Characterization of the heart looping phenotypes following AMBRA1 silencing in the myl7:EGFP zebrafish transgenic line. (A) Three-dimensional reconstructions of WT and ambra1a and ambra1b ATG-morphant hearts expressing the myocardial reporter transgene myl7:EGFP at 48 hpf. The panels show the various types of abnormal looping phenotypes found in morphants (incomplete D-loop, no loop, and complete/incomplete L-loop), in addition to the normal D-looping orientation of WT hearts. The dotted white line marks the midline axis of the embryos. Scale bar, 200 μm. (B) Percentage of D-loop, incomplete D-loop, complete or incomplete L-loop, and no-loop orientation of the developing heart at 48 hpf in WT embryos, AMBRA1 morphant embryos, and AMBRA1 morphant embryos co-injected with hAMBRA1 or mutated hAMBRA1PXP mRNAs. (C) Percentage of normal, abnormal, and dead 48 hpf embryos in the different conditions as above. For each condition, 200 embryos were analyzed. Statistical analysis was performed by chi-squared test. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. hAMBRA1PXP, human AMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites; EGFP, enhanced green fluorescent protein; ns, not significant. Color images are available online.
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