Figure 4

ISV malformation and aortic dilation in zebrafish cdkl1 morphants and crispants.

(A) ISV malformations in zebrafish embryos. Representative fluorescence images are shown for each phenotype; dorsal to the right. Cartoon illustrates aberrantly formed ISVs in 48-hpf fli-GFP embryos. (B) Quantification of ISV formation defects. (C) Coinjection of RNA encoding CDKL1^WT rescued ISV defects upon MO-induced Cdkl1 knockdown, while variants encoding CDKL1^Cys143Arg and CDKL1^Ser206Leu are unable to rescue ISV malformations. Overexpression of CDKL1^WT or its variants did not induce ISV defects. (D) Cdkl1 crispants (Cdkl1 Crispr) display ISV defects more often than do non-injected (NI) embryos or embryos injected with Cas9 only. Single ribonucleoprotein complexes including 2 guide RNAs (targeting Cdkl1 exons 6 and 7) and Cas9 were injected into the first cell of fertilized zebrafish eggs; as control, eggs were injected with Cas9 only. 1-way ANOVA with Šidák’s multiple-comparison tests were applied (B, C, and D). Bar graphs show means + SEM; n = 3 (B), n = 3–6 (C), and n = 4 (D) experiments. NS, P = 0.9806; ***P ≤ 0.001, ****P ≤ 0.0001. (E) Coinjection of RNA encoding CDKL1^WT rescued ISV defects upon CRISPR/Cas–mediated Cdkl1 knockdown, while neither mutant variant did so. n = 3 experiments. *P ≤ 0.05, **P = 0.0040, Brown-Forsythe and Welch’s ANOVA test with Dunnett’s T3 multiple-comparison test. (F) Cdkl1 morphants display aortic dilation. Fli-GFP zebrafish embryos were injected with control (CTRL) MO or Cdkl1 splMO. Representative image of the dorsal aorta in embryos 4 days post-fertilization. Red bracket indicates width of the aorta. (G) Quantification of aortic dilation. (H) Cdkl1 crispants display aortic dilation. Single ribonucleoprotein complexes including 2 guide RNAs (targeting Cdkl1 exons 6 and 7) and Cas9 were injected into the first cell of fertilized zebrafish eggs; as control, eggs were injected with Cas9 only. 2-tailed Mann-Whitney test was applied; n = 3 experiments; red lines show medians; ***P = 0.0003, ****P < 0.0001 (G and H). Circles in the graphs indicate individual experiments, and numbers of embryos analyzed are given (B–E, G, and H).