Bioengineering approach to study the role of cell migration during zebrafish heart regneration

Zebrafish heart regeneration remains one of the most interesting phenomena of the 21st century. Considering the extremely high rate of deaths due to cardiovascular diseases in the developed countries, 1 out of every 3 people, understanding natural cardiac regeneration would address a worldwide challenge. Even though many aspects of zebrafish heart regeneration have been elucidated, there are still many open questions to be answered. Among these, the work presented here focuses on understanding cell migration mechanisms of cardiomyocytes and epicardial cells during heart regeneration. The first approach involves the development of a cardiomyocyte-specific, photoinducible Cre/lox genetic labeling system and its use in lineage tracing of embryonic cardiomyocytes during heart development and regeneration. By using this method we showed that cardiomyocytes labeled in embryonic hearts survive and contribute to all myocardial layers of the adult zebrafish heart. Moreover, lineage tracing during regeneration showed that only cardiomyocytes immediately adjacent to the injury site contribute to the regeneration, and cardiomyocyte fate is extensively predetermined, with cells from each myocardial layer giving rise to cells that retain their layer identity in the regenerated myocardium. Then, we showed that by coupling this labeling system to three-photon microscopy activation, we can perform prospective labeling, and increase the spatial resolution of our labeling system. Three-photon illumination has been used for in vivo imaging of deep structures, but whether it can be used for photo-activation had never been tested. Here we showed, theoretically and experimentally for the first time, that three-photon illumination is suitable for activating molecules in deep tissues and improving our system in terms of spatial resolution and prospective labeling. The final approach consisted on developing an ex vivo experimental set up in order to investigate physical characteristics of epicardial cell migration during zebrafish heart regeneration. This method allowed us to measure physical features that are essential for cell migration such as migration velocity and traction forces of the epicardial explants obtained from zebrafish hearts. All the approaches developed in this thesis offer new bioengineering tools to study zebrafish heart regeneration, and reveal new insights on this process. Moreover, these techniques present wide applicability to perform lineage tracing of other cell types during zebrafish heart regeneration or in other biological processes.