Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation [Pires-daSilva & Sommer, Nat. Rev. Genet. 4, 39-49 (2003)]. Remarkably, in contrast to the large number of transcription factors present in animal genomes, only a few of these pathways operate during development [Sanz-Ezquerro, Münsterberg & Stricker, Front. Cell Dev. Biol. 5, 76 (2017)]. Moreover, most of them have been largely conserved during metazoan evolution [Babonis & Martindale, Philos. Trans. R. Soc. B 372, 20150477 (2017)]. How evolution has generated a vast diversity of animal morphologies with such a limited number of tools is still largely unknown. Here, we show that gain of interconnectivity between signaling pathways, and the GRNs they control, may have critically contributed to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF and Nodal signaling pathways during gastrulation in the invertebrate chordate amphioxus and zebrafish and compared the effects on gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through vertebrate-specific CREs. Moreover, in contrast to amphioxus, many of these CREs responded to multiple pathways in zebrafish, which reflects their high interconnectivity. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than tissues with more ancient origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and to the formation of new cell types and morphological novelties in this lineage. Overall design: RNA-seq and ATAC-seq for different treatments that stimulate/inhibits signaling pathways in zebrafish, xenopus tropicalis, amphioxus and ptychodera flava during embryonic development This submission corresponds to RNA-seq component of Ptychodera flava and Xenopus tropicalis data.