Evolution of cetacean-specific conserved non-coding elements suggests their role in the limb changes during secondary aquatic adaptation.

BACKGROUND: Limb morphology is particularly important for animals to inhabit different environments. Limb modifications (e.g., flipper-like forelimbs and hindlimb regression) are among the most critical secondary aquatic adaptation mechanisms enabling cetaceans to fully adapt to an aquatic environment. Exploring the molecular mechanisms underlying limb evolution in cetaceans has attracted considerable attention from evolutionary biologists. RESULTS: In the present study, conserved non-coding elements (CNEs) closely associated with limb development, which exhibited lineage-specific sequence divergence (nucleotide mutations and indels) in cetaceans, were identified using comparative genomics. These sequence divergences might have led to the loss of binding motifs for transcription factors involved in limb development and significant alterations in autoregulatory activity. A transgenic mouse was constructed to carry a cetacean-specific enhancer (i.e., hs1586), which exhibited a significant phenotypic difference in forelimb buds at embryonic day (E)10.5, supported by transcriptomic and epigenomic evidence. However, the phenotypic recovery after E11.5 suggested that enhancer redundancy in the mouse genome may have compensated for the effects caused by the incorporation of cetacean hs1586. This further suggests that the complex phenotypic changes of limbs in cetaceans are likely not driven by a single CNE but rather involve multiple CNEs and/or genes. CONCLUSIONS: In summary, our study supports the functional role of CNE sequence divergence and the complex mechanisms underlying limb morphology changes in cetaceans.