Deep homology of a cis-regulatory syntax and the evolutionary origin of the notochord.

Expression of in the notochord is regarded as a chordate novelty and links to the origin of the notochord, yet the evolution of this regulatory control remains unclear. Here, we uncovered a regulatory syntax (named SFZE) consisting of binding sites for four transcription factors in notochord enhancers of chordate genes. SFZE was also identified in potential enhancers in various non-chordate animals and even in , a unicellular relative to animals. These non-chordate SFZE-containing enhancers exhibited activity in the zebrafish notochord. Furthermore, the SFZE syntax in a non-chordate confers endoderm activity. Our results indicate the ancient association of SFZE with , likely predating the origin of animals. The emergence of notochordal expression could be attributed to co-option of upstream signals acting on the conserved SFZE syntax, which facilitates the origin of the notochord from rudimentary endodermal cells.