Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration.

Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how regenerating cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury. Here, we provide an in-depth analysis of the process of cardiomyocyte invasion using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area. Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages. Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins. The expression of mmp14b , which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue and along the apical surface of the wound. Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration. They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue.

Comparative single-cell profiling reveals distinct cardiac resident macrophages essential for zebrafish heart regeneration.

Zebrafish exhibit a robust ability to regenerate their hearts following injury, and the immune system plays a key role in this process. We previously showed that delaying macrophage recruitment by clodronate liposome (-1d_CL, macrophage-delayed model) impairs neutrophil resolution and heart regeneration, even when the infiltrating macrophage number was restored within the first week post injury (Lai et al., 2017). It is thus intriguing to learn the regenerative macrophage property by comparing these late macrophages vs. control macrophages during cardiac repair. Here, we further investigate the mechanistic insights of heart regeneration by comparing the non-regenerative macrophage-delayed model with regenerative controls. Temporal RNAseq analyses revealed that -1d_CL treatment led to disrupted inflammatory resolution, reactive oxygen species homeostasis, and energy metabolism during cardiac repair. Comparative single-cell RNAseq profiling of inflammatory cells from regenerative vs. non-regenerative hearts further identified heterogeneous macrophages and neutrophils, showing alternative activation and cellular crosstalk leading to neutrophil retention and chronic inflammation. Among macrophages, two residential subpopulations (hbaa+ Mac and timp4.3+ Mac 3) were enriched only in regenerative hearts and barely recovered after +1d_CL treatment. To deplete the resident macrophage without delaying the circulating macrophage recruitment, we established the resident macrophage-deficient model by administrating CL earlier at 8 d (-8d_CL) before cryoinjury. Strikingly, resident macrophage-deficient zebrafish still exhibited defects in revascularization, cardiomyocyte survival, debris clearance, and extracellular matrix remodeling/scar resolution without functional compensation from the circulating/monocyte-derived macrophages. Our results characterized the diverse function and interaction between inflammatory cells and identified unique resident macrophages prerequisite for zebrafish heart regeneration.

Modulation of VEGFA Signaling During Heart Regeneration in Zebrafish.

Over the last decades, myocardial infarction and heart failure have accounted every year for millions of deaths worldwide. After a coronary occlusion, the lack of blood supply to downstream muscle leads to cell death and scarring. To date, several pro-angiogenic factors have been tested to stimulate reperfusion of the affected myocardium, VEGFA being one of the most extensively studied. Given the unsuccessful outcomes of clinical trials, understanding how cardiac revascularization takes place in models with endogenous regenerative capacity holds the key to devising more efficient therapies. Here, we summarize the main findings on VEGFA's role during cardiac repair and regeneration, with a particular focus on zebrafish as a regenerative model. Moreover, we provide a comprehensive overview of available tools to modulate Vegfa expression and action in zebrafish regeneration studies. Understanding the role of Vegfa during zebrafish heart regeneration may help devise efficient therapies and circumvent current limitations in using VEGFA for therapeutic angiogenesis approaches.

Zebrafish Scube1 and Scube2 cooperate in promoting Vegfa signalling during embryonic vascularization.

AIMS: The secreted and membrane-anchored signal peptide-CUB-EGF domain-containing proteins (SCUBE) gene family composed of three members was originally identified from endothelial cells (ECs). We recently showed that membrane SCUBE2 binds vascular endothelial growth factor (VEGF) and acts as a co-receptor for VEGF receptor 2 to modulate EC migration, proliferation, and tube formation during postnatal and tumour angiogenesis. However, whether these SCUBE genes cooperate in modulating VEGF signalling during embryonic vascular development remains unknown. METHODS AND RESULTS: To further dissect the genetic interactions of these scube genes, transcription activator-like effector nuclease-mediated genome editing was used to generate knockout (KO) alleles of each scube gene. No overt vascular phenotypes were seen in any single scube KO mutants because of compensation by other scube genes during zebrafish development. However, scube1 and scube2 double KO (DKO) severely impaired EC filopodia extensions, migration, and proliferation, thus disrupting proper vascular lumen formation during vasculogenesis and angiogenesis as well as development of the organ-specific intestinal vasculature. Further genetic, biochemical, and molecular analyses revealed that Scube1 and Scube2 might act cooperatively at the cell-surface receptor level to facilitate Vegfa signalling during zebrafish embryonic vascularization. CONCLUSIONS: We showed for the first time that cooperation between scube1 and scube2 is critical for proper regulation of angiogenic cell behaviours and formation of functional vessels during zebrafish embryonic development.

Cardiomyocyte heterogeneity during zebrafish development and regeneration.

Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.

Genetic compensation triggered by mutant mRNA degradation.

Genetic robustness, or the ability of an organism to maintain fitness in the presence of harmful mutations, can be achieved via protein feedback loops. Previous work has suggested that organisms may also respond to mutations by transcriptional adaptation, a process by which related gene(s) are upregulated independently of protein feedback loops. However, the prevalence of transcriptional adaptation and its underlying molecular mechanisms are unknown. Here, by analysing several models of transcriptional adaptation in zebrafish and mouse, we uncover a requirement for mutant mRNA degradation. Alleles that fail to transcribe the mutated gene do not exhibit transcriptional adaptation, and these alleles give rise to more severe phenotypes than alleles displaying mutant mRNA decay. Transcriptome analysis in alleles displaying mutant mRNA decay reveals the upregulation of a substantial proportion of the genes that exhibit sequence similarity with the mutated gene's mRNA, suggesting a sequence-dependent mechanism. These findings have implications for our understanding of disease-causing mutations, and will help in the design of mutant alleles with minimal transcriptional adaptation-derived compensation.

Immune responses in cardiac repair and regeneration: a comparative point of view.

Immediately after cardiac injury, the immune system plays major roles in repair and regeneration as it becomes involved in a number of processes including damage-associated signaling, inflammation, revascularization, cardiomyocyte dedifferentiation and replenishment, and fibrotic scar formation/resolution. Recent studies have revealed that different immune responses occur in the various experimental models capable or incapable of cardiac regeneration, and that harnessing these immune responses might improve cardiac repair. In light of this concept, this review analyzes current knowledge about the immune responses to cardiac injury from a comparative perspective. Insights gained from such comparative analyses may provide ways to modulate the immune response as a potential therapeutic strategy for cardiac disease.

Conditional mutagenesis by oligonucleotide-mediated integration of loxP sites in zebrafish.

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration.

Reciprocal analyses in zebrafish and medaka reveal that harnessing the immune response promotes cardiac regeneration.

Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.

Fast revascularization of the injured area is essential to support zebrafish heart regeneration.

Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization.