Divergence of Tbx4 hindlimb enhancer HLEA underlies the hindlimb loss during cetacean evolution

The cetacean hindlimb skeleton massively decreased to only vestigial limb elements as cetaceans evolved from land to aquatic lifestyles; however, the molecular mechanism underlying this major morphological transition remains unclear. In this study, four deletions and specific substitutions were detected in cetacean hindlimb enhancer A (HLEA), an enhancer that can regulate Tbx4 expression in hindlimb tissues to control hindlimb development. Transcriptional activation of HLEA was significantly weaker in bottlenose dolphin than mice, and this was found to be closely associated with cetacean-specific deletions. Furthermore, deletions in cetacean HLEA might disrupt HOX and PITX1 binding sites, which are required for enhancer activation. The ancestral state of these deletions was investigated, and all four specific deletions were found to have occurred after the species diverged from their common ancestor, suggesting that the deletion occurred recently, during a secondary aquatic adaptation. Taking these findings together, we suggest that cetacean-specific sequence changes reduced the Tbx4 gene expression pattern, and consequently drove the gradual loss of hindlimb in cetaceans.