Genetic Interaction between pku300 and fbn2b Controls Endocardial Cell Proliferation and Valve Development in Zebrafish

Abstract

SummaryAbnormal cardiac valve morphogenesis is a common cause of human congenital heart disease. 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. We found that scotch tape te382 (sco te382 ) encodes a novel transmembrane protein that is crucial for endocardial cell proliferation and heart valve development. The zebrafish sco te382 mutant showed diminished endocardial cell proliferation, lack of heart valve leaflets and abnormal common cardinal and caudal veins. Positional cloning revealed a C946T nonsense mutation of a novel gene pku300 in the sco te382 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 ,570 kb away from pku300 in sco te382 mutants. The genetic mutant sco pku300 , derived from sco te382 , 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 sco te382 genetic mutants. pku300 transgenic expression in endocardial and endothelial cells, but not myocardial cells, partially rescued the atrial endocardial defects in sco te382 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 crucial 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.

Mechanosensitive Notch-Dll4 and Klf2-Wnt9 signaling pathways intersect in guiding valvulogenesis in zebrafish

“…To assess Dll4 expression in the prevalvular endocardium, we first compared the expression of a Dll4 reporter transgene Tg( Dll4:Gal4 ) pbb65 ; Tg( 5xUAS:EGFP ) nkuasgfp1a (hereafter referred to as Dll4 reporter) to that of the Notch activity reporter Tg( TP1:VenusPEST ) s940 ( Ninov et al., 2012 ) and canonical Wnt reporter Tg( 7xTCF-Xla.Siam:nlsmCherry ) ia5 (hereafter referred to as TCF reporter) ( Moro et al., 2012 ). The expression of the Dll4 reporter reflects the endogenous dll4 expression in the embryonic heart as previously described ( Wang et al., 2013 ) ( Figures S1 A and S1A′). Imaging of the atrioventricular endocardium at single-cell resolution revealed a dispersed pattern of Dll4 activity in prevalvular endocardial cells at 48 hpf.…”

Remodeling of Mitochondrial Flashes in Muscular Development and Dystrophy in Zebrafish

“…Whole-mount immunofluorescence was performed by a standard procedure [ 25 ]. Primary antibodies were used at different dilutions: anti-chicken myosin heavy chain (all fast isoforms) mouse monoclonal antibody with 1:10 dilution (F59; DSHB), anti-chicken myosin light chain 1 and 3f mouse monoclonal antibody with 1:10 dilution (F310; DSHB), anti-dystrophin 1:500 (Cat# D8043, MANDRA1; Sigma), anti-DRP1 1:400 (Cat# NB110-55288, Novas).…”

The Polycomb group gene rnf2 is essential for central and enteric neural system development in zebrafish

“…The neurons were detected by using rabbit anti-HuC/D (1:200) as primary antibody and DyLight 555 goat anti-mouse IgG (1:350, Abbkine, United States) as secondary antibody. For whole-mount immunostaining, embryos were digested in PBS containing 10 g/ml proteinase K, 0.1% Tween20 and blocked in PBS containing 10% normal goat serum, 0.5% DMSO and 0.3% Triton X-100 ( Wang et al, 2013 ). Images were taken with a confocal laser scanning microscope (Leica SP8 DLS).…”