Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

Figures

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  Figure 1. Expression patterns of pr130 in zebrafish. (A) Expression of pr130 at different stages in whole fish during zebrafish embryogenesis and in 72 hpf-heart; (B) Relative expression of pr130 in non-cardiac tissue and cardiac tissue of 72 hpf embryos was identified using real-time reverse transcription-polymerase chain reaction (RT-PCR); (C) The expression of pr130 in different tissues of adult zebrafish. Data represent the mean ± SD. **, p < 0.01.

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  Figure 2. Schematic diagram of pr130 knock-out in zebrafish using CRISPR/Cas9 system and analysis of PP2A activity. (A) Sequence of target site is underlined with black line and is shown in blue font in wild-type (WT) and protosacer adjacent motif (PAM) highlighted in red. Deletions and insertions are indicated by dashes and yellow highlight, respectively. Changed nucleotides are shown on the right side, “+” indicates insertion and “-” indicates deletion; (B) The sequences of WT, M1 and M2, the regions of insertion (M1) and deletion (M2) are indicated by red arrows; (C) A representative genotyping of ScaI digestion of PCR products amplified from genomic DNA, the products amplified from WT samples could be digested completely, whereas those of the homozygous mutants could not be digested, and those of the heterozygous mutants could be partially digested. +/+ indicates WT, (+/-) indicates heterozygous mutants, (-/-) indicates homozygous mutants; (D) The predicted amino acids of Pr130, in which showed a truncated protein, the first 49 aa (green) are identical to those of the WT Pr130 protein, which contains 23 (M1) or 3 (M2) miscoding amino acids (red); (E) Western blotting analysis of Pr130…

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  Figure 3. Effects of pr130 deletion on mortality and cardiac development. (A,B) pr130-/- embryos develop pericardial edema (arrow). A, WT; B, pr130-/-; (C) Survival curves; (D) Whole-mount in situ hybridization (WISH) using the heart probe cmlc2. Mutants showed a higher proportion of no-looping (arrows) compared with WT which showed normal heart looping; (E) Percentage of abnormal and normal heart looping. n = 100 embryos analyzed per panel. The symbols ** in the bar chart represent significant differences (p < 0.01).

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  Figure 4. Pr130 deletion causes defective function of heart. (A) The diastolic surface areas of ventricular and atrial; (B) Fractional shortening of ventricular; (C) Fractional area change of ventricular; (D) The systolic surface areas of atrial. V, ventricular; A, atrial. Data represent the mean ± SD. The symbols * and ** in the bar chart represent significant differences (p < 0.05 or p < 0.01). The numbers of zebrafish are indicated in the columns.

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  Figure 5. Decreased cardiomyocytes in pr130-/- zebrafish. (A) Representative images of H&E staining of adult heart tissue at 40× magnification. Scale bars, 200 µm; (B) Higher magnification of the boxed regions (400× magnification). The heart tissues of WT, M1, and M2 were from the same region of heart. Scale bars, 20 µm; (C) Pr130-/- exhibited reduced cardiomyocytes. Red arrows indicate the myocardium nucleus. Scale bars, 10 µm; (D) The cardiomyocytes in heart tissues of controls and pr130-/- adult zebrafish were quantified and presented in the bar graph. We calculated the number of cardiomyocytes in each of four chosen fields per fish at a magnification of 1000×. n = 16 hearts analyzed per panel. Data represent the mean ± SD. *, p < 0.05.

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  Figure 6. Ultrastructure of adult heart tissues observed using transmission electron microscopy. Pr130-/- zebrafish displayed disordered myocardium. In pr130-/- heart, the boundary of bright band (red boxes), M line (yellow arrows), and Z line (red arrows) were not clear. In some cases the gaps between some longitudinal myocardial fibers increased (yellow boxes and white arrows).

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  Figure 7. Increased apoptosis was observed in pr130-/-. (A–C) Representative images of Tunel staining sections of adult heart tissue of WT (A), M1 (B), and M2 (C). Black arrows indicate normal cells (blue), red arrows indicate apoptosis cells (brown); (D) Expression levels of apoptosis-associated genes from adult heart tissues. Data represent the mean ± SD. The symbols * and ** in the bar chart represent significant differences (p < 0.05 or p < 0.01).