| Deer antlers are the only mammalian appendages that are subject to an annual cycle of full renewal. It provides a unique model to explore the mechanisms underlying epimorphic regeneration, a phenomenon of de novo development of appendages distal to the level of amputation in mammals. The yearly renewal cycle of antlers briefly consists of four stages: rapid growth, calcification, antler skin (also called velvet) shedding and antler casting. During the rapid growth stage, antlers are composed of cartilage and bone, infiltrated with blood vessels and nerves networks and covered by a velvet skin. Rapid longitudinal elongation occurs at the growth zone located in the distal tip of each antler branch. Within this growth zone, progenitor cells in the antler perichondrium (AP) proliferate and differentiate externally into vevlet skin, and internally into chondroprogenitors and chondrocytes arranged in vertical trabeculae.Antlers have a unique velvet-like skin that is sparsely populated with hair and is known as velvet, However, studies have shown that antler external velvet components are not simple extensions of typical scalp skin enveloping pedicles. Regeneration of velvet skin commences with the wound healing over a pedicle stump by distal pedicle skin. The uniqueness in this case is, however, that when the centripetally migrating healing skin passes the point of distal pedicle periosteum (PP), the skin begins to change in nature from scalp type to velvet type. In contrast to pedicle skin (a typical scalp skin), antler velvet lacks a subcutaneous loose connective tissue layer, but has a much thicker epidermis and the ability to form new hair follicles. These newly formed follicles lack associated arrector pili muscles and sweat glands but possess large multilobed sebaceous glands. On the internal side, although the matrix of antler cartilage is biochemically similar to that of other hyaline cartilages, its unique feature is an extensive vascular network. These histological features make velvet and cartilage unique tissues types, underlying which large-scale changes in gene expression programs occur to create the new structures, through a series of proliferation, patterning, and differentiation events. However, little transcriptomic information is available for them.In this study, for velvet skin and cartilage from the antler of Red deer (Cervus elaphus), we performed de novo transcriptome assembly and analysis of total 4,958,615,700 nucleotides composed 55,095,730 clean short reads using RNA-Seq technology. After de novo assembly, with BLASTX alignment, assembled sequences were then annotated to NCBI nr, Gene ontology terms, Clusters of Orthologous Groups classifications, and Kyoto Encyclopedia of Genes and Genomes pathways databases. The results showed:1. De novo assembly of the clean reads by Trinity resulted in 63,550 unique all-unigenes. Velvet skin and catilage shared 33,295 all-unigenes,20,139 all-unigenes were vevlet-specific, and 10,116 were specially from cartilage.2. After blasted against the NCBI nr database.33,471 genes (52.67% of all unigenes) returned a BLAST result above the cut-off value (E-value 10-5). Similarly, up to 32,762 all-unigenes (51.55% of all unigenes) had Swissprot annotation, and 11,428 all-unigenes (17.98% of all unigenes) had COG annotation. Based on GO classifications,9,252 sequences (14.56%) were categorized into 52 functional groups. Meanwhile,25,577 all-unigenes (40.25%) were blasted into 241 KEGG pathways. All-unigenes have no hit in blast were predicted by ESTScan,691 putative novel genes were found and translated into peptide sequencs.3. Functional annotation and metabolic pathways analysis disclosed, during velvet and cartilage growth and development at the rapid growth stage, genes and pathways, involving cell, cellular skeleton, ribosomal structures, extracellular matrix, nucleitides and protein biosynthesis and transfer, translation, catelytic activity. And metabolic process, cell proliferation regulation, anti-apoptosis, play critical regulation functions.4. Through data mining,44 kinds of growth factors and their 22 kinds of receptors were discovered in antler velvet skin. Among them, insulin-like growth factor 2 had the highest expression level. Six growth factors and two kinds of recptors were selected and validated by real-time qPCR method.5. Anlter velvet skin and cartilage owned 6,961 and 2,776 tissue-specific genes, respectively. Within the shared genes,7,966 showed differentitial expression (log2Ratio≥1 and FDR≤0.001). Among them,5779 genes were up-regulated in velvet skin, and 2187 genes were up-regulated in cartilage. Through GO and KEGG enrichment analysis, these differential genes focused on cell components, cellular metabolics, proteins interaction, catalytic activity. 5328 genes involved were blasted against 236 pathways.These data represents the most comprehensive sequence resource available for the regenerating antler velvet skin and cartilage at the Red deer antler growth tip. and provide a basis for further research on deer antler molecular genetics and functional genomics. |
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