Adma and Ackr3a regulate cardiac lymphatic vessel formation and function during heart development and regeneration.

The lymphatic vessels in the heart, like other lymphatics in the body are essential to transport fluid, large molecules and immune cells. It remains unclear how these lymphatics form and whether defects in these lymphatic endothelial cells can cause congenital heart disease. To determine the gene expression patterns in these cardiac lymphatic endothelial cells in the heart during development and regeneration, we performed spatial transcriptomics using sequential fluorescence in situ hybridization (seqFISH) on the zebrafish heart. We discovered that adrenomedullin a (adma), a peptide hormone gene is mainly expressed in the epicardium, the outer layer of the heart, adjacent to cardiac lymphatics. The adma mutant fish develop severe edema and dilated outflow tracts with mis-patterned and reduced lymphatics, while their ventricular lymphatics are dilated with fewer sprouts, increased lyve1b expression, and altered lymphatic subpopulation composition. Loss of ackr3a, which encodes an Adm decoy receptor, leads to opposite phenotypes to adma mutants and increases lymphatic sprouts. The expression of ackr3a in the epicardium is important and its over-expression results in decreased lymphatics. During heart regeneration, adma and ackr3a are expressed in epicardium-derived cells within the wound. The lymphatic function in neutrophil clearance, is reduced in adma mutants but increased in ackr3a mutants. Our findings uncover the heterogeneity of cardiac lymphatic endothelial cells and the critical role of Adm signaling in cardiac lymphatic vessel formation.