Wang, J.; Brown, A. L.; Zhao, P.; Zheng, C. Z.; Chen, J.-N.; Zhou, B.; Liu, J.; Yokota, T.; Kaiser, A. D.; Park, S.-K.; Marsden, A. L.; Hsiai, T. K.

How chamber-specific hemodynamic shear forces coordinate the spatial and temporal patterning of cardiac valves remains poorly understood. The zebrafish atrioventricular (AV) valve transitions from 2-cusp to 4-cusp at 14 days post-fertilization (dpf) through commissural remodeling, differential cell proliferation, and chondrogenic matrix secretion. Orthogonal valvular axes are present at 25 hpf (hours), preceding endocardial cushion formation. AV canal remodeling aligns the shear stress with leaflet formation at 48 hpf and 14 dpf. At 28 hpf, the ventricle develops functional syncytium, while the atrial peristalsis remains and is necessary for the Klf2a-Snai1b signaling in Notch-negative and ccm1-positive endocardial cells. Without atrial contraction, the 2- to 4-cusp transition (86.7%) fails to occur. In the absence of ventricular contraction and relaxation, the formation of the first pair of leaflets (100%) does not initiate. As a corollary, we demonstrate that KLF4/SNAI1 expression at the commissure is conserved in the mammalian AV 2- to 4-cushion transition.