Regulatory rewiring of <i>Dand5</i> drove left-right organizer heterotopy and organ asymmetry evolution in chordates.

Abstract

Heterotopy-the spatial relocation of developmental processes-is a key mechanism driving morphological innovation, yet its underlying regulatory basis remains poorly understood. In chordates, the left-right (L-R) organizer shifted from the anterior domain as represented by amphioxus to the posterior end in vertebrates, accompanied by a coordinated relocation of the entire L-R gene regulatory network (GRN), including <i>Dand5</i> and its downstream genes <i>Nodal</i>, <i>Lefty,</i> and <i>Pitx</i>. Here, we dissect the regulation of <i>Dand5</i> in amphioxus and compare it with vertebrates. We show that L-R asymmetry of amphioxus <i>Dand5</i> is established transcriptionally and not posttranscriptionally as in vertebrates. Its transcription is directly activated by Hedgehog signaling and repressed by Wnt signaling, independent of vertebrate-specific inputs from Brachyury, and Notch and Wnt signaling. However, key elements required for postregulation of vertebrate <i>Dand5</i> asymmetry, including the responsiveness of its 3'-untranslated region (UTR) to Bicc1-mediated RNA repression and <i>Bicc1</i> expression in embryonic posterior end, seem already present in amphioxus. Our study therefore provides empirical evidence that repurposed regulatory sequences can drive heterotopy of developmental GRNs, offering a mechanistic explanation for the evolutionary shift of the LR signaling center and a foundation for exploring the diversification of LR organs across chordates.