A conserved Shh cis-regulatory module highlights a common developmental origin of unpaired and paired fins

Despite their evolutionary, developmental and functional importance, the origin of vertebrate paired appendages remains uncertain. In mice, a single enhancer termed ZRS is solely responsible for Shh expression in limbs. Here, zebrafish and mouse transgenic assays trace the functional equivalence of ZRS across the gnathostome phylogeny. CRISPR/Cas9-mediated deletion of the medaka (Oryzias latipes) ZRS and enhancer assays identify the existence of ZRS shadow enhancers in both teleost and human genomes. Deletion of both ZRS and shadow ZRS abolishes shh expression and completely truncates pectoral fin formation. Strikingly, deletion of ZRS results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and median fins and that paired fins likely emerged by the co-option of developmental programs established in the median fins of stem gnathostomes. Shh function was later reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness.This project received funding from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (grant agreement No. 740041), the European Union’s Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement #658521, the Spanish Ministerio de Economía y Competitividad (grants BFU2016-74961-P, BFU2014-53765-P and BFU2016-81887-REDT), the Andalusian Government (grant BIO-396), CNPq Universal Program Grant 403248/2016-7 and CAPES/Alexander von Humboldt Foundation fellowship (to I.S.). J.L. was supported by Becas Chile