Maternal alkbh4 mutation leads to epiboly delay. (A) CRISPR/Cas9-mediated deletion of alkbh4. Zebrafish alkbh4 contains 3 exons, and gRNA was designed to target the third exon. A specific genomic region was amplified from F2 individual embryo and sent for sequencing. The sequencing results of target sites are shown below, including the wild-type form and three kinds of alkbh4 mutant forms. (B) Maternal but not zygotic alkbh4 mutation results in epiboly initiation and progression delays. Wild-type, Zalkbh4, Malkbh4 and MZalkbh4 mutant embryos were collected after fertilization at the same time and then imaged at the indicated time points. (C) Epiboly initiation in (B) was measured by the percentage of embryos with yolk cell doming at 5 hpf. (D) Epiboly progression in (B) was measured by the average epiboly percentage of the embryos at 8 hpf. (E) In situ hybridization results of alkbh4 mRNA expression in wild-type and MZalkbh4 embryos at 2-cell and 25 hpf stages are shown. (F) CE movements are defective in MZalkbh4 and Malkbh4 mutant embryos. The mutant embryos were collected not only at the same time point but also at the morphologically comparable stages compared with wild-type embryos. dlx3b and ntla probes were used simultaneously for in situ hybridization. The ratios of embryos with representative pattern are indicated at the right corner of each picture. (G) The body length decreases in MZalkbh4 and Malkbh4 mutant larvae. Representative pictures of wild-type and mutant larvae at 5 dpf are shown. (H) The body lengths of wild-type and MZalkbh4 mutant larvae at 4 dpf, 5 dpf, 6 dpf and 7 dpf were measured by Image J software. The statistical results are shown here. Ne, the number of observed embryos. *, p<0.01. Scale bars: 100 μm in (B, E, F); 1 mm in (G).
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