In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.

In mammals, myocardial cell death due to infarction results in scar formation and little regenerative response. In contrast, zebrafish have a high capacity to regenerate the heart after surgical resection of myocardial tissue. However, whether zebrafish can also regenerate lesions caused by cell death has not been tested. Here, we present a simple method for induction of necrotic lesions in the adult zebrafish heart based on cryoinjury. Despite widespread tissue death and loss of cardiomyocytes caused by these lesions, zebrafish display a robust regenerative response, which results in substantial clearing of the necrotic tissue and little scar formation. The cellular mechanisms underlying regeneration appear to be similar to those activated in response to ventricular resection. In particular, the epicardium activates a developmental gene program, proliferates and covers the lesion. Concomitantly, mature uninjured cardiomyocytes become proliferative and invade the lesion. Our injury model will be a useful tool to study the molecular mechanisms of natural heart regeneration in response to necrotic cell death.

Das Herz eines Zebrafishs ist bemerkenswert, da es sich nach einer Verletzung vollständig regenerieren kann. Der Regenerationsprozess wird von Fibrose begleitet - der Bildung von überschüssigem Gewebe der extrazellulären Matrix (ECM). Anders als bei Säugetieren ist die Fibrose im Zebrafish nur transient. Viele Signalwege wurden identifiziert, die an der Herzregeneration beteiligt sind. Allerdings sind die Zelltypen, insbesondere Nicht-Kardiomyozyten, die für die Regulation des Regenerationsprozesses verantwortlich sind, weitgehend unbekannt. In dieser Arbeit haben wir systematisch alle Zelltypen des gesunden und des verletzten Zebrafischherzens mithilfe einer auf Mikrofluidik basierenden Hoch-Durchsatz- Einzelzell-RNA-Sequenzierung bestimmt. Wir fanden eine große Heterogenität von ECM-produzierenden Zellen, einschließlich einer Reihe neuer Fibroblasten, die nach einer Verletzung mit unterschiedlicher Dynamik auftreten. Wir konnten aktivierte Fibroblasten beschreiben und Fibroblasten-Subtypen mit einer pro-regenerativen Funktion identifizieren. Darüber hinaus haben wir eine Methode entwickelt, um die Transkriptomanalyse und die Rekonstruktion von Zell-Verwandtschaften auf Einzelzellebene zu kombinieren. Unter Verwendung der CRISPR-Cas9-Technologie führten wir zufällige Mutationen in bekannte und ubiquitär transkribierte DNA-Loci während der Embryonalentwicklung von Zebrafischen ein. Diese Mutationen dienten als zellspezifische, permanente und vererbbare “Barcodes”, die zu einem späteren Zeitpunkt erfasst werden konnten. Mit maßgeschneiderten Analysealgorithmen konnten wir dann Stammbäume der sequenzierten Einzelzellen erstellen. Mit dieser neuen Methode haben wir gezeigt, dass im sich regenerierenden Zebrafischherz ECM-produzierende Zellpopulationen entweder mit dem Epi- oder mit dem Endokardium verwandt sind. Zusätzlich entdeckten wir, dass vom Endokardium abgeleitete Zelltypen vom Wnt-Signalweg abhängig sind. / The zebrafish heart has the remarkable capacity to fully regenerate after injury. The regeneration process is accompanied by fibrosis - the formation of excess extracellular matrix (ECM) tissue, at the injury site. Unlike in mammals, the fibrosis of the zebrafish heart is only transient. While many pathways involved in heart regeneration have been identified, the cell types, especially non-myocytes, responsible for the regulation of the regenerative process have largely remained elusive. Here, we systematically determined all different cell types of both the healthy and cryo-injured zebrafish heart in its regeneration process using microfluidics based high-throughput single-cell RNA sequencing. We found a considerable heterogeneity of ECM producing cells, including a number of novel fibroblast cell types which appear with different dynamics after injury. We could describe activated fibroblasts that extensively switch on gene modules for ECM production and identify fibroblast sub- types with a pro-regenerative function. Furthermore, we developed a method that is capable of combining transcriptome analysis with lineage tracing on the single-cell level. Using CRISPR-Cas9 technology, we introduced random mutations into known and ubiquitously transcribed DNA loci during the zebrafish embryonic development. These mutations served as cell-unique, permanent, and heritable barcodes that could be captured at a later stage simultaneously with the transcriptome by high-throughput single-cell RNA sequencing. With custom tailored analysis algorithms, we were then able to build a developmental lineage tree of the sequenced single cells. Using this new method, we revealed that in the regenerating zebrafish heart, ECM contributing cell populations derive either from the epi- or the endocardium. Additionally, we discovered in a functional experiment that endocardial derived cell types are Wnt signaling dependent.