PROJECT SUMMARY AND ABSTRACT Over the past several decades the number of people in the United States with diabetes has steadily increased and reports from 2020 indicate nearly 10.5% of the population have the disease, leading to more than 270,000 deaths and an estimated cost of $327 billion. While tremendous effort is directed towards managing diabetes, there is no cure. One promising treatment is islet replacement therapy, addressing a major underlying issue in both type 1 and type 2 diabetes: dysfunctional and dying β cells. This treatment, however, is not widely accessible because of a lack of donor material. To overcome this limitation, insulin producing β cells can be generated from human pluripotent stem cells (hPSCs) in vitro, but despite great strides even the best protocols are unable to efficiently produce mature β cell populations for transplant. To improve this therapeutic strategy, it is essential to clarify mechanisms of pancreas development using in vivo models that will allow us to refine β cell differentiation protocols. Nearly all of the information used to formulate these protocols is derived from developmental studies in mice, which have provided a roadmap specifying the sequential addition or removal or signaling factors to promote β cell formation. For example, retinoic acid (RA) is included in early stages of all β cell differentiation protocols to specify pancreatic progenitors simulating numerous studies conducted in mice. Despite its importance at the onset of pancreas development, little is known about RA later in mouse or human pancreagenesis. To address this knowledge gap, we performed studies that identified a novel role for RA during endocrine progenitor specification where RA represses WNT signaling to promote β cell differentiation. Since RA clearly does not act alone in this process, I also began to examine the role of GATA factors during pancreas development as well, since previous work has demonstrated RA-GATA synergy in several models, including human β cell differentiations. Furthermore, haploinsufficiency of GATA6 in humans leads to pancreas agenesis in more than 50% of cases, yet this same deletion in mice and human β cell differentiations results in minor phenotypes, suggesting that background modifiers, like RA signaling, could be playing a major role. My preliminary studies described in this proposal demonstrate significant synergy between RA and GATA TFs in mice: simultaneous inhibition leads to drastically fewer and dysfunctional endocrine cells that contributes to severe physiological defects during development and adulthood. With guidance from these preliminary data, I will clarify the regulatory mechanisms defining endocrine specification, β cell development, and islet function by testing the hypothesis that synergy between RA signaling and GATA transcription factors is essential for embryonic pancreas development and adult islet function.