Abstract 2170: bungee - A Novel Regulator of Cardiac Valve Formation in Zebrafish
Congenital heart defects are the leading cause of death amongst newborns. Although within the group of cardiovascular malformations a high percentage accounts for defects of heart valves, so far little is known about disease causing genes and their respective function. To characterize new signaling pathways involved in cardiac valve formation, the zebrafish has become an excellent model organism. In a forward genetic screen of zebrafish mutants with defects in cardiac valve formation, we isolated the recessive, embryonic lethal mutant bungee (bngJH177). While first steps of cardiac development proceed normally in bng mutant embryos, around 72 hours post fertilization bng mutant hearts dilate and blood regurgitates, leading to a progressive loss of blood circulation. Histological analysis reveals that bng mutant hearts have a constricted bulbus arteriosus and widened atrio-ventricular canal (AVC), missing endocardial cushions. Altered expression of important endocardial and myocardial markers for heart valve development suggests that the failure of heart valve formation in bng mutant embryos is due to perturbed signalling between endocardial and myocardial cells within the early formation of the AVC. By positional cloning we revealed that the bungee phenotype is caused by a point mutation within a gene encoding a zebrafish serine/threonine protein kinase, leading to a substitution of a thyrosine to an asparagine within the highly conserved kinase domain. In vitro kinase assay displays a severely reduced kinase activity of bng mutant kinase. Injection of wild-type mRNA in bng mutant embryos restores the wild-type phenotype, while injection of a kinase-dead variant is unable to rescue the bng mutant phenotype. Additionally, morpholino-modified antisense oligonucleotides directed against a splice-donor site inside the kinase-domain phenocopies the bng mutant phenotype. By in situ hybridization we demonstrate that expression of the bungee protein kinase within the embryonic heart is restricted to the endocardial cell layer of the atrio-ventricular canal. Here we show for the first time that the bungee kinase activity is crucial for proper development of the atrioventricular canal and the formation cardiac valves.