Probing the efficacy and potency of cardiomyocyte regeneration in the zebrafish heart

与哺乳动物相比，斑马鱼表现出更高的再生能力。作为对损伤的反应，斑马鱼的心脏不会形成永久性疤痕，心肌细胞会重新进入细胞周期。然而，尽管斑马鱼的心脏再生经常被描述为“完全”，但实际上并不清楚该器官是否恢复到损伤前的状态。特别是，目前尚不清楚损伤的心肌细胞在多大程度上可以再生。我们发现，心肌细胞周期活动在伤口组织完全愈合之前就会恢复到基线水平，这表明没有永久性疤痕不能作为心肌完全再生的证据。考虑到斑马鱼心脏再生模型的普及，我们认为最终弄清楚心肌细胞实际上再生到什么程度是很重要的。我们已经建立了可靠的方法来评估心肌细胞的数量，使用立体学原理，从组织切片得出的数量。我们将确定心脏冷冻损伤后几个时间点的心肌细胞数量，因此我们不仅能够回答心肌细胞是否完全再生，而且能够回答发生这种情况的时间框架以及这与伤口组织消退的关系。此外，我们计划解决斑马鱼心脏再生研究中另一个有趣但尚未解决的基本问题。在许多再生系统中，细胞表现出有趣的细胞可塑性，包括成熟细胞向多能状态去分化的例子。虽然遗传谱系追踪显示分化的心肌细胞是心脏再生过程中新形成的心肌细胞的前体，但尚不清楚它们是否也能在再生过程中产生其他类型的细胞。这是因为迄今为止用于基于Cre-lox的心肌细胞谱系追踪的所有转基因系仅在心肌细胞中表达。因此，潜在的转分化到其他细胞类型将被错过。我们已经创造了一种新的转基因Cre应答系，它在心脏的主要细胞类型中表达。使用这个工具，我们将询问心肌细胞在再生过程中是否能促进内皮细胞、心外膜细胞或成纤维细胞谱系。总之，这里提出的工作将解决与斑马鱼心脏再生的细胞机制相关的两个基本开放问题，从而将允许对该模型的有用性进行大大改进的评估。

Compared to mammals, zebrafish display much elevated regenerative capacities. In response to injury, zebrafish hearts do not form permanent scars, and cardiomyocytes re-enter the cell cycle. Yet, although zebrafish heart regeneration is frequently described as "complete", it is actually unclear whether the organ recovers to a pre-injury status. In particular, it is unknown to which extent cardiomyocytes lost to injury are regenerated. We found that cardiomyocyte cell cycle activity returns to baseline levels much before wound tissue has been fully resolved, suggesting that absence of a permanent scar cannot be used as evidence for full myocardial regeneration. Considering the popularity of the zebrafish heart regeneration model, we think it is important to finally clarify to which extent cardiomyocytes actually regenerate. We have established reliable methods for assessment of cardiomyocyte numbers, using stereological principles to derive the number from tissue sections. We will determine cardiomyocyte numbers at several time points after heart cryoinjury, and thus we will not only be able to answer whether cardiomyocytes fully regenerate but also the time frame in which this happens and how this relates to wound tissue resolution.In addition, we plan to tackle another intriguing yet unanswered fundamental question in zebrafish heart regeneration research. In many regenerating systems, cells display intriguing cellular plasticity, including examples of dedifferentiation of mature cells to a multipotent state. While differentiated cardiomyocytes have been shown by genetic lineage tracing to be the precursors of newly forming cardiomyocytes during heart regeneration, it is unknown whether they can also give rise to other cells types during regeneration. This is because all transgenic lines that have so far been used for Cre-lox based cardiomyocyte lineage tracing were only expressed in cardiomyocytes. Thus, potential transdifferentiation to other cell types would have been missed. We have created a new transgenic Cre reponder line that is expressed in the major cell types of the heart. Using this tool we will ask whether cardiomyocytes can contribute to the endothelial, epicardial or fibroblast lineage during regeneration.Together the work proposed here will settle two fundamental open questions related to the cellular mechanisms of zebrafish heart regeneration, and thus will allow for a much-improved evaluation of the usefulness of this model.