Zebrafish and Silencing Affect Heart Development.

In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)-regulated autophagy and , both required for the autophagic process and during development, encode the protein AMBRA1, a positive regulator of early steps of autophagosome formation. As transcripts for both genes are expressed during embryogenesis in the heart region, in this work, we investigated the effects of and knockdown on heart development by means of morpholino oligonucleotides (MOs). Silencing of the two proteins by MOs directed against the ATG translation initiation codon affects cardiac morphogenesis, resulting in a small, string-like heart with pericardial edema, whereas treatment with splice-blocking MOs does not lead to overt cardiac phenotypes, thus revealing the relevance of maternally supplied transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount hybridization (WMISH) for myosin light chain 7 (), as well as ATG-MO microinjection in zebrafish transgenic line expressing green fluorescent protein in the heart, revealed defects with the heart jogging process followed by imperfect cardiac looping. Moreover, WMISH of homeodomain transcription factor 2 isoform c () transcripts showed both bilateral and reversed expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human () messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ATG-MOs with a mRNA mutated in the protein phosphatase 2a (PP2A) binding sites () were not able to rescue the cardiac phenotypes, at the difference from wild-type mRNA, and treatment of zebrafish embryos with the specific PP2A inhibitor cantharidin resulted in similar developmental cardiac defects. These results suggest a critical role for AMBRA1 in vertebrate heart development, likely involving the binding site for the PP2A phosphatase.