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The kupffers vesicle (KV) is the so-called left-right organizer in teleost fishes.KV is formed from dorsal forerunner cells (DFCs) and generates asymmetrical signals for breaking symmetry of embryos.It is unclear how DFCs or KV cells are prevented from intermingling with adjacent cells.In this study, we show that the Eph receptor gene ephb4b is highly expressed in DFCs while the ephrin ligand genes including efnb2b are expressed in cells next to the DFCs cluster during zebrafish gastrulation.Either ephb4b or efnb2b knockdown causes dispersal and fate conversion of DFCs, a smaller KV and randomization of laterality organs.The ephb4b knockout mutants display similar DFCs and laterality defects.Eph/ephrin signaling via activating RhoA activity mediates contact and repulsion between DFCs and neighboring cells during gastrulation, preventing intermingling of different cell populations.Therefore, our data uncover an important role of Eph/ephrin signaling in maintaining DFCs boundary and KV boundary for normal left-right asymmetrical development.