Functional evidence supports the potential role of Tbx4-HLEA in the hindlimb degeneration of cetaceans.

The evolution of limb morphology plays an important role in animal adaptation to different ecological niches. To fully adapt to aquatic life, cetaceans underwent hindlimb degeneration and forelimb transformed into flipper; however, the molecular mechanisms underlying the limb changes in cetaceans remain unclear. We previous study had shown that the Tbx4 hindlimb enhancer A (Tbx4-HLEA) in cetaceans exhibited specific deletions and nucleotide substitutions, with significantly reduced regulatory activity. To further investigate whether cetacean HLEA has a potential impact on hindlimb development in vivo, a knock-in mouse model was generated by knocking in the homologous cetacean HLEA in the present study. Phenotypic analysis showed a significant reduction in hindlimb bud development in homozygous knock-in mice at embryonic day (E)10.5; however, the phenotypic difference was rescued after E11.5. Transcriptomic and epigenetic analyses indicated that the cetacean HLEA acts as an enhancer in the mouse embryos and significantly reduces the transcriptional expression levels of Tbx4 at E10.5, supporting that downregulation of cetaceans HLEA regulatory activity reduces the expression of Tbx4. Additionally, both the number of activated non-coding elements and chromatin accessibility near Tbx4 were increased in homozygous knock-in mice at E11.5. The functional redundancy of enhancers compensated for the functional defect of cetacean HLEA, rescuing the expression level of Tbx4, and may account for the phenotype restoration after E11.5. In conclusion, our study suggested that the evolution of cetacean HLEA may be an important link with relevant molecular mechanism for the hindlimb degeneration.