Three highly related genes, Gata4, Gata5, and Gata6 (referred to here as Gata456) regulate essentially every aspect of cardiac biology, from generation of precardiac mesoderm, specification and differentiation of endocardial, epicardial, and myocardial progenitors, heart tube formation, growth and morphogenesis, septation and valve formation, cardioprotection and hypertrophy, and regeneration. How the three genes regulate the spatial, temporal, and tissue-specific genetic and epigenetic networks that underlie all of these disparate programs is poorly understood. Furthermore, mutations in each of the genes have individually been associated with human cardiomyopathies, including atrial and ventricular septal defects, tetralogy of Fallot, bicuspid aortic valve syndrome, and familial dilated cardiomyopathy. Other transcription factor genes, and some terminal differentiation markers are known to be regulated by Gata456, but a major gap in understanding is the identify of the key target genes that control intermediary functions such as lineage specification, growth, morphogenesis, and cardio-protection. We propose a new program as a “Pipeline of Discovery” to identify these downstream genes and probe their function in cardiogenesis and cardiac biology. The overall goal is to define the function of each Gata456 gene throughout development and adult life in various cardiac tissues including endocardium, myocardium, and epicardium. We seek to break the code for how the relative timing and location of expression for each gene impacts cell fate and survival, and organ morphogenesis and function. Complementary model systems exploit specific advantages and resolve species-specific distinctions: the zebrafish for understanding cardiogenesis including morphogenesis, and human pluripotent stem cells for understanding human cell identity and disease modeling. We have compiled a “toolbox” of zebrafish and hESC lines and an expert team of investigators to facilitate a comprehensive analysis of gain-and loss-of-function phenotypes, with a strong track record for such analyses and discovery of novel downstream targets. A breakthrough is needed to understand how Gata456 controls all the various aspects of cardiogenesis. We are finally in a position to define this code, by a systematic manipulation of each factor in different developmental and tissue contexts, leading to discovery of specific key downstream target genes that carry out these diverse functions. This project will not directly develop therapeutics for cardiac disease, but it will likely enhance development of cellular therapies. Chiefly, it will break ground beyond current descriptions of regulatory networks in two areas: 1) Defining the impact for loss or gain of individual Gata456 alleles at specific developmental stages and in specific tissues to precisely define functions in developing animals (zebrafish) and human cells (derived from human pluripotent cells). 2) Identifying the key downstream Gata456 ...