Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart. Distinct epicardial gene regulatory programmes drive development and regeneration of the zebrafish heart

Unlike the adult mammalian heart, which has limited regenerative capacity, the zebrafish heart can fully regenerate following injury. Reactivation of cardiac developmental programmes is considered key to successfully regenerating the heart, yet the regulatory elements underlying the response triggered upon injury and during development remain elusive. Organ-wide activation of the epicardium is essential for zebrafish heart regeneration and is considered a potential regenerative source to target in the mammalian heart. Here we compared the transcriptome and epigenome of the developing and regenerating zebrafish epicardium by integrating gene expression profiles with open chromatin ATAC-seq data. We identified epicardial enhancer elements with specific activity during development or during adult heart regeneration. By generating gene regulatory networks associated with epicardial development and regeneration, we inferred genetic programmes driving each of these processes, which were largely distinct. We identified Wt1a, Wt1b, and the AP-1 subunits Junbb, Fosab and Fosb as central regulators of the developing network, whereas Hif1ab, Nrf1, Tbx2b and Zbtb7a featured as putative central regulators of the regenerating epicardial network. Targeting hif1ab, nrf1, tbx2b and zbtb7a using CRISPR/Cas9 in injured hearts resulted in elevated epicardial cell numbers infiltrating the wound and excess fibrosis after cryoinjury, illustrating the functional importance of these regulatory factors during zebrafish heart regeneration. Our work reveals striking differences between the regulatory blueprint deployed during epicardial development and regeneration. These findings underline that heart regeneration goes beyond the reactivation of developmental programmes and provide important insights into epicardial regulation. Overall design: Bulk ATAC and RNA sequencing of purified zebrafish epicardial cells, either at 5 days-post-fertilization or at 3 days-post-cryoinjury or at 3 days-post-sham

与再生能力有限的成年哺乳动物心脏不同，斑马鱼心脏在受损后可实现完全再生。心脏发育程序的重新激活被认为是心脏成功再生的关键，但损伤触发的应答以及发育过程中的调控元件仍未明确。心外膜（epicardium）的全器官激活是斑马鱼心脏再生的必要条件，同时也被视为哺乳动物心脏中可靶向调控的潜在再生来源。本研究通过将基因表达谱与开放染色质ATAC测序（ATAC-seq）数据相结合，对比了发育阶段与再生阶段斑马鱼心外膜的转录组与表观基因组。本研究鉴定出在发育过程或成年心脏再生过程中具有特异性活性的心外膜增强子元件。通过构建与心外膜发育及再生相关的基因调控网络，我们推导得出驱动这两类过程的遗传程序，二者整体差异显著。我们鉴定出Wt1a、Wt1b以及激活蛋白1（AP-1）亚基Junbb、Fosab和Fosb作为发育调控网络的核心调控因子；而Hif1ab、Nrf1、Tbx2b与Zbtb7a则被推定为本研究再生心外膜网络的核心调控因子。在受损心脏中利用成簇规律间隔短回文重复序列/Cas9（CRISPR/Cas9）靶向编辑hif1ab、nrf1、tbx2b与zbtb7a后，冷冻损伤后浸润伤口的心外膜细胞数量升高，且出现过度纤维化，由此阐明了上述调控因子在斑马鱼心脏再生过程中的功能重要性。本研究揭示了心外膜发育与再生过程中所启用的调控蓝图之间存在显著差异。上述研究结果强调，心脏再生并非仅局限于发育程序的重新激活，同时也为心外膜调控机制提供了重要的研究视角。实验设计方案：对纯化后的斑马鱼心外膜细胞进行批量ATAC测序与RNA测序，样本分别取自受精后5天、冷冻损伤后3天以及假手术处理后3天的个体。