Multiomic data integration unveils molecular mechanisms underlying chestnut and skin tissue differentiation in Dezhou donkeys.

Donkey chestnuts are hairless, darkly pigmented keratinized structures located on the medial forelimbs, anatomically distinct from surrounding skin. Despite their unique morphology, the molecular mechanisms underlying their formation and function remain poorly understood. An integrated multi-omics approach was applied to chestnut and skin tissues collected from four Dezhou donkeys. Bulk RNA sequencing (RNA-seq) was performed on six samples to assess transcriptomic differences, followed by single-cell RNA sequencing (scRNA-seq) to resolve cellular composition, and proteomics to characterize protein expression patterns. Chestnut tissue displayed exceptionally high keratin expression, including KRT1, KRT5, KRT10, and KRT14 (TPM > 13,000), and was enriched in endothelial cells, pericytes, T cells, and mast cells. Proteomic analysis revealed significant upregulation of collagen I (COL1A1/COL1A2), indicative of active extracellular matrix remodeling. Pathway analysis identified enrichment in peroxisome activity, fatty acid metabolism, PPAR signaling, and PI3K-Akt networks. These findings collectively demonstrate that donkey chestnut is a metabolically active, specialized barrier tissue with a distinctive cellular and molecular profile. Its unique signatures related to keratinization, pigmentation, and immune cell enrichment suggest potential roles in barrier defense and anti-inflammatory regulation, providing a foundation for further investigation into the biological significance of this understudied structure.