SUMMARY Tetralogy of Fallot (TOF) is a critical congenital heart malformation that typically requires intervention soon after birth and affects about 1,660 newborns per year in the US. In TOF, blood flow through the pulmonary artery is reduced or blocked (due to pulmonary stenosis or atresia), while blood flow through the aorta is increased. Prenatally, TOF-induced abnormal blood flow progressively leads to pathological cardiac valve tissue remodeling. The semilunar (aortic and pulmonary) valve leaflets and the great arteries (the aorta and pulmonary artery) are particularly susceptible to abnormal blood flow. This is because fetal stages are a critical time when extracellular matrix components that dictate valve integrity and function, such as collagen and elastin, deposit and organize within valve tissues, and smooth muscle cells are differentiating and organizing in supporting arteries. However, how semilunar valves and great arteries remodel in response to TOF-induced abnormal hemodynamic loads during fetal stages remains unknown. To address this knowledge gap, this project will use a chick embryonic model of TOF that reproduces the characteristic heart morphologies and flow patterns found in human fetuses with TOF. Avian embryos are amenable to in vivo imaging and allow tightly controlled longitudinal studies in ovo. Moreover, valve leaflets in human and chick exhibit the same characteristic layers, which similarly arrange in response to blood flow cues during fetal development in human and pre-hatching in chick. The overarching hypothesis of this proposal is that before birth abnormal blood flow in TOF leads to aberrant, disorganized deposition of collagen and elastin fibers in semilunar valve leaflets, and abnormal smooth muscle cell localization in supporting great artery tissues. Three aims are proposed: 1) Determine characteristic blood flow patterns in fetal hearts with TOF using advanced in vivo imaging and computational modeling; 2) Assess abnormal tissue remodeling of semilunar valves and great arteries in TOF through a combination of advanced multiscale microscopy; 3) Develop predictive models of flow-induced semilunar valve remodeling in TOF. Through a novel combination of multiscale imaging and computational modeling, this project will elucidate how TOF-induced abnormal blood flow leads to (and exacerbates) semilunar valve and great artery tissue anomalies. In the future, findings from this project will improve fetal diagnosis and prediction of prenatal pathological valve remodeling, enabling better decisions of when and how to intervene to avoid or even reverse pathological valve remodeling, and will inform valve regeneration efforts.