ABSTRACT Genetic loss-of-function studies in model organisms have elucidated that the GATA family of transcription factors regulate crucial aspects of heart morphogenesis including early germ layer patterning and cardiac progenitor cell specification and differentiation. Importantly, heterozygotic mutations of the cardiogenic factor GATA6 in humans are associated with various forms of congenital heart disease (CHD) such as outflow tract and septal defects. The phenotypic diversity of CHD in patients containing these GATA6 mutations is likely due an unknown combination of variants in modifying or interacting genes that converge to influence the cardiac phenotype. A greater understanding of the GATA6 genetic regulatory network that controls human heart development is thus essential for advancing therapies. Here, I propose utilizing cardiac-directed differentiating human pluripotent stem cells (hPSCs) as a system to study GATA6 function. Preliminary evidence showed that GATA6-/- hPSCs failed to generate cardiomyocytes or express markers of cardiac progenitors, and had reduced cardiac mesoderm marker expression during cardiac-directed differentiation compared to wild-type (WT) controls. Furthermore, GATA6+/- hPSCs generated cardiomyocytes less efficiently and had reduced cardiac progenitor marker gene expression compared to WT cells, revealing a phenotype in GATA6 heterozygotes that has not been reported in previous animal studies. Based on these data, I aim to study GATA6 function during cardiac mesoderm patterning and GATA6 haploinsufficiency during cardiogenesis through two Specific Aims. In Aim 1, I plan to discover the function of GATA6 during the earliest stages of human cardiogenesis. Two candidate downstream targets of GATA6 activity identified from RNA-seq data, LGR5 and RIPPLY1, will first be studied through genetic gain- and loss-of-function studies. GATA6 ChIP-seq and ATAC-seq will also be performed in GATA6-/- and WT cells during early stages of cardiac differentiation to identify direct targets of GATA6 and regulatory genes affected by chromatin inaccessibility, respectively. In Aim 2, I propose to define the consequence of human GATA6 haploinsufficiency during cardiogenesis. An induced pluripotent stem cell (iPSC) line was created from a CHD patient containing a heterozygous mutation in GATA6 (c.1071delG) that differentiates to cardiomyocytes less efficiently than a WT iPSC line. Here, engineered GATA6+/- hESCs and patient-derived GATA61071delG/+ iPSCs will be differentiated to cardiomyocytes and examined at distinct stages of cardiogenesis to carefully define the cardiac phenotype. GATA6+/- cells have increased expression of retinoic acid (RA) signaling related genes, which may underlie the cardiac phenotype. RA signaling will thus be inhibited with the goal of rescuing the phenotype of GATA6 haploinsufficiency. Finally, the transcriptomes of differentiating GATA6+/- hESC and patient-derived iPSC lines will be analyzed using RNA-seq t...