The remarkable morphological diversity observed across the animal kingdom is a result of the evolutionary modifications of developmental processes. A striking example of such diverse morphologies is the tetrapod limb where profound phenotypic adaptations enabled species to colonize diverse terrestrial, aquatic, and aerial habitats. This organ thus provides a compelling model for examining the cellular mechanisms driving such diversification. Among mammals, powered flight evolved uniquely within the order Chiroptera, commonly known as bats. This remarkable ability is attributed to their wing structure, which is characterized by massively elongated digits connected by a specialized tissue membrane, the chiropatagium, a distinctive feature found only in this species. However, the molecular origins and the extent of developmental reprogramming necessary to achieve this dramatic morphological adaptation remain poorly understood. To address this, we employed comparative single-cell and functional genomics approaches to investigate limb developmental cell states in both mice and bats. Our analysis revealed a significant conservation of cell states and processes across species. In contrast to previous hypotheses, this also included the process of interdigital apoptosis, which typically eliminates interdigital tissue in species with separated digits. Through microdissection of embryonic chiropatagium and subsequent single-cell transcriptomics profiling of this tissue, we discovered that the chiropatagium originates from fibroblasts that are distinct from apoptosis-related interdigital cells. We furthermore revealed that these fibroblasts in the distal, autopodial limb region repurpose a developmental program otherwise found in the proximal limb. Genes expressed in this chiropatagium cell program showed enrichment in functions related to cell proliferation, migration, and extracellular matrix organization, highlighting the significance of these processes in wing development. Functional genomics and gene network analyses identified the developmental transcription factors MEIS2 and TBX3 as key regulators of this cell program. Importantly, ectopic expression of these genes in distal mesenchymal cells of transgenic mice led to the activation of genes linked to chiropatagium development. As a result, mutant limbs exhibited significant morphological changes, including increased cell number, enhanced extracellular matrix content, and retention of interdigital tissue. Lastly, the bat MEIS2 and TBX3 regulatory landscapes revealed significant differences in enhancer activity and 3D chromatin structure, suggesting species-specific genomic features that mediate their activity during wing formation. Altogether, our findings unravel fundamental molecular mechanisms underlying bat wing development and demonstrate how drastic morphological changes, such as the emergence of a wing, can arise from the repurposing of existing developmental programs.
