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Zebrafish can regenerate their damaged hearts throughout their lifespan. It is, however, unknown, whether regeneration remains effective when challenged with successive cycles of cardiac damage in the same animals. Here, we assessed ventricular restoration after two, three and six cryoinjuries interspaced by recovery periods. Using transgenic cell-lineage tracing analysis, we demonstrated that the second cryoinjury damages the regenerated area from the preceding injury, validating the experimental approach. We identified that after multiple cryoinjuries, all hearts regrow a thickened myocardium, similarly to hearts after one cryoinjury. However, the efficiency of scar resorption decreased with the number of repeated cryoinjuries. After six cryoinjuries, all examined hearts failed to completely resolve the fibrotic tissue, demonstrating reduced myocardial restoration. This phenotype was associated with enhanced recruitment of neutrophils and decreased cardiomyocyte proliferation and dedifferentiation at the early regenerative phase. Furthermore, we found that each repeated cryoinjury increased the accumulation of collagen at the injury site. Our analysis demonstrates that the cardiac regenerative program can be successfully activated many times, despite a persisting scar in the wounded area. This finding provides a new perspective for regenerative therapies, aiming in stimulation of organ regeneration in the presence of fibrotic tissue in mammalian models and humans. [ABSTRACT FROM AUTHOR]

Zebrafish heart regeneration depends on cardiac cell proliferation, epicardium activation and transient reparative tissue deposition. The contribution and the regulation of specific collagen types during the regenerative process, however, remain poorly characterized. Here, we identified that the non-fibrillar type XII collagen, which serves as a matrix-bridging component, is expressed in the epicardium of the zebrafish heart, and is boosted after cryoinjury-induced ventricular damage. During heart regeneration, an intense deposition of Collagen XII covers the outer epicardial cap and the interstitial reparative tissue. Analysis of the activated epicardium and fibroblast markers revealed a heterogeneous cellular origin of Collagen XII. Interestingly, this matrix-bridging collagen co-localized with fibrillar type I collagen and several glycoproteins in the post-injury zone, suggesting its role in tissue cohesion. Using SB431542, a selective inhibitor of the TGF-β receptor, we showed that while the inhibitor treatment did not affect the expression of collagen 12 and collagen 1a2 in the epicardium, it completely suppressed the induction of both genes in the fibrotic tissue. This suggests that distinct mechanisms might regulate collagen expression in the outer heart layer and the inner injury zone. On the basis of this study, we postulate that the TGF-β signaling pathway induces and coordinates formation of a transient collagenous network that comprises fibril-forming Collagen I and fiber-associated Collagen XII, both of which contribute to the reparative matrix of the regenerating zebrafish heart. [ABSTRACT FROM AUTHOR]

Background Epimorphic regeneration of a missing appendage in fish and urodele amphibians involves the creation of a blastema, a heterogeneous pool of progenitor cells underneath the wound epidermis. Current evidence indicates that the blastema arises by dedifferentiation of stump tissues in the vicinity of the amputation. In response to tissue loss, silenced developmental programs are reactivated to form a near-perfect copy of the missing body part. However, the importance of chromatin regulation during epimorphic regeneration remains poorly understood. Results Here, we show that specific components of the Nucleosome Remodeling and Deacetylase complex (NuRD) are required for fin regeneration in zebrafish. We found that transcripts of the chromatin remodeler chd4a/Mi-2, the histone deacetylase hdac1/HDAC1/2, the retinoblastoma-binding protein rbb4/RBBP4/7, and the metastasis-associated antigen mta2/MTA were specifically co-induced in the blastema during adult and embryonic fin regeneration and displayed a similar spatial and temporal expression pattern. In addition, chemical inhibition of Hdac1 and morpholino-mediated knockdown of chd4a, mta2, and rbb4 impaired regenerative outgrowth, resulting in reduction of blastema cell proliferation and in differentiation defects. Conclusion Altogether, our data suggest that specialized NuRD components are induced in the blastema during fin regeneration and are involved in blastema cell proliferation and redifferentiation of osteoblast precursor cells. These results provide in vivo evidence for the involvement of key epigenetic factors in the cellular reprogramming processes, occurring during epimorphic regeneration in zebrafish. [ABSTRACT FROM AUTHOR]

Zebrafish heart regeneration relies on the capacity of cardiomyocytes to proliferate upon injury. To understand the principles of this process after cryoinjury-induced myocardial infarction, we established a spatio-temporal map of mitotic cardiomyocytes and their differentiation dynamics. Immunodetection of phosphohistone H3 and embryonic ventricular heavy chain myosin highlighted two distinct regenerative processes during the early phase of regeneration. The injury-abutting zone comprises a population of cardiac cells that reactivates the expression of embryo-specific sarcomeric proteins and it displays a 10-fold higher mitotic activity in comparison to the injury-remote zone. The undifferentiated cardiomyocytes resemble a blastema-like structure between the original and wound tissues. They integrate with the fibrotic tissue through the fibronectin-tenascin C extracellular matrix, and with the mature cardiomyocytes through upregulation of the tight junction marker, connexin 43. During the advanced regenerative phase, the population of undifferentiated cardiomyocytes disperses within the regenerating myocardium and it is not detected after the termination of regeneration. Although the blastema represents a transient landmark of the regenerating ventricle, the remaining mature myocardium also displays an enhanced mitotic index when compared to uninjured hearts. This suggests an unexpected contribution of a global proliferative activity to restore the impaired cardiac function. Based on these findings, we propose a new model of zebrafish heart regeneration that involves a combination of blastema-dependent epimorphosis and a compensatory organ-wide response. [ABSTRACT FROM AUTHOR]