Live imaging of the immune response to heart injury in larval zebrafish reveals a multi-stage model of neutrophil and macrophage migration

Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in vivo in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our in vivo imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our in vivo imaging led to the finding of a novel wound responsive mpeg1+ neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation

Pannexin 1 drives efficient epithelial repair after tissue injury

Epithelial tissues such as lung and skin are exposed to the environment and therefore particularly vulnerable to damage during injury or infection. Rapid repair is therefore essential to restore function and organ homeostasis. Dysregulated epithelial tissue repair occurs in several human disease states, yet how individual cell types communicate and interact to coordinate tissue regeneration is incompletely understood. Here, we show that pannexin 1 (Panx1), a cell membrane channel activated by caspases in dying cells, drives efficient epithelial regeneration after tissue injury by regulating injury-induced epithelial proliferation. Lung airway epithelial injury promotes the Panx1-dependent release of factors including ATP, from dying epithelial cells, which regulates macrophage phenotype after injury. This process, in turn, induces a reparative response in tissue macrophages that includes the induction of the soluble mitogen amphiregulin, which promotes injury-induced epithelial proliferation. Analysis of regenerating lung epithelium identified Panx1-dependent induction of Nras and Bcas2, both of which positively promoted epithelial proliferation and tissue regeneration in vivo. We also established that this role of Panx1 in boosting epithelial repair after injury is conserved between mouse lung and zebrafish tailfin. These data identify a Panx1-mediated communication circuit between epithelial cells and macrophages as a key step in promoting epithelial regeneration after injury

Investigating the role of macrophages in larval zebrafish heart regeneration

Acute myocardial infarction occurs when a coronary artery becomes occluded following atherosclerotic plaque rupture. Consequently, the myocardium suffers ischemic cell death, manifesting as a region of necrotic cardiomyocytes in the left ventricle. Unfortunately, the human heart is unable to replace these lost cardiomyocytes, and this can lead to heart failure. In contrast, zebrafish can completely regenerate their hearts after cardiac injury via dedifferentiation and proliferation of surviving cardiomyocytes. Macrophages are a required cell type for successful zebrafish heart regeneration and so may offer a roadmap to the optimisation of human myocardial repair. However, macrophages exhibit highly plastic and heterogenous phenotypes and are thought to influence healing through a variety of means. To accelerate the study of macrophages in zebrafish heart regeneration, our laboratory has developed a larval zebrafish model of cardiac regeneration. The model uses a laser to induce cardiac injury in 3 days post fertilisation larvae which recover in just 48 hours, as opposed to 2 months in adults. Larval zebrafish are also small and transparent, facilitating unparalleled in vivo imaging. Together, these qualities facilitate rapid and detailed investigation into both heart regeneration and the role of macrophages in heart regeneration. In this study I have therefore performed detailed characterisations of larval zebrafish heart regeneration, macrophage recruitment and the role of macrophages in larval zebrafish heart regeneration. I hypothesised that macrophages would be required for the regeneration of the injured larval heart. To test this hypothesis, I first used a combination of whole larva live imaging and photoconversion-based lineage tracing to map macrophage migration from primary haematopoietic sites to the injured heart. These data showed macrophages to be recruited first from the pericardium and then the caudal haematopoietic tissue, preferentially using the abluminal wall of blood vessels to migrate to the site of injury. I next used macrophage-less irf8-/- larvae and metronidazole-nitroreductase macrophage ablation to test the requirement for macrophages for several important regenerative processes. My data showed macrophages to be required for wound debridement and cardiomyocyte proliferation but not structural and functional recovery of the heart. Using in vivo imaging and a variety of pharmacological and recombinant protein interventions, I found epicardial Vegfaa to be required for cardiomyocyte proliferation and macrophages to be required for early epicardial expansion. Finally, I showed Vegfaa to increase endocardial notch signalling, which is known to drive cardiomyocyte proliferation, thus offering a mechanism for how macrophages may influence this process. These findings reveal a previously unrecognised role of macrophages in epicardial activation, providing a novel target for beneficial therapeutic intervention in the future

Aristocracy in Early T'ang Period.

An amendment to this paper has been published and can be accessed via a link at the top of the paper

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair