PROJECT ABSTRACT The lymphatic system is a complex vascular and tissue network that transports the lymph fluid containing proteins, macromolecules, and extravasated cells from peripheral tissues back to the circulation. The flow of lymph into lymphatic vessels is largely driven by local changes in interstitial pressure, which signals to lymphatic endothelial cells (LECs) through anchoring filaments in the surrounding extra cellular matrix. In response to increased interstitial pressure, anchoring filaments pull LECs to widen the overlapping cell-cell junctions and allow the flow of lymph into lymphatic vessels. Perturbations in development can lead to congenital lymphatic malformations and malignancies, while disruptions in post-natal lymphatic function can lead to pathologic lymphatic fluid accumulation and chronic immune and digestive problems. Despite the essential role of LECs in lymphatic system development and tissue fluid homeostasis, the signals that specify and maintain LEC identify are not well described. Previous studies have shown that LECs differentiate from venous endothelial cells and that homeodomain transcription factor Prox1 serves as a master regulator of this lineage conversion process. During embryogenesis, a subset of venous endothelial cells of the cardinal vein express Prox1 and migrate out to form rudimentary lymphatic vessels. It is known that these Prox1-positive cells downregulate the expression of genes associated with venous endothelial identify and upregulate gene expression signatures consistent with LECs. However, the exact mechanisms underlying Prox1 mediated LEC differentiation are not known. We previously showed that Prox1 physically interacts with venous endothelial cell fate regulator, COUP-TFII. This preliminary work suggests that in addition to driving venous endothelial cell differentiation, COUP-TFII, also works in concert with Prox1 to establish and maintain the LEC lineage, which is the basis for the hypothesis of the proposed study. The concept that the key molecular regulator of venous endothelial cell identity plays an essential role in lymphatic development is of great interest and highlights the close histogenetic relationship between the two vascular systems. By dissecting the molecular interactions between Prox1 and COUP-TFII during LEC development, this proposal aims to elucidate the gene regulatory networks that orchestrate endothelial cell differentiation and advance our understanding of arteriovenous-lymphatic vascular development and disease.