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

摘要 在诸如心肌梗塞（MI）的损伤之后，成人心脏仅具有最小的 再生能力。与人类和大多数哺乳动物相比，成年斑马鱼具有 具有显著的解决纤维化瘢痕形成和损伤后再生心肌的能力。既然有 目前还没有MI诱导损伤的临床再生疗法，因此仍然非常需要 生物医学研究界了解亲再生机制，使强大的 斑马鱼心脏再生与这个再生过程无关的是心外膜， 心脏的最外层组织。通过提供关键的旁分泌信号和促再生细胞类型， 心外膜促进损伤部位的心肌细胞（CM）增殖和血管再生。的 尽管如此，这些分子和细胞过程的精确机制在很大程度上是未知的，因此， 提出了许多关于斑马鱼心脏再生调节的问题。解决这些 问题，我们进行了单细胞RNA测序的分离心外膜细胞从受伤， 未受伤的成年斑马鱼心脏初步分析鉴定了一个成年心外膜祖细胞 在某些实施方案中，细胞是具有分化成血管周围细胞以促进血管生成的潜力的亚群。在这里， 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， 这些祖细胞对心脏再生的影响。进一步的分析表明，Tgfb途径调节了 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途径在再生中的作用， 心外膜使用遗传获得/功能丧失测定和修饰的RNA注射。总之，这个项目 可能最终为靶向心外膜祖细胞的促再生潜力的策略提供信息， 改善MI患者心脏修复和功能。

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.