Dissecting epicardial contributions to zebrafish heart regeneration at the single-cell level

摘要 在损伤后，如心肌梗塞(MI)，成年人的心脏只有极少量的 再生能力。与人类和大多数哺乳动物不同，成年斑马鱼拥有 具有显著的消融纤维化瘢痕和损伤后再生心肌的能力。因为有 目前尚无临床再生疗法治疗心肌梗死所致的损伤，仍有很大的需求。 生物医学研究界了解促进再生的机制，使 斑马鱼心脏再生。在这个再生过程中不可或缺的是心外膜，它是 心脏最外层的组织层。通过提供关键的旁分泌信号和促进再生的细胞类型， 心外膜促进心肌细胞(CM)的增殖和损伤部位的血运重建。那 可以说，这些分子和细胞过程的确切机制在很大程度上是未知的，因此 对斑马鱼心脏再生的调控提出了许多问题。要解决这些问题 问题：我们对损伤后的心外膜细胞进行了单细胞RNA测序。 未受伤的成年斑马鱼心脏。初步分析鉴定为成人心外膜祖细胞 有可能分化为血管周围细胞以促进血管生成的亚群。这里, 通过基因消融和谱系追踪，我们的目标是确定细胞和分子的贡献 这些祖细胞可以促进心脏再生。进一步的分析表明，TGFb途径调节着 这些心外膜祖细胞的增殖和分化。为了更好地了解 潜在的调控机制，我们将进一步确定TGFb途径在 采用遗传功能得/失分析和改良的RNA注射。总而言之，这个项目 可能最终为靶向心外膜祖细胞促再生潜能的策略提供信息 改善心肌梗塞患者的心脏修复和功能。

Abstract After an injury, such as myocardial infarction (MI), the adult human heart possesses only minimal regenerative capabilities. In contrast to humans and most mammals, the adult zebrafish possesses a remarkable ability to resolve fibrotic scarring and regenerate cardiac muscle after injury. Since there are currently no clinical regenerative therapies for MI-induced injuries, there persists a great need in the biomedical research community to understand the pro-regenerative machinery that enable robust zebrafish heart regeneration. Indispensable to this regenerative process is the epicardium, which is the outermost tissue layer of the heart. By contributing key paracrine signals and pro-regenerative cell types, the epicardium promotes cardiomyocyte (CM) proliferation and revascularization of the injury site. That being said, the precise mechanisms of these molecular and cellular processes are largely unknown, thus raising many questions in regard to the regulation of zebrafish heart regeneration. To address these questions, we performed single-cell RNA sequencing of isolated epicardial cells from injured and uninjured adult zebrafish hearts. Preliminary analysis identified an adult epicardial progenitor cell subpopulation that has the potential to differentiate into perivascular cells to facilitate angiogenesis. Here, through genetic ablation and lineage tracing, we aim to determine the cellular and molecular contributions of these progenitors to heart regeneration. Further analysis suggests that the Tgfb pathway regulates the proliferation and differentiation of these epicardial progenitors. To gain a greater understanding of the underlying regulatory mechanisms, we will further define the regenerative role of the Tgfb pathway in the epicardium using genetic gain/loss-of function assays and modified RNA injections. In sum, this project may ultimately inform strategies to target the pro-regenerative potential of epicardial progenitors to improve heart repair and function in MI patients.