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Fig. 4 id2b-/- adults exhibit thinner atrioventricular valve leaflets and prominent retrograde blood flow. (A) Two sgRNAs, represented by short vertical lines, were designed to create id2b-/- mutants. Co-injection of the two sgRNAs with zCas9 protein induces a 157 bp truncation in the exon 1 of id2b, which can be detected by genotyping primers marked with arrows. This genetic modification leads to the formation of a premature stop codon and the subsequent loss of the helix-loop-helix (HLH) domain. Right, quantitative real-time PCR (qRT-PCR) analysis of id2b and id2a mRNA levels in id2b+/+ and id2b-/- adult hearts. (B) No discernible morphological differences were observed between id2b+/+ and id2b-/- larvae at both 72 hr post-fertilization (hpf) and 96 hpf. (C) Kaplan-Meier survival curve analysis and log-rank test of id2b+/+ (n=50) and id2b-/- (n=46). Wpf, weeks post-fertilization. (D) Pericardial edema and an enlarged atrium are evident in a subset of id2b-/- adults. V, ventricle; A, atrium. (E) id2b-/- adults developed thinner atrioventricular valve leaflets (denoted by arrowheads) compared to id2b+/+. Enlarged views of boxed areas are shown in the bottom panels. (F, G) Echocardiograms of adult id2b+/+ (F) and id2b-/- (G) hearts. Unidirectional blood flow was observed in the id2b+/+ heart, while retrograde blood flow was evident in the id2b-/- heart. (H) Ratio of retrograde flow area over inflow area shows a significant increase in retrograde flow in id2b-/- (n=13) compared to id2b+/+ (n=10). Data are presented as mean ± s.e.m. Unpaired two-tailed Student’s t-tests were used to determine statistical significance. *p<0.05, **p<0.01, ***p<0.001. Scale bars, 500 μm (B and D, bottom), 2 mm (D, top), 200 μm (E).