Mechanosensitive Notch-Dll4 and Klf2-Wnt9 Signaling Pathways Intersect in Guiding Valvulogenesis in Zebrafish

Abstract

In the zebrafish embryo, the onset of blood flow generates fluid shear stress on endocardial cells, which are specialized endothelial cells that line the interior of the heart. This mechanical stimulation activates signaling pathways that play crucial roles in atrioventricular valvulogenesis. High levels of fluid shear stress activate both Notch and Klf2 signaling in endocardial cells, causing some to ingress into the cardiac jelly and initiate valvulogenesis. But it remains unclear why only specific, individual cells respond this way, and how the mechanosensitive Notch and Klf2 pathways contribute to the process. Here, we show that lateral inhibition between endocardial cells, mediated by Notch, singles out Delta-like 4-positive endocardial cells. These cells ingress into the cardiac jelly where they form an abluminal cell population. High Notch activity prevents this cell behavior. We also find that Delta-like 4-positive cells only ingress in response to Wnt9a, which is produced in parallel through an Erk5-Klf2-Wnt9a signaling cascade also activated by blood flow. Our work explains how mechanical stimulation activates parallel mechanosensitive signaling pathways that cooperate and produce binary effects by driving endocardial cells towards either luminal or abluminal fates. Ultimately, these cell fate decisions sculpt cardiac valve leaflets.