Proper growth, septation, and maturation of the cardiac outflow tract (OFT) into valved aortic and pulmonary outlets are essential for oxygenated circulation after birth. 1-2% of live births and up to 30% of pre-term fetal deaths have congenital heart defects, many of which affect the remodeling of the valvuloseptal primordial tissues, called the proximal and distal outflow cushions. Despite much effort uncovering the genetic basis of early OFT cushion formation, this understanding has not explained the clinically relevant phases of growth, condensation and elongation into valves and septa. One reason for this appears to be the domination of conditional and collective signaling mechanisms that are well accessible by genetic approaches. Mechanical forces (shear stress, pressure, tension) are ever present during this complex period of OFT growth and remodeling, but to date no studies have investigated these key interactions, especially for their contributions to OFT defects. We believe that clinically relevant OFT remodeling arise from improper cushion endocardial and/or mesenchymal sensation of and/or response to their local mechanical environment, which in turn drives the incorrect signaling programs. The Butcher lab has pioneered innovative technology 1) to quantify local in vivo mechanical forces within this OFT region and register them with local in situ gene/protein expression, 2) to not-invasively visualize and precisely ablate intracardiac tissues without collateral damage in vivo, and 3) to directly test mechanobiological mechanisms of endocardial cushion growth and remodeling ex vivo. The preliminary data in this proposal present evidence of two mechanoregulated molecular switches that potentiate between OFT cushion proliferation and differentiation, which motivates the novel hypothesis that local mechanosensaton operates molecular switches to control sizing, shape, and stratification of the outflow valves and septa. Aim 1 will implement innovative non- invasive laser photoablations of the formed proximal or distal cushions of the avian OFT to create genetically unbiased clinically relevant outflow tract malformations. We will then quantitatively analyze and register their hemodynamic, morphological and phenotypic changes. We will further apply novel deconvolution integration of sc-Seq and slide-seq to reveal unprecedented spatio-temporal resolution of the cellular course of malformation, and elaborate how known and newly discovered molecular regulatory programs associate with local mechanical stress changes. Aim 2 will test the mechanistic causailty of the mechanotransduction operated molecular switches in the OFT cushion endocardium via shear stress patterns. Aim 3 will test the operation of different mechanobiogical switches in cushion mesenchyme via tension/compression. using high throughput ex vivo organ cultures. The findings from these studies will substantally advance our understanding of mechanoregulation and conditional signalin...