Signaling pathways control a large number of gene regulatory networks (GRNs) during animal development, acting as major tools for body plan formation. Remarkably, in contrast to the large number of transcription factors present in animal genomes, only a few of these pathways operate during development. Moreover, most of them are largely conserved along metazoan evolution. 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 played a critical contribution to the origin of vertebrates. We perturbed the retinoic acid, Wnt, FGF and Nodal signaling pathways during gastrulation in amphioxus and zebrafish and comparatively examined its effects in gene expression and cis-regulatory elements (CREs). We found that multiple developmental genes gain response to these pathways through novel CREs in the vertebrate lineage. Moreover, in contrast to amphioxus, many of these CREs are highly interconnected and respond to multiple pathways in zebrafish. Furthermore, we found that vertebrate-specific cell types are more enriched in highly interconnected genes than those tissues with more ancestral origin. Thus, the increase of CREs in vertebrates integrating inputs from different signaling pathways probably contributed to gene expression complexity and the formation of new cell types and morphological novelties in this lineage.
Overall design: RNA-seq experiments in zebrafish and amphioxus embryos after modifying different signaling pathways
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