Deer antlers are amazing natural appendages that grow faster than any other known mammalian bone. Antler growth occurs at the tip and is initially cartilage, which is later replaced by bone tissue. However, little is known regarding the precise role of cooperation between cell lineages and functional genes in regulating antler growth, and molecular mechanisms responsible for rapid growth remain elusive. In this study, we use an RNA-Seq approach to elucidate the full spectrum of cell lineages, functional genes, and their cooperative interactions during antler growth. We identify Sox9 as a pivotal transcription factor during chondrogenesis and skeletal development expressed in the chondrocyte lineage from the multipotent mesenchymal precursor stage through most subsequent cell differentiation stages with a particularly strong activity in proliferating and prehypertrophic chondrocytes. Furthermore, we analyze the miRNA expression patterns at initial growth stage and rapid growth stage and identify several miRNAs that involve in regulating antler chondrogenesis and rapid growth. Among these miRNAs, miR-140 plays pivotal role during antler growth by targeting Sox9 and vice versa.
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References
1.
AkiyamaH., ChaboissierM.C., MartinJ.F., SchedlA., and de CrombruggheB. (2002). The transcription factor Sox9 has essential roles in successive steps of the chondrocyte differentiation pathway and is required for expression of Sox5 and Sox6. Genes Dev, 16, 2813–2828.
2.
AltschulS.F., GishW., MillerW., MyersE.W., and LipmanD.J. (1990). Basic local alignment search tool. J Mol Biol, 215, 403–410.
3.
ArtigasN., UreñaC., Rodríguez-CarballoE., RosaJ.L., and VenturaF. (2014). Mitogen-activated protein kinase (MAPK)-regulated interactions between Osterix and Runx2 are critical for the transcriptional osteogenic program. J Biol Chem, 289, 27105–27117.
4.
BanksJ.W., and NewbreyW.J. (1983). Antler development as a unique modification of mammalian endochondral ossification. In: Antler Developments in Cervidae. BanksR.D., ed. (Kingsville, TX: Cesar Kleburg Wildlife Research Institute), pp. 279–306.
5.
BanksW.J., and NewbreyJ.W. (1982). Light microscopic studies of the ossification process in developing antlers. In: Antler Development in Cervidae. BanksR.D., ed. (Kingsville, TX: Caesar Kleberg Wildlife Research Institute), pp. 231–260.
6.
BenjaminM., and EvansE.J. (1990). Fibrocartilage. J Anat, 171, 1–15.
7.
BiW., DengJ.M., ZhangZ., BehringerR.R., and de CrombruggheB. (1999). Sox9 is required for cartilage formation. Nat Genet, 22, 85–89.
8.
ChapmanD.I. (1975). Antlers—bones of contention. Mammal Rev, 5, 121–172.
9.
ChenC., RidzonD.A., BroomerA.J., ZhouZ., LeeD.H., NguyenJ.T., et al. (2005). Real-time quantification of microRNAs by stem-loop RT-PCR. Nucleic Acids Res, 33, e179.
10.
DąbrowskaN., KiełbowiczZ., NowackiW., BajzertJ., ReichertP., BieżyńskiJ., et al. (2015). Antlerogenic stem cells: molecular features and potential in rabbit bone regeneration. Connect Tissue Res, 19, 1–14.
11.
DaiL., ZhangX., HuX., ZhouC., and AoY. (2012). Silencing of microRNA-101 prevents IL-1β-induced extracellular matrix degradation in chondrocytes. Arthritis Res Ther, 14, R268.
12.
DyP., WangW., BhattaramP., WangQ., WangL., BallockR.T., et al. (2012). Sox9 directs hypertrophic maturation and blocks osteoblast differentiation of growth plate chondrocytes. Dev Cell, 22, 597–609.
13.
EmbreeM.C., ChenM., PylawkaS., KongD., IwaokaG.M., KalajzicI., et al. (2016). Exploiting endogenous fibrocartilage stem cells to regenerate cartilage and repair joint injury. Nat Commun, 7, 13073.
14.
EyreD. (2002). Collagen of articular cartilage. Arthritis Res, 4, 30–35.
15.
EyreD.R., and MuirH. (1977). Quantitative analysis of types I and II collagens in human intervertebral discs at various ages. Biochim Biophys Acta, 492, 29–42.
16.
FaucheuxC., NesbittS.A., HortonM.A., and PriceJ.S. (2001). Cells in regenerating deer antler cartilage provide a microenvironment that supports osteoclast differentiation. J Exp Biol, 204, 443–455.
17.
FosterJ.W., Dominguez-SteglichM.A., GuioliS., KwokC., WellerP.A., StevanovicM., et al. (1994). Campomelic dysplasia and autosomal sex reversal caused by mutations in an SRY-related gene. Nature, 372, 525–530.
18.
GrabherrM.G., HaasB.J., YassourM., LevinJ.Z., ThompsonD.A., AmitI., et al. (2011). Full-length transcriptome assembly from RNA-Seq data without a reference genome. Nat Biotechnol, 29, 644–652.
19.
GuL., MoE., ZhuX., JiaX., FangZ., SunB., et al. (2008). Analysis of gene expression in four parts of the red-deer antler using DNA chip microarray technology. Anim Biol, 58, 67–90.
HenryS.P., LiangS., AkdemirK.C., and de CrombruggheB. (2012). The postnatal role of Sox9 in cartilage. J Bone Miner Res, 27, 2511–2525.
22.
HuW., LiM., HuR., LiT., and MengX. (2015). microRNA-18b modulates insulin-like growth factor-1 expression in deer antler cell proliferation by directly targeting its 3′ untranslated region. DNA Cell Biol, 34, 282–289.
23.
HuoY.S., HuoH., and ZhangJ. (2014). The contribution of deer velvet antler research to the modern biological medicine. Chin J Integr Med, 20, 723–728.
24.
KarlsenT.A., JakobsenR.B., MikkelsenT.S., and BrinchmannJ.E. (2014). microRNA-140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN. Stem Cells Dev, 23, 290–304.
25.
KobayashiS., TakebeT., InuiM., IwaiS., KanH., ZhengY.W., et al. (2011). Reconstruction of human elastic cartilage by a CD44+ CD90+ stem cell in the ear perichondrium. Proc Natl Acad Sci U S A, 108, 14479–14484.
26.
KronenbergH.M. (2003). Developmental regulation of the growth plate. Nature, 423, 332–336.
27.
LeL.T., SwinglerT.E., and ClarkI.M. (2013). Review: the role of microRNAs in osteoarthritis and chondrogenesis. Arthritis Rheum, 65, 1963–1974.
28.
LeeS., YoonD.S., PaikS., LeeK.M., JangY., and LeeJ.W. (2014). microRNA-495 inhibits chondrogenic differentiation in human mesenchymal stem cells by targeting Sox9. Stem Cells Dev, 15, 1798–808.
29.
LiC., and ChuW. (2016). The regenerating antler blastema: the derivative of stem cells resident in a pedicle stump. Front Biosci (Landmark Ed), 21, 455–467.
30.
LiC., ClarkD.E., LordE.A., StantonJ.A., and SuttieJ.M. (2002). Sampling technique to discriminate the different tissue layers of growing antler tips for gene discovery. Anat Rec, 268, 125–130.
31.
LiC., and SuttieJ.M. (1994). Light microscopic studies of pedicle and early first antler development in red deer (Cervus elaphus). Anat Rec, 239, 198–215.
32.
LiC., ZhaoH., LiuZ., and McMahonC. (2014). Deer antler—a novel model for studying organ regeneration in mammals. Int J Biochem Cell Biol, 56, 111–122.
33.
LiuC.F., and LefebvreV. (2015). The transcription factors SOX9 and SOX5/SOX6 cooperate genome-wide through super-enhancers to drive chondrogenesis. Nucleic Acids Res, 43, 8183–8203.
34.
LiuM., YaoB., ZhangH., GuoH., HuD., WangQ., and ZhaoY. (2014). Identification of novel reference genes using sika deer antler transcriptome expression data and their validation for quantitative gene expression analysis. Genes Genom, 36, 573–582.
35.
MackieE.J., TatarczuchL., and MiramsM. (2011). The skeleton: a multi-functional complex organ: the growth plate chondrocyte and endochondral ossification. J Endocrinol, 211, 109–121.
36.
MarioniJ.C., MasonC.E., ManeS.M., StephensM., and GiladY. (2008). RNA-Seq: an assessment of technical reproducibility and comparison with gene expression arrays. Genome Res, 18, 1509–1517.
37.
MizunoM., KobayashiS., TakebeT., KanH., YabukiY., MatsuzakiT., et al. (2014). Brief report: reconstruction of joint hyaline cartilage by autologous progenitor cells derived from ear elastic cartilage. Stem Cells, 32, 816–821.
38.
MolnárA., GyurjánI., KorposE., BorsyA., StégerV., BuzásZ., et al. (2007). Identification of differentially expressed genes in the developing antler of red deer Cervus elaphus. Mol Genet Genomics, 277, 237–248.
39.
MortazaviA., WilliamsB.A., MccueK., SchaefferL., and WoldB. (2008). Mapping and quantifying mammalian transcriptomes by RNA-Seq. Nat Methods, 5, 1–8.
40.
NagalakshmiU., WangZ., WaernK., ShouC., RahaD., GersteinM., et al. (2008). The transcriptional landscape of the yeast genome defined by RNA sequencing. Science, 10, 57–63.
41.
NakamuraY., HeX., KatoH., WakitaniS., KobayashiT., WatanabeS., et al. (2012). Sox9 is upstream of microRNA-140 in cartilage. Appl Biochem Biotechnol, 166, 64–71.
42.
NishimuraR., WakabayashiM., HataK., MatsubaraT., HonmaS., WakisakaS., et al. (2012). Osterix regulates calcification and degradation of chondrogenic matrices through matrix metalloproteinase 13 (MMP13) expression in association with transcription factor Runx2 during endochondral ossification. J Biol Chem, 287, 33179–33190.
43.
NishioY., DongY., ParisM., O'KeefeR.J., SchwarzE.M., and DrissiH. (2006). Runx2-mediated regulation of the zinc finger Osterix/Sp7 gene. Gene, 372, 62–70.
44.
OhC.D., LuY., LiangS., Mori-AkiyamaY., ChenD., de CrombruggheB., et al. (2015). Distinct transcriptional programs underlie Sox9 regulation of the mammalian chondrocyte. PLoS One, 9, e107577.
45.
OhbaS., HeX., HojoH., and McMahonA.P. (2015). Distinct transcriptional programs underlie Sox9 regulation of the mammalian chondrocyte. Cell Rep, 12, 229–243.
46.
OrtuñoM.J., SusperreguiA.R., ArtigasN., RosaJ.L., and VenturaF. (2013). Osterix induces Col1a1 gene expression through binding to Sp1 sites in the bone enhancer and proximal promoter regions. Bone, 52, 548–556.
47.
ParkJ., JeonB., KangS., OhM., KimM., JangS., et al. (2015). Study on the changes in enzyme and insulin-like growth factor-1 concentrations in blood serum and growth characteristics of velvet antler during the antler growth period in Sika deer (Cervus nippon). Asian-Australas J Anim Sci, 28, 1303–1308.
48.
PriceJ., and AllenS. (2004). Exploring the mechanisms regulating regeneration of deer antlers. Philos Trans R Soc Lond B Biol Sci, 359, 809–822.
49.
PriceJ.S., AllenS., FaucheuxC., AlthnaianT., and MountJ.G. (2005). Deer antlers: a zoological curiosity or the key to understanding organ regeneration in mammals?. J Anat, 207, 603–618.
50.
RobertsS., MenageJ., SandellL.J., EvansE.H., and RichardsonJ.B. (2009). Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation. Knee, 16, 398–404.
51.
SchmittgenT.D., and LivakK.J. (2008). Analyzing real-time PCR data by the comparative C (T) method. Nat Protoc, 3, 1101–1108.
52.
SchroederA., MuellerO., StockerS., SalowskyR., LeiberM., GassmannM., et al. (2006). The RIN: an RNA integrity number for assigning integrity values to RNA measurements. BMC Mol Biol, 7, 3.
53.
ShangJ., LiuH., and ZhouY. (2013). Roles of microRNAs in prenatal chondrogenesis, postnatal chondrogenesis and cartilage-related diseases. J Cell Mol Med, 17, 1515–1524.
54.
UngerS., SchererG., and Superti-FurgaA. (2013). Campomelic Dysplasia (GeneReviews). Seattle, WA: University of Washington.
55.
WagnerT., WirthJ., MeyerJ., ZabelB., HeldM., ZimmerJ., et al. (1994). Autosomal sex reversal and campomelic dysplasia are caused by mutations in and around the SRY-related gene SOX9. Cell, 79, 1111–1120.
56.
WangL., FengZ., WangX., WangX., and ZhangX. (2010). DEGseq: an R package for identifying differentially expressed genes from RNA-Seq data. Bioinformatics, 26, 136–138.
57.
WangZ., GersteinM., and SnyderM. (2009). RNA-Seq: a revolutionary tool for transcriptomics. Nat Rev Genet, 10, 57–63.
58.
WuC., TianB., QuX., LiuF., TangT., QinA., et al. (2014). MicroRNAs play a role in chondrogenesis and osteoarthritis (review). Int J Mol Med, 34, 13–23.
59.
YangB., GuoH., ZhangY., ChenL., YingD., and DongS. (2011a). MicroRNA-145 regulates chondrogenic differentiation of mesenchymal stem cells by targeting Sox9. PLoS One, 6, e21679.
60.
YangJ., QinS., YiC., MaG., ZhuH., ZhouW., et al. (2011b). MiR-140 is co-expressed with Wwp2-C transcript and activated by Sox9 to target Sp1 in maintaining the chondrocyte proliferation. FEBS Lett, 585, 2992–2997.
61.
YangL., TsangK.Y., and TangH.C. (2014). Hypertrophic chondrocytes can become osteoblasts and osteocytes in endochondral bone formation. Proc Natl Acad Sci U S A, 111, 12097–12102.
62.
YaoB., ZhaoY., WangQ., ZhangM., LiuM., LiuH., et al. (2012a). De novo characterization of the antler tip of Chinese Sika deer transcriptome and analysis of gene expression related to rapid growth. Mol Cell Biochem, 364, 93–100.
63.
YaoB., ZhaoY., ZhangH., ZhangM., LiuM., LiuH., et al. (2012b). Sequencing and de novo analysis of the Chinese Sika deer antler-tip transcriptome during the ossification stage using Illumina RNA-Seq technology. Biotechnol Lett, 34, 813–822.
64.
ZhaoY., YaoB., ZhangM., WangS., ZhangH., and XiaoW. (2013). Comparative analysis of differentially expressed genes in Sika deer antler at different stages. Mol Biol Rep, 40, 1665–1676.
65.
ZhouX., ZhangZ., FengJ.Q., DusevichV.M., SinhaK., ZhangH., et al. (2010). Multiple functions of Osterix are required for bone growth and homeostasis in postnatal mice. Proc Natl Acad Sci U S A, 107, 12919–12924.
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