Abstract

The antler primary growth center is located in the distal tip of the growing antler with five consecutive tissue layers under the velvet skin. Because of blastema progenitor cells of the outermost reserve mesenchymal layer with multipotent differentiation capacity, antler regeneration recapitulates embryonic skeletal development through the endochondral ossification (ECO) process. However, the precise molecular mechanism governing cell fate decisions and tissue morphogenesis across the antler growth center remains incompletely understood. To further decipher this modified endochondral ossification from the perspective of proteome dynamics, we first profile the deer antler proteome atlas with 8,173 proteins, covering the whole five layers that comprehensively represent the mesenchymal condensation, rapid mesenchymal proliferation, chondrocyte hypertrophy, and cartilage mineralization. By integrating three clustering methods, including WGCNA, Fuzzy C-means clustering, and monotonic feature selector, we elucidate proteome dynamics within the growth center, identifying key factors and elucidating biological processes through GO enrichment analysis. Integrated clustering analyses revealed distinct protein expression modules corresponding to the sequential stages of ECO. Proteins highly expressed in the RM layer were significantly enriched in processes related to mesenchymal cell proliferation and stemness. The transition to PC and CA zones was marked by a coordinated upregulation of proteins involved in extracellular matrix organization, collagen fibril assembly, and chondrocyte differentiation. In the MC layer, we observed an enrichment of proteins associated with cartilage mineralization and chondrocyte hypertrophy. Notably, our deep proteomic approach enabled the identification of low-abundance regulatory proteins, including key chondrogenic transcription factors such as SOX9, SOX6, and RUNX3, whose expression patterns correlated precisely with their expected roles in chondrogenesis. Furthermore, we identified a unique signature of 11 embryonic and 23 mesenchymal stem cell markers in the RM layer, suggesting a distinct intermediate stem cell state driving regeneration. Comparative analysis with existing transcriptomic data revealed that mRNA levels exhibit greater fluctuation than their corresponding protein abundances, highlighting the critical role of post-transcriptional regulation in orchestrating the smooth progression of ECO. This study provides the most comprehensive, spatially resolved proteomic resource for the deer antler growth center to date. Our findings delineate a precise spatiotemporal cascade of protein dynamics that governs the entire endochondral ossification process, from progenitor cell proliferation to terminal mineralization. The identification of key regulatory hubs and the evidence for significant post-transcriptional control offer new insights into bone development.