Abstract Body: Background: Congenital bicuspid valve leaflets create aberrant biomechanical microenvironments that accelerate valvular calcification in humans. How hemodynamic shear forces coordinate the spatial and temporal patterning of valvular cusp development remains poorly understood. The zebrafish atrioventricular (AV) valve transitions from two- to four-cusp at 14 days post-fertilization (dpf), serving as a simplified model for flow-mediated patterning of valvular leaflets.

Hypothesis: Chamber-specific (ventricular vs. atrial) shear stress mediates the formation of distinct pairs of the AV leaflets.

Method: We used transgenic reporter lines, laser microdissection-assisted RNA seq, and Stereo-seq validation to elucidate the mechanism of 2- to 4- cusp transistion. We then test our hypothesis using in vivo light-sheet imaging, computational modeling, and mutants of chamber-specific myosin heavy chains (wea - myh6 and haf - myh7).

Results: Zebrafish AV valve 2- to 4-cusp transistion occurs 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 shear stress with the valvular axes prior to leaflet formation at 48 hpf and 14 dpf (Figure 1). At 28 hpf, the ventricle develops functional syncytium, while the atrial contraction remains peristaltic. Mutants with non-contracting ventricle (haf) and atrium (wea) reveal that atrial peristalsis is necessary for the Klf2a-Snai1b signaling in Notch-negative and ccm1-positive endocardial cells to initiate the 2- to 4-cusp transition. Meanwhile, ventricular contraction is required for initiating the first pair of AV leaflets. As a coronary, we demonstrate that KLF4/SNAI1 expression at the commissure is conserved in the mammalian AV 2- to 4-cushion transition (Figure 2).