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

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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.