FREE SHIPPING over $40
Horse Supplements USA English
Horse Supplements Canada EnglishSuppléments Équins Canada French
My Account
View Cart My Horses Track Orders Account Details My Subscriptions Update Address Payment Methods
Register/Login
0
Home/Equine Research Bank/Genes (Basel)/Identification of Novel Equine (Equus caballus) Tendon Marke...
Genes2016; 7(11); 97; doi: 10.3390/genes7110097

Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing.

Abstract: Although several tendon-selective genes exist, they are also expressed in other musculoskeletal tissues. As cell and tissue engineering is reliant on specific molecular markers to discriminate between cell types, tendon-specific genes need to be identified. In order to accomplish this, we have used RNA sequencing (RNA-seq) to compare gene expression between tendon, bone, cartilage and ligament from horses. We identified several tendon-selective gene markers, and established eyes absent homolog 2 () and a G-protein regulated inducer of neurite outgrowth 3 () as specific tendon markers using RT-qPCR. Equine tendon cells cultured as three-dimensional spheroids expressed significantly greater levels of than , and stained positively for EYA2 using immunohistochemistry. EYA2 was also found in fibroblast-like cells within the tendon tissue matrix and in cells localized to the vascular endothelium. In summary, we have identified and as specific molecular markers of equine tendon as compared to bone, cartilage and ligament, and provide evidence for the use of EYA2 as an additional marker for tendon cells in vitro.
Get Full Text
Publication Date: 2016-11-10 PubMed ID: 27834918PubMed Central: PMC5126783DOI: 10.3390/genes7110097Google Scholar: Lookup
The Equine Research Bank provides access to a large database of publicly available scientific literature. Inclusion in the Research Bank does not imply endorsement of study methods or findings by Mad Barn.
LinkedIn
  • Journal Article

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article focuses on identifying specific molecular markers for equine (horse) tendons using RNA sequencing, to better distinguish them from other musculoskeletal tissues. This is key to cell and tissue engineering that seek to understand specific cell types. The researchers found that the genes eyes absent homolog 2 (EYA2) and a G-protein regulated inducer of neurite outgrowth 3 (GPRIN3) can be used as specific tendon markers.

RNA Sequencing and the Search for Tendon Markers

  • The research began with the challenge that, although several genes are specifically expressed in tendons, these same genes also show expression in other musculoskeletal tissues. This overlap makes it difficult for researchers to accurately discriminate between cell types in cellular and tissue engineering studies.
  • To solve this issue, the researchers employed RNA sequencing (RNA-seq), a powerful tool for studying gene expression. They compared the gene expression profiles of tendon, bone, cartilage, and ligament tissues harvested from horses.

Identification of Tendon-Specific Markers

  • Through this process of gene comparison, researchers successfully identified several gene markers that are selectively expressed in tendons. In particular, two genes, EYA2 and GPRIN3, stood out as unique and specific tendon markers.
  • After their identification via RNA-seq, these markers were further confirmed using RT-qPCR, a technique used to measure the quantity of specific RNAs (in this case, EYA2 and GPRIN3).

Verification of Tendon Markers and Their Implications

  • The team also found that equine tendon cells, when cultured in a three-dimensional environment, expressed significantly more EYA2 than GPRIN3. This observation was corroborated by immunohistochemistry staining, which also confirmed the presence of EYA2 in the cultured cells.
  • EYA2 was also detected in fibroblast-like cells (a cell type commonly found in tendons) within the tendon tissue matrix, as well as in cells situated in the vascular endothelium. This indicates EYA2’s potential for being a useful marker to identify and study tendon cells both in vivo and in vitro.
  • In conclusion, the identification of EYA2 and GPRIN3 as specific molecular markers for equine tendons represents a significant advance in the field of tissue engineering and can facilitate further studies into understanding the biology of tendons.

Cite This Article

APA
Kuemmerle JM, Theiss F, Okoniewski MJ, Weber FA, Hemmi S, Mirsaidi A, Richards PJ, Cinelli P. (2016). Identification of Novel Equine (Equus caballus) Tendon Markers Using RNA Sequencing. Genes (Basel), 7(11), 97. https://doi.org/10.3390/genes7110097

Publication

Genes
ISSN: 2073-4425
NlmUniqueID: 101551097
Country: Switzerland
Language: English
Volume: 7
Issue: 11
PII: 97

Researcher Affiliations

Kuemmerle, Jan M
  • Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. jkuemmerle@vetclinics.uzh.ch.
  • Equine Hospital, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland. jkuemmerle@vetclinics.uzh.ch.
Theiss, Felix
  • Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. ftheiss@vetclinics.uzh.ch.
  • Equine Hospital, Vetsuisse Faculty, University of Zurich, CH-8057 Zurich, Switzerland. ftheiss@vetclinics.uzh.ch.
Okoniewski, Michal J
  • Scientific IT Services, Swiss Federal Institute of Technology, CH 8092 Zurich, Switzerland. michalo@id.ethz.ch.
Weber, Fabienne A
  • Institute of Laboratory Animal Science, University of Zurich, CH-8057 Zurich, Switzerland. fabienne.weber@lasc.uzh.ch.
Hemmi, Sonja
  • Division of Trauma Surgery, Center for Clinical Research, University Hospital Zurich, University of Zurich, Sternwartstrasse 14, CH-8091 Zurich, Switzerland. Sonja.hemmi@usz.ch.
Mirsaidi, Ali
  • Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. ali.mirsaidi@cabmm.uzh.ch.
Richards, Peter J
  • Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. peter.richards@cabmm.uzh.ch.
Cinelli, Paolo
  • Center for Applied Biotechnology and Molecular Medicine, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland. paolo.cinelli@usz.ch.
  • Division of Trauma Surgery, Center for Clinical Research, University Hospital Zurich, University of Zurich, Sternwartstrasse 14, CH-8091 Zurich, Switzerland. paolo.cinelli@usz.ch.

Conflict of Interest Statement

The authors declare no conflict of interest.

References

This article includes 41 references
  1. Cassel M, Baur H, Hirschmu00fcller A, Carlsohn A, Fru00f6hlich K, Mayer F. Prevalence of Achilles and patellar tendinopathy and their association to intratendinous changes in adolescent athletes.. Scand J Med Sci Sports 2015 Jun;25(3):e310-8.
    doi: 10.1111/sms.12318pubmed: 25212527google scholar: lookup
  2. Reardon RJ, Boden LA, Mellor DJ, Love S, Newton JR, Stirk AJ, Parkin TD. Risk factors for superficial digital flexor tendinopathy in Thoroughbred racehorses in steeplechase starts in the United Kingdom (2001-2009).. Vet J 2013 Mar;195(3):325-30.
    doi: 10.1016/j.tvjl.2012.06.033pubmed: 22840205google scholar: lookup
  3. Williams RB, Harkins LS, Hammond CJ, Wood JL. Racehorse injuries, clinical problems and fatalities recorded on British racecourses from flat racing and National Hunt racing during 1996, 1997 and 1998.. Equine Vet J 2001 Sep;33(5):478-86.
    doi: 10.2746/042516401776254808pubmed: 11558743google scholar: lookup
  4. Lui PP, Maffulli N, Rolf C, Smith RK. What are the validated animal models for tendinopathy?. Scand J Med Sci Sports 2011 Feb;21(1):3-17.
    doi: 10.1111/j.1600-0838.2010.01164.xpubmed: 20673247google scholar: lookup
  5. Smith RK. Mesenchymal stem cell therapy for equine tendinopathy.. Disabil Rehabil 2008;30(20-22):1752-8.
    doi: 10.1080/09638280701788241pubmed: 18608378google scholar: lookup
  6. Smith R, McIlwraith W, Schweitzer R, Kadler K, Cook J, Caterson B, Dakin S, Heinegu00e5rd D, Screen H, Stover S, Crevier-Denoix N, Clegg P, Collins M, Little C, Frisbie D, Kjaer M, van Weeren R, Werpy N, Denoix JM, Carr A, Goldberg A, Bramlage L, Smith M, Nixon A. Advances in the understanding of tendinopathies: a report on the Second Havemeyer Workshop on equine tendon disease.. Equine Vet J 2014 Jan;46(1):4-9.
    doi: 10.1111/evj.12128pubmed: 24329581google scholar: lookup
  7. Bullough R, Finnigan T, Kay A, Maffulli N, Forsyth NR. Tendon repair through stem cell intervention: cellular and molecular approaches.. Disabil Rehabil 2008;30(20-22):1746-51.
    doi: 10.1080/09638280701788258pubmed: 18720123google scholar: lookup
  8. Andia I, Latorre PM, Gomez MC, Burgos-Alonso N, Abate M, Maffulli N. Platelet-rich plasma in the conservative treatment of painful tendinopathy: a systematic review and meta-analysis of controlled studies.. Br Med Bull 2014 Jun;110(1):99-115.
    doi: 10.1093/bmb/ldu007pubmed: 24795364google scholar: lookup
  9. Mu00fcller SA, Todorov A, Heisterbach PE, Martin I, Majewski M. Tendon healing: an overview of physiology, biology, and pathology of tendon healing and systematic review of state of the art in tendon bioengineering.. Knee Surg Sports Traumatol Arthrosc 2015 Jul;23(7):2097-105.
    doi: 10.1007/s00167-013-2680-zpubmed: 24057354google scholar: lookup
  10. Obaid H, Connell D. Cell therapy in tendon disorders: what is the current evidence?. Am J Sports Med 2010 Oct;38(10):2123-32.
    doi: 10.1177/0363546510373574pubmed: 20699425google scholar: lookup
  11. Young M. Stem cell applications in tendon disorders: a clinical perspective.. Stem Cells Int 2012;2012:637836.
    doi: 10.1155/2012/637836pmc: PMC3289928pubmed: 22448174google scholar: lookup
  12. Smith RK, Korda M, Blunn GW, Goodship AE. Isolation and implantation of autologous equine mesenchymal stem cells from bone marrow into the superficial digital flexor tendon as a potential novel treatment.. Equine Vet J 2003 Jan;35(1):99-102.
    doi: 10.2746/042516403775467388pubmed: 12553472google scholar: lookup
  13. Becerra P, Valdu00e9s Vu00e1zquez MA, Dudhia J, Fiske-Jackson AR, Neves F, Hartman NG, Smith RK. Distribution of injected technetium(99m)-labeled mesenchymal stem cells in horses with naturally occurring tendinopathy.. J Orthop Res 2013 Jul;31(7):1096-102.
    doi: 10.1002/jor.22338pubmed: 23508674google scholar: lookup
  14. Guest DJ, Smith MR, Allen WR. Monitoring the fate of autologous and allogeneic mesenchymal progenitor cells injected into the superficial digital flexor tendon of horses: preliminary study.. Equine Vet J 2008 Mar;40(2):178-81.
    doi: 10.2746/042516408X276942pubmed: 18267891google scholar: lookup
  15. Murray SJ, Santangelo KS, Bertone AL. Evaluation of early cellular influences of bone morphogenetic proteins 12 and 2 on equine superficial digital flexor tenocytes and bone marrow-derived mesenchymal stem cells in vitro.. Am J Vet Res 2010 Jan;71(1):103-14.
    doi: 10.2460/ajvr.71.1.103pmc: PMC4246500pubmed: 20043789google scholar: lookup
  16. Sole A, Spriet M, Padgett KA, Vaughan B, Galuppo LD, Borjesson DL, Wisner ER, Vidal MA. Distribution and persistence of technetium-99 hexamethyl propylene amine oxime-labelled bone marrow-derived mesenchymal stem cells in experimentally induced tendon lesions after intratendinous injection and regional perfusion of the equine distal limb.. Equine Vet J 2013 Nov;45(6):726-31.
    doi: 10.1111/evj.12063pubmed: 23574488google scholar: lookup
  17. Kleinman HK, Philp D, Hoffman MP. Role of the extracellular matrix in morphogenesis.. Curr Opin Biotechnol 2003 Oct;14(5):526-32.
    doi: 10.1016/j.copbio.2003.08.002pubmed: 14580584google scholar: lookup
  18. Richardson LE, Dudhia J, Clegg PD, Smith R. Stem cells in veterinary medicine--attempts at regenerating equine tendon after injury.. Trends Biotechnol 2007 Sep;25(9):409-16.
    doi: 10.1016/j.tibtech.2007.07.009pubmed: 17692415google scholar: lookup
  19. Taylor SE, Vaughan-Thomas A, Clements DN, Pinchbeck G, Macrory LC, Smith RK, Clegg PD. Gene expression markers of tendon fibroblasts in normal and diseased tissue compared to monolayer and three dimensional culture systems.. BMC Musculoskelet Disord 2009 Feb 26;10:27.
    doi: 10.1186/1471-2474-10-27pmc: PMC2651848pubmed: 19245707google scholar: lookup
  20. Brandau O, Meindl A, Fu00e4ssler R, Aszu00f3di A. A novel gene, tendin, is strongly expressed in tendons and ligaments and shows high homology with chondromodulin-I.. Dev Dyn 2001 May;221(1):72-80.
    doi: 10.1002/dvdy.1126pubmed: 11357195google scholar: lookup
  21. Kardasinski M, Thum T. Cardiac fibroblasts on the fast track--scleraxis: from Achilles' heel to anti-fibrotic therapy.. J Mol Cell Cardiol 2009 Aug;47(2):174-6.
    doi: 10.1016/j.yjmcc.2009.05.006pubmed: 19463829google scholar: lookup
  22. Shukunami C, Takimoto A, Oro M, Hiraki Y. Scleraxis positively regulates the expression of tenomodulin, a differentiation marker of tenocytes.. Dev Biol 2006 Oct 1;298(1):234-47.
    doi: 10.1016/j.ydbio.2006.06.036pubmed: 16876153google scholar: lookup
  23. Jelinsky SA, Archambault J, Li L, Seeherman H. Tendon-selective genes identified from rat and human musculoskeletal tissues.. J Orthop Res 2010 Mar;28(3):289-97.
    doi: 10.1002/jor.20999pubmed: 19780194google scholar: lookup
  24. Mantione KJ, Kream RM, Kuzelova H, Ptacek R, Raboch J, Samuel JM, Stefano GB. Comparing bioinformatic gene expression profiling methods: microarray and RNA-Seq.. Med Sci Monit Basic Res 2014 Aug 23;20:138-42.
    pmc: PMC4152252pubmed: 25149683doi: 10.12659/MSMBR.892101google scholar: lookup
  25. Peffers M, Liu X, Clegg P. Transcriptomic signatures in cartilage ageing.. Arthritis Res Ther 2013 Aug 23;15(4):R98.
    doi: 10.1186/ar4278pmc: PMC3978620pubmed: 23971731google scholar: lookup
  26. Peffers MJ, Fang Y, Cheung K, Wei TK, Clegg PD, Birch HL. Transcriptome analysis of ageing in uninjured human Achilles tendon.. Arthritis Res Ther 2015 Feb 18;17(1):33.
    doi: 10.1186/s13075-015-0544-2pmc: PMC4355574pubmed: 25888722google scholar: lookup
  27. Theiss F, Mirsaidi A, Mhanna R, Ku00fcmmerle J, Glanz S, Bahrenberg G, Tiaden AN, Richards PJ. Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold inu00a0vitro.. Biomaterials 2015 Nov;69:99-109.
    doi: 10.1016/j.biomaterials.2015.08.013pubmed: 26283157google scholar: lookup
  28. Gartland A, Rumney RM, Dillon JP, Gallagher JA. Isolation and culture of human osteoblasts.. Methods Mol Biol 2012;806:337-55.
    pubmed: 22057462doi: 10.1007/978-1-61779-367-7_22google scholar: lookup
  29. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for Illumina sequence data.. Bioinformatics 2014 Aug 1;30(15):2114-20.
    doi: 10.1093/bioinformatics/btu170pmc: PMC4103590pubmed: 24695404google scholar: lookup
  30. Anders S, Huber W. Differential expression analysis for sequence count data.. Genome Biol 2010;11(10):R106.
    doi: 10.1186/gb-2010-11-10-r106pmc: PMC3218662pubmed: 20979621google scholar: lookup
  31. Anders S, McCarthy DJ, Chen Y, Okoniewski M, Smyth GK, Huber W, Robinson MD. Count-based differential expression analysis of RNA sequencing data using R and Bioconductor.. Nat Protoc 2013 Sep;8(9):1765-86.
    doi: 10.1038/nprot.2013.099pubmed: 23975260google scholar: lookup
  32. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data.. Bioinformatics 2010 Jan 1;26(1):139-40.
    doi: 10.1093/bioinformatics/btp616pmc: PMC2796818pubmed: 19910308google scholar: lookup
  33. Mirsaidi A, Tiaden AN, Richards PJ. Preparation and osteogenic differentiation of scaffold-free mouse adipose-derived stromal cell microtissue spheroids (ASC-MT).. Curr Protoc Stem Cell Biol 2013 Nov 13;27:2B.5.1-2B.5.12.
    doi: 10.1002/9780470151808.sc02b05s27pubmed: 24510285google scholar: lookup
  34. McIlwraith CW, Frisbie DD, Kawcak CE, Fuller CJ, Hurtig M, Cruz A. The OARSI histopathology initiative - recommendations for histological assessments of osteoarthritis in the horse.. Osteoarthritis Cartilage 2010 Oct;18 Suppl 3:S93-105.
    doi: 10.1016/j.joca.2010.05.031pubmed: 20864027google scholar: lookup
  35. Yao L, Bestwick CS, Bestwick LA, Maffulli N, Aspden RM. Phenotypic drift in human tenocyte culture.. Tissue Eng 2006 Jul;12(7):1843-9.
    doi: 10.1089/ten.2006.12.1843pubmed: 16889514google scholar: lookup
  36. Xu PX, Cheng J, Epstein JA, Maas RL. Mouse Eya genes are expressed during limb tendon development and encode a transcriptional activation function.. Proc Natl Acad Sci U S A 1997 Oct 28;94(22):11974-9.
    doi: 10.1073/pnas.94.22.11974pmc: PMC23674pubmed: 9342347google scholar: lookup
  37. Iida N, Kozasa T. Identification and biochemical analysis of GRIN1 and GRIN2.. Methods Enzymol 2004;390:475-83.
    pubmed: 15488195doi: 10.1016/S0076-6879(04)90029-8google scholar: lookup
  38. Fan X, Brass LF, Poncz M, Spitz F, Maire P, Manning DR. The alpha subunits of Gz and Gi interact with the eyes absent transcription cofactor Eya2, preventing its interaction with the six class of homeodomain-containing proteins.. J Biol Chem 2000 Oct 13;275(41):32129-34.
    doi: 10.1074/jbc.M004577200pubmed: 10906137google scholar: lookup
  39. Marioni JC, Mason CE, Mane SM, Stephens M, Gilad Y. RNA-seq: an assessment of technical reproducibility and comparison with gene expression arrays.. Genome Res 2008 Sep;18(9):1509-17.
    doi: 10.1101/gr.079558.108pmc: PMC2527709pubmed: 18550803google scholar: lookup
  40. Bi Y, Ehirchiou D, Kilts TM, Inkson CA, Embree MC, Sonoyama W, Li L, Leet AI, Seo BM, Zhang L, Shi S, Young MF. Identification of tendon stem/progenitor cells and the role of the extracellular matrix in their niche.. Nat Med 2007 Oct;13(10):1219-27.
    doi: 10.1038/nm1630pubmed: 17828274google scholar: lookup
  41. Thorpe CT, Peffers MJ, Simpson D, Halliwell E, Screen HR, Clegg PD. Anatomical heterogeneity of tendon: Fascicular and interfascicular tendon compartments have distinct proteomic composition.. Sci Rep 2016 Feb 4;6:20455.
    doi: 10.1038/srep20455pmc: PMC4740843pubmed: 26842662google scholar: lookup

Citations

This article has been cited 6 times.
  1. Ramos-Mucci L, Sarmiento P, Little D, Snelling S. Research perspectives-Pipelines to human tendon transcriptomics.. J Orthop Res 2022 May;40(5):993-1005.
    doi: 10.1002/jor.25315pubmed: 35239195google scholar: lookup
  2. Lee S, Baker ME, Clinton M, Taylor SE. Use of Omics Data in Fracture Prediction; a Scoping and Systematic Review in Horses and Humans.. Animals (Basel) 2021 Mar 30;11(4).
    doi: 10.3390/ani11040959pubmed: 33808497google scholar: lookup
  3. Paterson YZ, Cribbs A, Espenel M, Smith EJ, Henson FMD, Guest DJ. Genome-wide transcriptome analysis reveals equine embryonic stem cell-derived tenocytes resemble fetal, not adult tenocytes.. Stem Cell Res Ther 2020 May 19;11(1):184.
    doi: 10.1186/s13287-020-01692-wpubmed: 32430075google scholar: lookup
  4. Liao X, Falcon ND, Mohammed AA, Paterson YZ, Mayes AG, Guest DJ, Saeed A. Synthesis and Formulation of Four-Arm PolyDMAEA-siRNA Polyplex for Transient Downregulation of Collagen Type III Gene Expression in TGF-u03b21 Stimulated Tenocyte Culture.. ACS Omega 2020 Jan 28;5(3):1496-1505.
    doi: 10.1021/acsomega.9b03216pubmed: 32010823google scholar: lookup
  5. Smith JL, Wilson ML, Nilson SM, Rowan TN, Oldeschulte DL, Schnabel RD, Decker JE, Seabury CM. Genome-wide association and genotype by environment interactions for growth traits in U.S. Gelbvieh cattle.. BMC Genomics 2019 Dec 4;20(1):926.
    doi: 10.1186/s12864-019-6231-ypubmed: 31801456google scholar: lookup
  6. Jo CH, Lim HJ, Yoon KS. Characterization of Tendon-Specific Markers in Various Human Tissues, Tenocytes and Mesenchymal Stem Cells.. Tissue Eng Regen Med 2019 Apr;16(2):151-159.
    doi: 10.1007/s13770-019-00182-2pubmed: 30989042google scholar: lookup

Secure Payments

PayPal Visa Mastercard American Express

Satisfaction Guarantee

We offer a 60-day guarantee to ensure you are completely satisfied.

Fast & Free Shipping

We provide fast and free shipping on all orders over $40.

Use Nutrition Calculator
Check if your horse's diet meets their nutrition requirements with our easy-to-use tool Check your horse's diet with our easy-to-use tool
Talk to a Nutritionist
Discuss your horse's feeding plan with our experts over a free phone consultation Discuss your horse's diet over a phone consultation
Submit Diet Evaluation
Get a customized feeding plan for your horse formulated by our equine nutritionists Get a custom feeding plan formulated by our nutritionists