BMPR2 affects valve development via ECM–receptor interaction in zebrafish

Figures

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  FIGURE 1. Schematic diagram of bmpr2a/b gene knockout in zebrafish. (A,B) Schematic diagrams of sgRNA targeting for bmpr2a and bmpr2b gene knockout, respectively. The red background and font represent exon 8 DNA, the green background and font represent exon 9 DNA, the purple background represents the protospacer adjacent motif (PAM), and the yellow background represents the target sequences. (C,D) Alignment between the wild-type and bmpr2a and bmpr2b knockout genomes using SnapGene software. The red rectangle and font represent exon 8, the black rectangle and font represent intron 8, and the green rectangle and font represent exon 9. (E) Protein alignment of human BMPR2 (NP_001195), zebrafish Bmpr2a (NP_001034906), and zebrafish Bmp2b (NP_001034896). The black boxes denote the protein kinase domain; the red lines indicate the deleted sequence of the Bmpr2a protein in the bmpr2a knockout sequence; the blue double arrows indicate the deleted sequence of the Bmpr2b protein in the bmpr2b knockout mutant; the black arrows (pointing to D482 and D485) indicate two amino acid residues that directly interact with the ligand ACVRL1. (F,G) qRT-PCR detected the validation of bmpr2a and bmpr2b gene…

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  FIGURE 2. Ventricular morphology and heart parameters analysis via M-mode at 48 and 72 hpf. (A) M-modes from movies of embryo hearts at 48 hpf revealed cardiac physiological functions. (B,C) Optical recordings of diastolic interval and systolic interval at 48 hpf, respectively. (D) Heart rate was measured from the pacemaker activity at 48 hpf. (E) Heart period was measured as the interval between the start of one diastole and the beginning of the next at 48 hpf. (F) Morphological analysis from the M-mode screenshot displayed the ventricular diastolic and systolic diameter at 48 hpf. (G) Statistical results of ventricular systolic diameter at 48 hpf. (H) Statistical results of ventricular diastolic diameter. (I) Fractional shortening (FS) provides an estimate of the ejection volume at 48 hpf. (J–N) Statistical results of the heart rate, heart period, ventricular systolic diameter, ventricular diastolic diameter, and FS at 72 hpf. HP, heart period; SI, systolic interval; DI, diastolic interval; DD, diastolic diameter; SD, systolic diameter; FS, fractional shortening. Data are presented as the mean ± SD (n > 10). One-way ANOVA was used to compare the significance between each group. ns, p >…

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  FIGURE 3. bmpr2a −/− ;bmpr2b −/− affects the myofibril substructures. (A) IF-detected subcellular localization of Myl7 exhibited striated bands at 48 hpf. The white bracket lines with double-ended arrows indicate the band length; the blue bracket lines with double-ended arrows indicate the band width. Scale bar, 2 μm. (B,C) Measurement of the width and length in A, respectively. Data are presented as the mean ± SD. One-way ANOVA was used to compare the significance between the groups. ns, p > 0.05; *, p < 0.05; **, p < 0.01.

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  FIGURE 4. bmpr2a −/− ;bmpr2b −/− affects the valve development. (A) Tg (flia:GFP) zebrafish line showing the overall structure of the heart at 144 hpf. The red dashed line indicates the heart structure; the blue dashed line indicates the valve structure. A, atrium; V, ventricle; BA, bulbus arteriosus. Scale bar: 100 µm. (B,C) Quantification of the left valve and right valve diameter in A. L-Va (left valve) is represented by yellow dotted line in A; R-Va (right valve) is represented by the red dotted line in A. n ≥ 6. (D) WISH detected the expression of nfact1, fn1b, has2, and klf2a in mutant zebrafish at 52 hpf. (E–H) qRT-PCR detected the expression of nfact1, fn1b, has2, and klf2a in the mutant zebrafish at 52 hpf. Data are presented as the means ± SD. The Kruskal–Wallis test was used to compare the statistical significance of differences among the groups. ns, p > 0.05; *, p < 0.05; **, p < 0.01.

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  FIGURE 5. Transcriptomic analysis of differentially expressed genes in bmpr2a/b mutant zebrafish. (A) Venn analysis of the DEGs of the three groups, namely, bmpr2a −/− ;bmpr2b +/+ , bmpr2a +/+ ;bmpr2b −/− , and bmpr2a −/− ;bmpr2b −/− . The Venn DEGs were named the Venn_365 subset. (B) Heatmap of the Venn_365 subset. (C,D) KEGG and GO enrichment analyses of the Venn_365 subset, respectively.

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  FIGURE 6. Molecular mechanism verification: bmpr2a −/− ;bmpr2b −/− affects heart contraction at 48 hpf. (A) Heatmap of genes that affect cardiac contraction. (B) qRT-PCR detected the expression of the cardiac contraction genes. (C) WISH verified the expression of the cardiac contraction genes. (D) Western blotting detected the expression of the cardiac protein Myl7. Lane 1, wild-type group (bmpr2a +/+ ;bmpr2b +/+ ); lane 2, bmpr2a −/− ;bmpr2b +/+ group; lane 3, bmpr2a +/+ ;bmpr2b −/− group; lane 4, bmpr2a −/− ;bmpr2b −/− group. Data are presented as the mean ± SD. The Kruskal–Wallis test was used to compare the statistical significance of differences among the groups. ns, p > 0.05; *, p < 0.05.

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  FIGURE 7. bmpr2a/b affects the valve development via the ECM–receptor interaction. (A) Heatmap of the ECM–receptor interaction, valve markers, EndMT-related genes, and mesenchymal cell-related genes. (B,C) qRT-PCR detected the expression of the ECM–receptor interaction genes. (D) Western blotting detected the expression of the ECM–receptor interaction protein Fn1. Lane 1, wild-type group (bmpr2a +/+ ;bmpr2b +/+ ); lane 2, bmpr2a −/− ;bmpr2b +/+ group; lane 3, bmpr2a +/+ ;bmpr2b −/− group; lane 4, bmpr2a −/− ;bmpr2b −/− group. (E) qRT-PCR detected the expression of the EndMT-related genes cdh1, cdh17, and snail. (F) Western blotting detected the expression of the EndMT-related protein Cdh1. (G) qRT-PCR detected the expression of the mesenchymal cell-related genes cdh2 and vim. (H) Western blotting detected the expression of the mesenchymal cell-related proteins Cdh2 and Vim. The Kruskal–Wallis test was used to compare the statistical significance of differences among the groups. Data are presented as the mean ± SD. ns, p > 0.05; *, p < 0.05; **, p < 0.01.

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  FIGURE 8. PPI analyses of the regulatory network of valve development genes and cardiac contraction genes. (A) PPI analyses of the regulatory network of valve development genes, ECM–receptor interaction genes, EndMT-related genes, and mesenchymal cell-related genes. (B) PPI analyses of the regulatory network of cardiac contraction genes. (The PPI website is String: https://cn.string-db.org/cgi/input?sessionId=bVkLDdkSKKje&input_page_active_form=multiple_identifiers).

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  FIGURE 9. Diagram illustrating the molecular mechanism of bmpr2a/b affecting heart looping, cardiac contraction, and valve development; ↓ indicates the genes with downregulated expression in bmp2a/b-deficient zebrafish; ↑ indicates the genes with upregulated expression in bmp2a/b-deficient zebrafish; the genes enclosed in brackets are the main genes that regulate the expression of the genes in the circles, which were predicted by the PPI regulatory network (Figure 8).