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

Abstract

Vertebrate limbs have undergone profound morphological diversification, enabling adaptations to a broad spectrum of ecological niches. In marine mammals, the evolution of highly specialized flipper-like forelimbs represents a profound structural transformation associated with aquatic habitats. This adaptation has been hypothesized to result, in part, from the inhibition of interphalangeal cell apoptosis during limb development, although the underlying genetic mechanism remains poorly understood. This study investigated the evolutionary dynamics and functional consequences of three key bone morphogenetic protein genes, BMP2, BMP4, and BMP7, which regulate apoptosis in interphalangeal mesenchymal stromal cells during embryonic limb development to ensure proper differentiation of interphalangeal tissues. Comparative genomic analysis revealed 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 TGF-β propeptide functional domain in BMP4. In vitro assays confirmed that cetacean-specific BMP4 mutations significantly disrupted normal cell apoptosis and proliferation and altered the transcription and protein expression of downstream apoptosis-related factors, including cytochrome c (Cyt c), BCL2 associated X, and B-cell lymphoma 2, within the BMP signaling pathway. The significant influence of BMP4 mutations on apoptotic inhibition highlights a potential role in the development of limb bud mesenchymal tissue and the emergence of the flipper forelimb phenotype in cetaceans.

Keywords: BMP4; Cetaceans; Flipper limb; Functional change.

Figure 1

Specific amino acid alterations in cetaceans A: Left: Phylogenetic tree of species in this study. Right: Blue boxes highlight lineage-specific mutations, all located within or adjacent to functional domains of the genes. B: 3D structural prediction of cetacean BMP proteins (BMP2, BMP4, and BMP7). Red represents TGF-β propeptide functional domain, green indicates cetacean-specific amino acid sites.

Figure 2

Assessment of apoptosis and proliferation in NIH3T3 cells overexpressing BMP4 plasmids A: Flow cytometry profiles of cells transfected with mBMP4, dBMP4, or mut-mBMP4. B: Quantification of apoptotic populations using Annexin V/PI dual staining, distinguishing live cells (Annexin V−/PI−), early apoptotic cells (Annexin V+/PI−), and late apoptotic cells (Annexin V+/PI+). C: CCK-8 assay measuring cell proliferation in BMP4-transfected NIH3T3 cells. Data are presented as mean±SD, ^*: P<0.05; ^**: P<0.01; ^***: P<0.001.

Figure 3

Transcriptional expression levels of BMP4 in cetaceans and mutant constructs A: Relative mRNA expression of BMP4 in cells transfected with mBMP4, mut-mBMP4, and dBMP4 constructs. B–D: Expression levels of three candidate apoptotic genes in NIH3T3 cells transfected with vector, mBMP4, mut-mBMP4, and dBMP4. Data are presented as mean±SD, ns: Not significant; ^*: P<0.05; ^**: P<0.01; ^***: P<0.001.

Figure 4

Protein expression of BMP4 and apoptosis-related proteins in transfected NIH3T3 cells A: Western blot analysis of BMP4 and apoptosis-related proteins (Bax and Cyt c) in NIH3T3 cells transfected with mBMP4, mut-mBMP4, or dBMP4 plasmids. Cells expressing dBMP4 exhibited significantly reduced levels of pro-apoptotic proteins. B–D: Quantification of BMP4, Bax, and Cyt c protein expression. Data are presented as mean±SD, ^*: P<0.05.

Figure 5

Schematic representation of BMP signaling and intrinsic apoptotic pathways Left: Schematic of BMP signaling pathway during cetacean limb bud development, incorporating cetacean embryo adapted from Gavazzi et al. (2023). Right: Schematic of intrinsic apoptotic pathway based on Tang et al. (2019). CS17: Carnegie stage 17; AER: Apical ectodermal ridge. Blue and red labels are candidate factors from this study, and arrows represent regulatory relationships.