Highlights d Flt4 is suppressed by fluid shear stress within the atrioventricular valve leaflet d Fluid shear stress activates Notch and Klf2a signaling in an independent manner d The Klf2a and Notch pathways repress Flt4 d Notch and Flt4 work antagonistically to shape the atrioventricular valve

Highlights d Atrioventricular valvulogenesis in zebrafish is regulated by Notch and Klf2 pathways d Notch-mediated lateral inhibition singles out Delta-like-4positive endocardial cells d These cells ingress into the cardiac jelly in response to Erk5-Klf2-Wnt9a signaling d Frizzled9b makes presumptive ingressing endocardial cells competent to respond to Wnt9a

Steinberg's differential adhesion hypothesis suggests that adhesive mechanisms are important for sorting of cells and tissues during morphogenesis (Steinberg, 2007). During zebrafish vasculogenesis, endothelial cells sort into arterial and venous vessel beds but it is unknown whether this involves adhesive mechanisms. Claudins are tight junction proteins regulating the permeability of epithelial and endothelial tissue barriers. Previously, the roles of claudins during organ development have exclusively been related to their canonical functions in determining paracellular permeability. Here, we use atomic force microscopy to quantify claudin-5-dependent adhesion and find that this strongly contributes to the adhesive forces between arterial endothelial cells. Based on genetic manipulations, we reveal a non-canonical role of Claudin-5a during zebrafish vasculogenesis, which involves the regulation of adhesive forces between adjacent dorsal aortic endothelial cells. In vitro and in vivo studies demonstrate that loss of claudin-5 results in increased motility of dorsal aorta endothelial cells and in a failure of the dorsal aorta to lumenize. Our findings uncover a novel role of claudin-5 in limiting arterial endothelial cell motility, which goes beyond its traditional sealing function during embryonic development.

Aims The present study aims to characterise the genetic risk architecture of bicuspid aortic valve (BAV) disease, the most common congenital heart defect. Methods and results We carried out a genome-wide association study (GWAS) including 2,236 BAV patients and 11,604 controls. This led to the identification of a new risk locus for BAV on chromosome 3q29. The single nucleotide polymorphism (SNP) rs2550262 was genome-wide significant BAV-associated (P = 3.49 × 10−08) and was replicated in an independent case-control sample. The risk locus encodes a deleterious missense variant in MUC4 (p.Ala4821Ser), a gene that is involved in epithelial-to-mesenchymal transformation. Mechanistical studies in zebrafish revealed that loss of Muc4 led to a delay in cardiac valvular development suggesting that loss of MUC4 may also play a role in aortic valve malformation. The GWAS also confirmed previously reported BAV risk loci at PALMD (P = 3.97 × 10−16), GATA4 (P = 1.61 × 10−09), and TEX41 (P = 7.68 × 10−04). In addition, the genetic BAV architecture was examined beyond the single-marker level revealing that a substantial fraction of BAV heritability is polygenic and approximately 20% of the observed heritability can be explained by our GWAS data. Furthermore, we used the largest human single cell atlas for foetal gene expression and show that the transcriptome profile in endothelial cells is a major source contributing to BAV pathology. Conclusion Our study provides a deeper understanding of the genetic risk architecture of BAV formation on the single-marker and polygenic level.

No abstract