Deep homology of a brachyury regulatory syntax and origin of the notochord

The brachyury gene encodes a T-box transcription factor (TF) that is crucial for the development of the notochord, a novel trait of chordates ¹ . Brachyury expression in axial mesodermal cells (notochord progenitors) is regarded as a chordate innovation ² , yet it remains unclear how the chordate ancestor acquired this expression domain. By examining the sequences of previously identified notochord enhancers of several chordate brachyury genes ^3–5 , we uncovered a regulatory syntax consisting of binding sites for four TFs (Su(H), Foxh1, Zic, and Ets) with a strict order and orientation. We also identified this syntax, here named SFZE, in potential cis -regulatory modules (CRMs) of brachyury orthologs in various non-chordate animals and even in Capsaspora , a close unicellular relative to animals. Reporter assays demonstrated that SFZE-containing CRMs from non-chordate organisms exhibited regulatory activity in the zebrafish notochord, and mutagenesis of the TF sites reduced the activity. Furthermore, the SFZE syntax in sea urchin confers its endoderm activity, with the TF sites functionally decoupled during gastrulation. These findings indicate that the association of the SFZE syntax with brachyury is ancient, likely predating the origin of animals. The emergence of axial brachyury expression is therefore probably not the result of a newly acquired notochord enhancer, but is instead likely attributed to co-option of upstream signals acting on the conserved SFZE syntax, which facilitates the origin of the notochord from rudimentary endodermal cells.