Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume

Atrial natriuretic peptide (nppa/anf) and brain natriuretic peptide (nppb/bnp) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy; however, their genomic location in cis has impeded formal analysis. Using genome editing, we have generated mutants for nppa and nppb, and found that single mutants were indistinguishable from wild type, whereas nppa/nppb double mutants displayed heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4, tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirmed cardiac jelly expansion in nppa/nppb double mutants. Finally, bmp4 knockdown rescued the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.