The lymphatic vascular system plays an essential role in the transport of interstitial fluid and lipids, and in the induction of adaptive immune responses in vertebrates. Normal functioning of the lymphatic vascular system depends upon intraluminal lymphatic valves (LV) that facilitate propulsive flow of lymph fluid in collecting lymphatic vessels. Defects in LV development and function results in accumu- lation of lymph in tissues or body cavities resulting in lymphedema, chylothorax and chylous ascites. From a medical perspective, understanding the molecular mechanisms that regulate the development and function of LV is critical, yet our knowledge of these mechanisms remains limited. We have re- ported previously that RASA1, which inhibits activation of the intracellular Ras signal transduction pathway, is required for the development and maintenance of LV. In addition, others have reported that the receptor tyrosine kinase, EPHB4, is required for LV development. However, the precise mo- lecular mechanisms by which RASA1 and EPHB4 regulate LV are unknown. A long-term goal of the King laboratory is to understand the role of the Ras signaling pathway in different physiological sys- tems in health and disease. The overall objective of this application, which is consistent with this long- term goal, is to understand how RASA1 regulates the development and function of LV. Our central hypothesis is that RASA1, through physical interaction with EPHB4, promotes the export of collagen IV from LV-forming (LVF) lymphatic endothelial cells (LEC) and mature LEC for deposition in the ex- tracellular matrix core of developing and established LV leaflets respectively. The rationale for these studies is that they will inform upon the molecular mechanisms by which RASA1 and EPHB4 regulate the development and function of LV. We plan to test our central hypothesis and, thereby, attain the objective of this application by pursuing the following two specific aims: In the first aim, we will use different molecular cell biologic, mouse genetic, and physiological approaches to understand the mo- lecular mechanism by which RASA1 loss results in failed development and maintenance of LV. In the second aim, we will use similar approaches to understand the role of EPHB4 in the development of LV and the mechanisms involved. The proposed studies are innovative because of the novel method- ologies employed and the concept that an EPHB4-RASA1 axis is essential for the development and function of LV acting to export collagen IV from LVF LEC and LV LEC. The studies are significant be- cause of their potential to lead to new therapies for the prevention and treatment of LV abnormalities in humans with inherited mutations in RASA1 and EPHB4 genes.