GSE97860
cis-regulatory logic of Shh expression reveals common history of unpaired and paired fins
Despite their evolutionary, developmental, and functional importance the origin of vertebrate paired appendages remains a mystery. Classical ideas center on three hypotheses: that paired fins relate to gill arches, are derived from a fin fold, or that median fins evolved first and paired fins arose by co-option of ancient genetic patterning modules. Importantly, the third hypothesis makes specific predictions about the function and phylogenetic history of cis-regulatory elements (CREs) involved in appendage patterning. Here we show that a major enhancer for Sonic hedgehog (Shh, a key diffusible morphogen essential for proper growth and patterning of limbs and paired fins) is deeply shared between paired and unpaired fins in fish and with the limbs of mice. In mice, a single CRE termed ZRS is solely responsible for coordinating Shh expression in limbs. We use transgenic assays in zebrafish and mouse to trace the functional equivalence of the ZRS across gnathostome phylogeny and its likely absence in agnathans. CRISPR/Cas9-mediated deletion of the medaka core ZRS sequence, inspection of shh epigenetic landscape and in vivo enhancer assays reveal the existence of ZRS shadow enhancers in both teleost and human genomes. Strikingly, a complete deletion of the medaka ZRS, an experiment expected to only affect the paired fins, results in an almost complete ablation of the dorsal fin. This finding indicates that a ZRS-Shh regulatory module is shared by paired and medial fins, and that paired fins likely emerged by the co-option of developmental programs established in the median fins of early vertebrates. Later on, this critical ZRS-driven Shh function was reinforced in pectoral fin development with the recruitment of shadow enhancers, conferring additional robustness. Overall design: 4C-seq experiments from Shh promoter and ZRS enhancer in mouse and medaka.