA-rescued efcab1^−/−)