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Background: With the unprecedented rapid growth rate (up to 2.75 cm/day), velvet antler is an invaluable model for the identification of potent growth factors and signaling networks for extremely fast growing tissues, mainly cartilage. Antler growth center (AGC) locates in its tip and consists of five tissue layers: reserve mesenchyme (RM), precartilage (PC), transition zone (TZ), cartilage (CA) and mineralized cartilage (MC). The aim of this study was to investigate the transcription dynamics in the AGC using RNA-seq technology., Results: Five tissue layers in the AGC were collected from three 3-year-old male sika deer using our previously reported sampling method (morphologically distinguishable). After sequencing (15 samples; triplicates/tissue layer), we assembled a reference transcriptome de novo and used RNA-seq to measure gene expression profiles across these five layers. Nine differentially expressed genes (DEGs) were selected from our data and subsequently verified using qRT-PCR. The results showed a high consistency with the RNA-seq results (R 2  = 0.80). Nine modules were constructed based on co-expression network analysis, and these modules contained 370 hub genes. These genes were found to be mainly involved in mesenchymal progenitor cell proliferation, chondrogenesis, osteogenesis and angiogenesis. Combination of our own results with the previously published reports, we found that Wnt signaling likely plays a key role not only in stimulating the antler stem cells or their immediate progeny, but also in promoting chondrogenesis and osteogenesis during antler development., Conclusion: We have successfully assembled a reference transcriptome, generated gene expression profiling across the five tissue layers in the AGC, and identified nine co-expressed modules that contain 370 hub genes and genes predorminantly expressed in and highly relevant to each tissue layer. We believe our findings have laid the foundation for the identification of novel genes for rapid proliferation and chondrogenic differentiation of antler cells.

Deer antlers are the only mammalian organs that can fully regenerate after being lost and provide a valuable model for cartilage development. As one of the best-studied epigenetic mechanisms, DNA methylation is known to engage in organ and tissue development. This study aimed to investigate the role of DNA methylation in antler chondrogenesis by comparing whole-genome DNA methylation between precartilage and cartilage. Quantitative reverse transcription PCR (RT-qPCR) showed significant differences in the expression levels of DNA methyltransferase genes (DNMT1, DNMT3A, and DNMT3B) between precartilage and cartilage. Subsequently, we obtained DNA methylation profiles of antler precartilage and cartilage tissues by whole-genome bisulfite sequencing. Although sequencing data indicated that overall methylation levels at CpG and non-CpG sites were similar between precartilage and cartilage, 140,784 differentially methylated regions (DMRs, P < 0.05) and 3,941 DMR-related genes were identified. Gene ontology (GO) analysis of DMR-related genes demonstrated some significantly enriched GO terms (P < 0.05) related to chondrogenesis, including insulin receptor binding, collage trimer, integrin binding, and extracellular matrix structural constituent. Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of DMR-related genes uncovered that the PI3K/AKT, cortisol synthesis and secretion, glycosaminoglycan biosynthesis-keratan sulfate, Hippo, and NF-κB signaling pathways might play a pivotal role in the transition of precartilage to cartilage. Moreover, we found that 25 DMR-related genes, including CD44, IGF1, ITGAV, ITGB1, RUNX1, COL2A1, COMP, and TAGLN, were most likely involved in antler chondrogenesis. In conclusion, this study revealed the genome-wide DNA methylation patterns of antler precartilage and cartilage, which may contribute to understanding the epigenetic regulation of antler chondrogenesis., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

This review describes the formation, structure, and function of bony compartments in antlers, horns, ossicones, osteoderm and the os penis/os clitoris (collectively referred to herein as AHOOO structures) in extant mammals. AHOOOs are extra-skeletal bones that originate from subcutaneous (dermal) tissues in a wide variety of mammals, and this review elaborates on the co-development of the bone and skin in these structures. During foetal stages, primordial cells for the bony compartments arise in subcutaneous tissues. The epithelial-mesenchymal transition is assumed to play a key role in the differentiation of bone, cartilage, skin and other tissues in AHOOO structures. AHOOO ossification takes place after skeletal bone formation, and may depend on sexual maturity. Skin keratinization occurs in tandem with ossification and may be under the control of androgens. Both endochondral and intramembranous ossification participate in bony compartment formation. There is variation in gradients of density in different AHOOO structures. These gradients, which vary according to function and species, primarily reduce mechanical stress. Anchorage of AHOOOs to their surrounding tissues fortifies these structures and is accomplished by bone-bone fusion and Sharpey fibres. The presence of the integument is essential for the protection and function of the bony compartments. Three major functions can be attributed to AHOOOs: mechanical, visual, and thermoregulatory. This review provides the first extensive comparative description of the skeletal and integumentary systems of AHOOOs in a variety of mammals., (© 2020 Cambridge Philosophical Society.)

Deer antlers are a bony organ solely able to acquired distinct unique attributes during evolution and all these attributes are against thus far known natural rules. One of them is as the fastest animal growing tissue (2 cm/day), they are remarkably cancer-free, despite high cell division rate. Although tumor-like nodules on the long-lived castrate antlers in some deer species do occur, but they are truly benign in nature. In this review, we tried to find the answer to this seemingly contradictory phenomenon based on the currently available information and give insights into possible clinic application. The antler growth center is located in its tip; the most intensive dividing cells are resident in the inner layer of reserve mesenchyme (RM), and these cells are more adopted to osteosarcoma rather than to normal bone tissues in gene expression profiles but acquire their energy mainly through aerobic oxidative phosphorylation pathway. To counteract propensity of neoplastic transformation, antlers evolved highly efficient apoptosis exactly in the RM, unparalleled by any known tissues; and annual wholesale cast to jettison the corps. Besides, some strong cancer suppressive genes including p53 cofactor genes and p53 regulator genes are highly positively selected by deer, which would have certainly contributed to curb tumorigenesis. Thus far, antler extracts and RM cells/exosomes have been tried on different cancer models either in vitro or in vivo, and all achieved positive results. These positive experimental results together with the anecdotal folklore that regular consumption of velvet antler is living with cancer-free would encourage us to test antlers in clinic settings., (© 2023. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

The annual regrowth of deer antlers provides a valuable model for studying organ regeneration in mammals. We describe a single-cell atlas of antler regrowth. The earliest-stage antler initiators were mesenchymal cells that express the paired related homeobox 1 gene ( PRRX1 mesenchymal cells). We also identified a population of "antler blastema progenitor cells" (ABPCs) that developed from the + mesenchymal cells and directed the antler regeneration process. Cross-species comparisons identified ABPCs in several mammalian blastema. In vivo and in vitro ABPCs displayed strong self-renewal ability and could generate osteochondral lineage cells. Last, we observed a spatially well-structured pattern of cellular and gene expression in antler growth center during the peak growth stage, revealing the cellular mechanisms involved in rapid antler elongation.PRRX1 + mesenchymal cells and directed the antler regeneration process. Cross-species comparisons identified ABPCs in several mammalian blastema. In vivo and in vitro ABPCs displayed strong self-renewal ability and could generate osteochondral lineage cells. Last, we observed a spatially well-structured pattern of cellular and gene expression in antler growth center during the peak growth stage, revealing the cellular mechanisms involved in rapid antler elongation.

As the only known mammalian organ that can fully and annually regenerate, deer antler has significant advantages over lower-order animal models when investigating the control of stem-cell-based organ regeneration. Antler regeneration is known to be initiated and maintained by neural-crest-derived stem cells in different states of activation. Antler stem cells can therefore be used as a model to study proteins and pathways involved in the maintenance of a stem cell niche and their activation and differentiation during organ formation. In this study, the MSC markers CD73, CD90, and CD105 were examined within the antler tip. Label-free quantification was performed to investigate the protein profiles of antler stem cells under different stages of activation and included dormant pedicle periosteum (DPP), antler growth center (GC), post-active stem cells from mid-beam antler periosteum (MAP), and deer facial periosteum (FP) as a control ( n = 3 per group). PEAKS and IPA software were used to analyze the proteomic data. Our research confirmed the central role of stem cell activation in the development of this mammalian organ by localizing the MSC markers within the antler growth center. Label-free quantification revealed that the greatest number of unique proteins (87) was found in the growth center. There were only 12 proteins found with expression levels that significantly differed between DPP and FP. Protein profiles of these two groups indicated that antler stem cells may use similar mechanisms to maintain dormancy within a stem cell niche. The number of significantly regulated proteins among DPP, MAP, and GC was 153. Among them, the majority were upregulated in the growth center. Activation of antler stem cells was associated with many biological processes and signaling pathways, such as Hippo and canonical Wnt signaling. This work identifies the key pathways, molecular/cellular functions, and upstream regulators involved in mammal organ regeneration. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the iProX partner repository with the dataset identifier PXD016824.

Deer antlers are amazing appendages with the fastest growth rate among mammalian organs. Antler growth is driven by the growth center through a modified endochondral ossification process. Thus, identification of signaling pathways functioning in antler growth center would help us to uncover the underlying molecular mechanism of rapid antler growth. Furthermore, exploring and dissecting the molecular mechanism that regulates antler growth is extremely important and helpful for identifying methods to enhance long bone growth and treat cartilage- and bone-related diseases. In this study, we build a comprehensive intercellular signaling network in antler growth centers from both the slow growth stage and rapid growth stage using a state-of-art RNA-Seq approach. This network includes differentially expressed genes that regulate the activation of multiple signaling pathways, including the regulation of actin cytoskeleton, calcium signaling, and adherens junction. These signaling pathways coordinately control multiple biological processes, including chondrocyte proliferation and differentiation, matrix homeostasis, mechanobiology, and aging processes, during antler growth in a comprehensive and efficient manner. Therefore, our study provides novel insights into the molecular mechanisms regulating antler growth and provides valuable and powerful insight for medical research on therapeutic strategies targeting skeletal disorders and related cartilage and bone diseases.