Pancreas agenesis (PA) is a developmental disorder characterized by either a reduction or complete lack of pancreatic mass. Notably, heterozygous loss of GATA6 accounts for >60% of all human pancreas agenesis cases; however, the pancreatic phenotype within each individual can be incompletely penetrant, ranging from severe neonatal diabetes to mild adult onset diabetes due to beta cell dysfunction. These findings suggest that additional genetic modifiers may contribute to the pathogenesis caused by the reduction in GATA6 expression. During the previous funding period, the collaboration between the Gadue and Sussel labs leveraged the strengths of human induced pluripotent stem cell (hiPSC)-based pancreas differentiation and in vivo murine models of development to discover robust synergy between GATA6 and retinoic acid (RA) signaling in regulating several stages of pancreas development. Our findings suggest two novel concepts: (1) an unappreciated role for RA signaling during endocrine progenitor specification and (2) synergy between RA and specifically GATA6 (but not GATA4) is required to specify beta cells in both mice and humans. The primary goals of this renewal application are to use these complementary genetic model systems to better define this synergy and elucidate the mechanisms by which the intersection of RA signaling and GATA6 regulate pancreas development. Furthermore, we will explore how human mutations in GATA6 disrupt this interaction to influence disease severity. Our published studies combined with new preliminary data in both the mice and human models has led us to hypothesize that this conserved synergy between RA signaling and GATA6 gene regulation is essential for pancreas development and the combined disruption of these pathways contributes to pancreas and islet cell development. We will test this hypothesis with the following specific aims: 1) Establish the synergistic roles of RA/GATA6 during pancreatic endocrine and β cell development and identify the specific stages of the hiPSC-derived pancreas differentiation protocol that require a combination of RA signaling and GATA6 function for optimal beta cell development; 2) Determine the precise molecular mechanism(s) underlying the intersection of RARa and GATA6 in the regulation gene expression pathways that promote pancreatic islet differentiation; and 3) Define how patient specific GATA6 disease mutations affect RA/GATA6 synergy and downstream pathways during pancreas development. The experiments proposed in this application will provide substantial novel insight into the conserved regulatory pathways that are required for appropriate pancreas development and islet cell differentiation and will further inform how mutations in GATA6 cause a wide range of pancreatic phenotypes in humans.