robo4 deficiency results in aberrant blood flow in the adult zebrafish. a, A mutant line for robo4 was generated using CRISPR–Cas9. A 7-bp deletion was induced in exon 6 (robo4∆7 ). b, robo4 expression was analyzed by qRT–PCR on four adult hearts per genotype (three technical replicates averaged, both male and female) and then biological replicates were averaged (horizontal line). Results are shown normalized to β-actin mRNA expression. The graph shows the averaged technical replicates for four (n = 4) biological replicates per genotype run on the same plate. Asterisks signify significant differences to wild-type per a two-tailed Welch’s t test, P = 0.024 for wild-type and heterozygous mutants, P = 0.0082 for wild-type and homozygous mutants, *P < 0.05; **P < 0.01. c, Representative color Doppler echocardiograms for wild-type zebrafish show blood flow during systole (blue) and during diastole (red). A representative pulsed-wave Doppler image of a wild-type zebrafish shows normal flow pattern. Representative color Doppler echocardiograms for robo4∆7/∆7 mutant zebrafish show blood flow during systole (blue) and regurgitation (red) during diastole from the bulbus arteriosus (BA) into the ventricle (V). The white dotted line marks the ventricle from the bulbus arteriosus. A representative pulsed-wave Doppler image of robo4∆7/∆7 zebrafish shows regurgitant flow through the ventriculo-bulbar valve (orange arrow). The yellow arrow marks normal flow through the ventriculo-bulbar valve. d, Approximately 11 of 41 (26.8%) of robo4∆7 adult mutants (heterozygous and homozygous, both male and female) exhibit aberrant echocardiograms with regurgitation or turbulence, while only 4 of 45 (8.88%) of wild-type adult fish (clutch mate and AB fish, both male and female) showed this phenotype. Statistical differences were calculated per Fisher’s exact test, one-tailed P value, *P = 0.028 (P < 0.05 is considered statistically significant). The tester was blinded to genotype during the echocardiography procedure and analysis. e, Histological staining (H&E and Masson’s trichrome) of the ventricle and bulbus arteriosus for wild-type and robo4∆7/∆7 zebrafish. V, ventricle; BA, bulbus arteriosus.

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