A cetacean-specific CNE suppresses osteogenesis to drive appendicular modification in secondary aquatic adaptation

Cetaceans represent a hallmark of secondary aquatic adaptation, accompanied by extensive appendicular remodeling, whose underlying regulatory mechanisms remain challenging to resolve. Here, we investigated a cetacean-specific conserved non-coding element, CNE300, which exhibits accelerated evolution and reduced regulatory activity in cell-based assays. Homozygous cetacean CNE300 knock-in mice showed significantly shortened appendicular long bones and trabecular microarchitectural alterations, including reduced trabecular density. These phenotypic changes were concordant with key features of cetacean limb evolution. Transcriptomic profiling of embryonic limb buds identified a distinctive transcriptional bias toward cartilage differentiation programs in limb developmental genes, and postnatal endochondral ossification exhibited reduced osteogenic activity with normal bone resorption, suggesting that reduced osteogenesis might be a major contributor to the skeletal alterations observed in knock-in mice and natural cetacean limbs. Our findings demonstrated that CNE300 functions as a regulatory element driving appendicular remodeling and provide direct mechanistic insight into the molecular basis underlying cetacean limb evolution.