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. 2019 Jun 20;2:226. doi: 10.1038/s42003-019-0462-y

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© The Author(s) 2019

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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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^−/−)