Project Summary Coronary and cardiac lymphatic vessels constitute the vasculature of the heart and provide a supporting environment for heart development and myocardial regeneration. Coronary arteries play critical roles in maintaining heart functions by providing a continuous supply of oxygen and nutrients. The lymphatic vasculature is a specialized network of vessels that drains fluid from tissues and enables immune-cell trafficking and surveillance throughout the body. Although recent clinical observations suggest that lymphatic obstruction might be associated with heart diseases and myocardial edema decreases cardiac output and can cause interstitial fibrosis, the importance of the cardiac lymphatic system has been largely underappreciated. It is not clear how cardiac lymphatic vessels form and function during heart development, repair, and regeneration. We have characterized cardiac lymphatic vessel formation using zebrafish as a model for their remarkable regenerative capacity. In this proposal, we will test the hypothesis that coronary arteries can provide guidance for the formation of lymphatic vessels during zebrafish heart development and regeneration and Cxcr4a and Ackr3a positively and negatively regulate these processes. We will determine the functions of Ackr3a in coronary and cardiac lymphatic vessel development and heart regeneration. Using novel zebrafish CRISPR mutants we generated, we will analyze new candidate genes in coronary and cardiac lymphatic vessel development. With these zebrafish as tools, we will assess whether cardiac lymphatic vessels can affect heart regeneration by modulating fluid homeostasis and immune cell clearance. Our proposed research will not only provide insights into the molecular mechanisms of coronary and cardiac lymphatic vessel formation during heart development and in response to cardiac injury, it might lead to promising targets for future therapeutic approaches.