Adaptive evolution of BMP4 as a potential mechanism for flipper forelimb changes in cetaceans

Vertebrate limb morphology and physiological functions have undergone extreme adaptations to complex environments. Marine mammals, in particular, have evolved highly specialized limb structures to adapt to aquatic habitats. Although the inhibition of interphalangeal cell apoptosis has been suggested to drive the specialization of their forelimbs into a convergent flipper phenotype, the underlying genetic mechanism remains poorly understood. In this study, we focused on three critical bone morphogenetic protein genes (i.e. BMP2, BMP4, and BMP7) which are responsible for initiating and sustaining apoptosis in interphalangeal mesenchymal stromal cells during embryonic limb development to ensure proper differentiation of interphalangeal tissues. It was revealed a significantly accelerated evolution for BMP4 and BMP7 in the cetacean ancestral lineage, with two positively selected sites (V79I and H247R) involved in cetacean-specific amino acid substitutions located in the TGFβ_propeptide functional domain in BMP4. In vitro assays further confirmed that cetacean-specific BMP4 mutations significantly disrupted normal cell apoptosis and proliferation, and altered transcription and protein expression of downstream apoptosis-related factors including cyt C, Bax, and Bcl-2 within the BMP signaling pathway. The significant influence of BMP4 mutations on inhibiting apoptotic processes implied their potential role in the development of limb bud mesenchymal tissue and the formation of flipper forelimb phenotype in cetaceans.