What genomic changes led to the origin of vertebrates remains a mystery. On the one hand, animal evolution is thought to be driven mostly by changes in the cis-regulatory regions of a shared conserved and toolkit of developmental genes. On the other hand, vertebrates experienced two rounds of whole genome duplication (WGD) that increased their gene repertoire, particularly of regulatory genes controlling embryo development. To shed light into the origin and evolution of the vertebrate regulatory genome, we have generated an unprecedented transcriptomic and epigenomic resource for the non-duplicated genome of the European amphioxus, a closely related invertebrate chordate. These data include RNA-seq for more than 35 developmental stages and adult tissues, CAGE-seq, ChIP-seq, bisulphite-seq and ATAC-seq for several developmental stages and adult tissues. By comparing these data sets with equivalent novel and previously available data for various vertebrate species, especially zebrafish, we uncovered multiple conserved and vertebrate-specific regulatory landmarks. We first identify a conserved chordate phylotypic stage, a developmental period in which different chordate species show the highest gene expression similarity. We also shed light on the origin of enhancer demethylation in vertebrates, by identifying, for the first time in an invertebrate species, differentially methylated enhancers. Furthermore, we show that conserved clusters of co-expressed and tissue-specific genes display similar enrichments for cis-regulatory motifs between amphioxus and vertebrates. Finally, we study the impact of vertebrate WGDs on the evolution of gene regulation, providing the first genome-wide quantitative assessment of sub-functionalization and neo-functionalization processes after the vertebrate WGDs changing the way in which these evolutionary mechanisms have been traditionally understood. Overall design: ATAC-seq assays in different developmental stages of european amphioxus, zebrafish and medaka