Generation of the vmhc^KI–EGFP zebrafish KI line. (A) Schematic diagram of the KI strategy based on the HR pathway triggered by the CRISPR/Cas9 system. The TAA stop codon (in red) is included in the guide RNA target sequences. Protospacer adjacent motif (PAM) sequences are marked in green. The donor vector used for HR contains the left homologous arm, P2A-EGFP, and right homologous arm. Restriction enzymes sites used for left and right homologous arms cloning are marked in blue. (B) Correct integration of P2A-EGFP into the C-terminal of the endogenous vmhc locus was confirmed by genotyping PCR and Sanger sequencing in the F1 stable KI embryos.

Enhanced green fluorescent protein in the vmhc^KI–EGFP zebrafish KI line recapitulates endogenous expression patterns of the vmhc gene. (A) EGFP patterns in the vmhc^KI–EGFP KI fish line from 16 to 18 somite stage (SS) to 48 hpf. Scale bar, 100 μm. White arrows point to specific patterns of the EGFP signal. (B) Endogenous expression patterns of the vmhc gene from 16 to 18 somite stage (SS) to 48 hpf revealed by WISH. Black arrows point to the specific expression pattern of vmhc endogenous transcripts. The dotted line outlines the shape of the atrium. Scale bar, 100 μm. (C) Western blotting and quantification analysis of the Vmhc protein expression in the vmhc^KI–EGFP KI fish line compared to wild-type (WT) control. N = 4. Unpaired 2-tailed Student’s t-test. V, ventricle. A, atrium.

CPAAHTs exposure induced ectopic expression of vmhc gene in the atrium and caused cardiac-looping defects to dose-dependently. (A,B) Representative images of heart-looping morphology (A) and quantification analysis (B) of the percentage of the vmhc^KI–EGFP KI embryos with corresponding cardiac-looping structure after exposure to different concentrations of CPAAHTs compared to untreated control at 48 hpf. The arrow points to the ectopic expression of the EGFP signal in the atrium. The dotted line outlines the shape of the atrium. V, ventricle. A, atrium. N = 129–315, chi-squared test.

CPAAHTs exposure suppressed the expression of genes required for myocardium differentiation and LR asymmetry. (A,B) Dorsal view of WISH images (A) and quantitative RT-PCR analysis (B) of transcription factors nkx2.5, gata4, mef2ca, and tbx20 in 0.8 μg/μl CPAAHTs treated embryos compared with untreated controls at 8-somite stage (SS). Arrows point to expected expression patterns for the corresponding genes. N = 3. Unpaired 2-tailed Student’s t-test. (C,D) Dorsal view of WISH images (C) and quantitative RT-PCR analysis (D) of Nodal-related gene spaw and its downstream target genes in 0.8 μg/μl CPAAHTs treated embryos compared with untreated controls at 25- to 28-SS. Arrows point to the expected expression patterns for the corresponding genes. N = 3. Unpaired 2-tailed Student’s t-test.