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

Abstract

Atrial natriuretic peptide (<i>nppa/anf</i>) and brain natriuretic peptide (<i>nppb/bnp</i>) 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 <i>in cis</i> has impeded formal analysis. Using genome editing, we have generated mutants for <i>nppa</i> and <i>nppb</i>, and found that single mutants were indistinguishable from wild type, whereas <i>nppa</i>/<i>nppb</i> double mutants displayed heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of <i>bmp4</i>, <i>tbx2b</i>, <i>has2</i> and <i>versican</i> 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 <i>nppa</i>/<i>nppb</i> double mutants. Finally, <i>bmp4</i> knockdown rescued the expansion of <i>has2</i> expression and cardiac jelly in double mutants. This definitively shows that <i>nppa</i> and <i>nppb</i> function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber.