PROJECT SUMMARY/ABSTRACT Hemochorial placentation occurs in many mammalian species including primates and rodents. It ensures the most intimate contact between maternal and embryonic compartments and requires specialized adjustments. Among these adjustments is the need for extensive remodeling of the maternal uterine spiral arteries. Uterine vascular modifications are required for the delivery of nutrients to the fetus. Mechanisms controlling uterine spiral artery remodeling remain poorly understood. Central to the vascular remodeling process is a specialized population of trophoblast cells referred to as invasive trophoblast cells (generic term) or alternatively as extravillous trophoblast (EVT) cells (human-specific term). Impairments in differentiation of the invasive trophoblast/EVT cell lineage, intrauterine trophoblast cell invasion, and/or trophoblast cell-directed uterine cell modulation contribute to early pregnancy loss, placental dysfunction, pregnancy-related diseases, and a range of postnatal health issues. Our long-term goal is to elucidate regulatory processes controlling development of the invasive trophoblast/EVT cell lineage and their role in establishing the uterine-placental interface. Placentation in the human and rat are both characterized by deep intrauterine trophoblast cell invasion and extensive trophoblast cell guided spiral artery remodeling. Our research strategy is based on our prior establishment and extensive characterization of in vitro and in vivo models for studying deep hemochorial placentation in the rat and the utilization of human placentation site specimens and human trophoblast stem cells for investigating gene regulatory networks controlling EVT cell development. In this proposal we focus on two regulatory pathways controlling invasive trophoblast/EVT cell differentiation: i) phosphatidylinositol 3- kinase (PI3K)/AKT pathway; ii) CBP/EP300-interacting transactivator with Glu/Asp rich carboxy-terminal domain 2 (CITED2)-regulated pathway. These regulatory pathways are viewed as entry points and guides to mechanisms controlling development of the invasive trophoblast/EVT cell lineage. The proposed research provides an innovative approach to studying deep hemochorial placentation. Collectively, the research is directed toward elucidating molecular mechanisms underlying physiologically relevant processes that ensure successful hemochorial placentation. This approach will lead to the identification of conserved regulatory pathways controlling the invasive trophoblast/EVT cell lineage, which will create opportunities and a platform for understanding the pathogenesis of placental anomalies leading to pregnancy disorders.