Abnormal cardiac valve morphogenesis is a common cause of human congenital heart diseases. The molecular mechanisms regulating
endocardial cell proliferation and differentiation into cardiac valves remain largely unknown, although great progress has
been made on the endocardial contribution to the atrioventricular cushion and valve formation. Here, we found that scotch tapete382 (scote382) encoded a novel transmembrane protein critical for endocardial cell proliferation and heart valve development. The zebrafish
scote382 mutant showed diminished endocardial cell proliferation, lack of heart valve leaflets, abnormal common cardinal and caudal
veins. Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the scote382 locus, which encoded a 540 amino-acid protein on cell membranes with one putative transmembrane domain and three IgG domains.
A known G3935T missense mutation of fbn2b was also found <570kb away from pku300 in scote382 mutants. The genetic mutant scopku300, derived from scote382, only had the C946T mutation of pku300 and showed reduced numbers of atrial endocardial cells and an abnormal common cardinal vein. Morpholino knockdown of fbn2b led to fewer atrial endocardial cells and an abnormal caudal vein. Knockdown of both pku300 and fbn2b phenocopied these phenotypes in scote382 genetic mutants. pku300 transgenic expression in endocardial/endothelial cells, but not myocardial cells, partially rescued the atrial endocardial
defects in scote382 mutants. Mechanistically, pku300 and fbn2b were required for endocardial cell proliferation, endocardial Notch signaling, and the proper formation of endocardial cell
adhesion and tight junctions, all of which are critical for cardiac valve development. We conclude that pku300 and fbn2b represent the few genes capable of regulating endocardial cell proliferation and signaling in zebrafish cardiac valve development.