Urochordates are the closest relatives of vertebrates and at the larval stage, possess a characteristic bilateral chordate body plan. In vertebrates, the genes that orchestrate embryonic patterning are in part regulated by highly conserved non-coding elements (CNEs), yet these elements have not been identified in urochordate genomes. Consequently the evolution of the cis-regulatory code for urochordate development remains largely uncharacterised. Here, we use genome-wide comparisons between C. intestinalis and C. savignyi to identify putative urochordate cis-regulatory sequences. Ciona conserved non-coding elements (ciCNEs) are associated with largely the same key regulatory genes as vertebrate CNEs. Furthermore, some of the tested ciCNEs are able to activate reporter gene expression in both zebrafish and Ciona embryos, in a pattern that at least partially overlaps that of the gene they associate with, despite the absence of sequence identity. We also show that the ability of a ciCNE to up-regulate gene expression in vertebrate embryos can in some cases be localised to short sub-sequences, suggesting that functional cross-talk may be defined by small regions of ancestral regulatory logic, although functional sub-sequences may also be dispersed across the whole element. We conclude that the structure and organisation of cis-regulatory modules is very different between vertebrates and urochordates, reflecting their separate evolutionary histories. However, functional cross-talk still exists because the same repertoire of transcription factors has likely guided their parallel evolution, exploiting similar sets of binding sites but in different combinations.
Vertebrates share many aspects of early development with our closest chordate ancestors, the tunicates. However, whilst the repertoire of genes that orchestrate development is essentially the same in the two lineages, the genomic code that regulates these genes appears to be very different, even though it is highly conserved within vertebrates themselves. Using comparative genomics, we have identified a parallel developmental code in tunicates and confirmed that this code, despite a lack of sequence conservation, associates with a similar repertoire of genes. However, the organisation of the code spatially is very different in the two lineages, strongly suggesting that most of it arose independently in vertebrates and tunicates, and in most cases lacking any direct sequence ancestry. We have assayed elements of the tunicate code, and found that at least some of them can regulate gene expression in zebrafish embryos. Our results suggest that regulatory code has arisen independently in different animal lineages but possesses some common functionality because its evolution has been driven by a similar cohort of developmental transcription factors. Our work helps illuminate how complex, stable gene regulatory networks evolve and become fixed within lineages.