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Fig. 1 - Supplemental 1 (A–C) Metrn genes CRISPR/Cas9 mediated knockout generation and validation. (D) Metrns loss-of-function effect on heart morphology. (A) Targeted metrns loci in the zebrafish genome for knockout generation. Red highlights the sgRNA homologous sequence, and PAM sequences are highlighted in green. The generated mutation is indicated below by dashed lines, and the number of deleted nucleotides is indicated after the D sign. (B) Metrn, metrnla, and metrnlb expression in triplMut embryos from two-cell stage to 1 day post fertilization (dpf) is highly reduced or undetectable by in situ hybridization (lateral views). (C) Metrns expression levels are reduced in triplMut embryos as shown by qRT-PCR analysis of metrn, metrnla, and metrnlb expression level at 14 hpf for metrn and metrnla and at 48 hpf for metrnlb in wild type (WT) and triplMut embryos. (Student t-test, ***p-value: 3.1e-06 for metrn; *p-value: 0.014 for metrnla; *p-value: 0.04 for metrnlb). Error bars indicate standard deviation. (D) Quantification in percentage of the number of embryos with S-looped, D-looped, or mild/no looped heart phenotypes at 2 dpf upon injection of metrnla mRNA, metrn mRNA, or both in triplMut embryos. Displayed p-values compared to WT: ****p-value:<1.0e-5 for metrnla mRNA, ****p-value:<1.0e-5 for metrn mRNA, ****p-value:<1.0e-5 for metrn +metrnla mRNA. Scale bar in (B) 250 μm. Source data for Figure 1—figure supplement 1 provided in “Figure 1—figure supplement 1—source data 1”.