Cer, Nodal, Lefty and Pitx are key players during vertebrate left-right (LR) axis determination.Some of these genes have also been identified in several invertebrates and indicated a role in their LR axis formation.But the function and regulatory relationship of these genes have not been well studied in invertebrates to date.In the present study, using Talen-mediated knockdown and Hsp70 promoter-induced ectopic over-expression methods,we demonstrated that all of these four genes are required for establishing the LR axis in the basal chordate amphioxus.