AbstractIntraluminal lymphatic valves (LVs) and lymphovenous valves (LVVs) are critical to ensure the unidirectional flow of lymphatic fluid. Morphological abnormalities in these valves always cause lymph or blood reflux, and result in lymphedema. However, the underlying molecular mechanism of valve development remains poorly understood. We here report the implication of Efnb2-Ephb4-Rasa1 regulated Erk signaling axis in lymphatic valve development with identification of two new valve structures. Dynamic monitoring of phospho-Erk activity indicated that Erk signaling is spatiotemporally inhibited in some lymphatic endothelial cells (LECs) during the valve cell specification. Inhibition of Erk signaling via simultaneous depletion of zygotic erk1 and erk2 or treatment with MEK inhibitor selumetinib causes lymphatic vessel hypoplasia and lymphatic valve hyperplasia, suggesting opposite roles of Erk signaling during these two processes. ephb4b mutants, efnb2a;efnb2b or rasa1a;rasa1b double mutants all have defective LVs and LVVs and exhibit blood reflux into lymphatic vessels with an edema phenotype. Importantly, the valve defects in ephb4b or rasa1a;rasa1b mutants are mitigated with high-level gata2 expression in the presence of MEK inhibitors. Therefore, Efnb2-Ephb4 signaling acts to suppress Erk activation in valve-forming cells to promote valve specification upstream of Rasa1. Not only do our findings reveal a molecular mechanism of lymphatic valve formation, but also provide a basis for the treatment of lymphatic disorders.