In zebrafish, two paralogous genes, activating molecule in beclin-1 (BECN1)–regulated autophagy ambra1a and ambra1b, 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 ambra1a and ambra1b 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 ambra1 transcripts for heart development. Co-injection of both ATG-MOs determines a more severe cardiac phenotype, with prominent pericardial edema. Whole-mount in situ hybridization (WMISH) for myosin light chain 7 (myl7), as well as ambra1 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 (pitx2c) transcripts showed both bilateral and reversed pitx2c expression in morphants. The morphants' cardiac phenotypes were effectively rescued by co-injection of MOs with human AMBRA1 (hAMBRA1) messenger RNA (mRNA), pointing at the conservation of Ambra1 functions during evolution. Co-injections of ambra1 ATG-MOs with a hAMBRA1 mRNA mutated in the protein phosphatase 2a (PP2A) binding sites (hAMBRA1^PXP) were not able to rescue the cardiac phenotypes, at the difference from wild-type hAMBRA1 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.

中文翻译：

---

斑马鱼 ambra1a 和 ambra1b 沉默影响心脏发育。


在斑马鱼中，beclin-1 (BECN1) 中的两个旁系同源基因激活分子调节自噬ambra1a和ambra1b ，两者都是自噬过程和发育过程所必需的，编码蛋白质 AMBRA1，这是自噬体形成早期步骤的正调节因子。由于这两个基因的转录物在心脏区域的胚胎发生过程中表达，因此在这项工作中，我们通过吗啉代寡核苷酸（MO）研究了ambra1a和ambra1b敲低对心脏发育的影响。针对 ATG 翻译起始密码子的 MO 沉默这两种蛋白会影响心脏形态发生，导致出现心包水肿的小线状心脏，而剪接阻断 MO 的治疗不会导致明显的心脏表型，从而揭示了相关性母体提供的用于心脏发育的ambra1转录本。两种 ATG-MO 的共同注射决定了更严重的心脏表型，具有明显的心包水肿。肌球蛋白轻链 7 ( myl7 ) 的整体原位杂交 (WMISH) 以及在心脏中表达绿色荧光蛋白的斑马鱼转基因系中进行ambra1 ATG-MO 显微注射，揭示了心脏慢跑过程中的缺陷，随后出现不完美的心脏循环。此外，同源域转录因子 2 亚型 c ( pitx2c ) 转录物的 WMISH 显示在 morphant 中双侧和反向的pitx2c表达。通过将 MO 与人AMBRA1 ( hAMBRA1 ) 信使 RNA (mRNA) 共注射，可有效挽救 morphant 的心脏表型，这表明 Ambra1 功能在进化过程中得到了保守。 与野生型hAMBRA1 mRNA 不同，将ambra1 ATG-MO 与蛋白磷酸酶 2a (PP2A) 结合位点 ( hAMBRA1 ^PXP ) 突变的hAMBRA1 mRNA 共注射不能挽救心脏表型，并且对斑马鱼进行治疗具有特异性 PP2A 抑制剂斑蝥素的胚胎会导致类似的发育性心脏缺陷。这些结果表明 AMBRA1 在脊椎动物心脏发育中发挥着关键作用，可能涉及 PP2A 磷酸酶的结合位点。