Transcriptomic and functional analysis reveals dermal SHH potentially regulates feathered-foot morphogenesis in Wuding chickens through the Wnt/β-catenin pathway

The feathered-foot trait, present in some chicken breeds like the Wuding, provides a unique evolutionary model to study the developmental divergence of avian skin appendages. This study integrated transcriptomic profiling with in vitro cellular assays to uncover the molecular basis of this morphological innovation. RNA sequencing of shank skin derived from feathered feet (FC) and scaled feet (SC) in Wuding chickens at embryonic day 12 (E12) identified 1,119 differentially expressed genes (DEGs). Functional enrichment highlighted key pathways in feather follicle development, notably the Wnt/β-catenin, BMP and Hedgehog signaling cascades. Subsequent protein–protein interaction network analysis identified Sonic hedgehog ( SHH ) as a key regulatory hub gene. To explore its functional impact on the feathered-foot development in Wuding chicken, this study manipulated SHH expression in dermal fibroblasts at E7. Ectopic SHH overexpression activated the canonical Wnt/β-catenin pathway and significantly enhanced fibroblast proliferation and cell cycle progression. Conversely, RNAi-mediated SHH knockdown produced significant suppressive effects. Mechanistically, SHH promoted cellular proliferation, accompanied by the activation of the JAK2/STAT3 and AKT/mTOR signaling axes, delayed senescence by modulating P53 , P21 and SIRT1 expression, and inhibited apoptosis potentially via elevating the BCL2/BAX ratio and suppressing Caspase-3. Collectively, these findings establish that dermal SHH promotes a pro-proliferative, anti-senescent dermal microenvironment conducive to feather follicle development, potentially through the coordinated activation of the Wnt/β-catenin pathway in Wuding chicken. This work elucidates a core genetic module controlling skin appendage fate and offers insights into the evolution of feather diversity.