Advertisement

Cell and Tissue Research

, Volume 342, Issue 3, pp 363–376 | Cite as

Remodeling of the notochord during development of vertebral fusions in Atlantic salmon (Salmo salar)

  • Elisabeth Ytteborg
  • Jacob Seilø Torgersen
  • Mona E. Pedersen
  • Grete Baeverfjord
  • Kirsten O. Hannesson
  • Harald Takle
Regular Article

Abstract

Histological characterization of spinal fusions in Atlantic salmon (Salmo salar) has demonstrated shape alterations of vertebral body endplates, a reduced intervertebral space, and replacement of intervertebral cells by ectopic bone. However, the significance of the notochord during the fusion process has not been addressed. We have therefore investigated structural and cellular events in the notochord during the development of vertebral fusions. In order to induce vertebral fusions, Atlantic salmon were exposed to elevated temperatures from fertilization until they attained a size of 15 g. Based on results from radiography, intermediate and terminal stages of the fusion process were investigated by immunohistochemistry and real-time quantitative polymerase chain reaction. Examination of structural extracellular matrix proteins such as Perlecan, Aggrecan, Elastin, and Laminin revealed reduced activity and reorganization at early stages in the pathology. Staining for elastic fibers visualized a thinner elastic membrane surrounding the notochord of developing fusions, and immunohistochemistry for Perlecan showed that the notochordal sheath was stretched during fusion. These findings in the outer notochord correlated with the loss of Aggrecan- and Substance-P-positive signals and the further loss of vacuoles from the chordocytes in the central notochord. At more progressed stages of fusion, chordocytes condensed, and the expression of Aggrecan and Substance P reappeared. The hyperdense regions seem to be of importance for the formation of notochordal tissue into bone. Thus, the remodeling of notochord integrity by reduced elasticity, structural alterations, and cellular changes is probably involved in the development of vertebral fusions.

Keywords

Notochord Perlecan Spinal fusions Substance P Atlantic salmon Salmo salar (Teleostei) 

Abbreviations

AF

Anulus fibrosus

bp

Base pair

Col2

Collagen type 2

DAPI

4,6-Diamidino-2-phenylindole

Ef1a

Elongation factor 1

GBM

Glomerular kidney membrane

IDD

Intervertebral disc degeneration

IVD

Intervertebral disk

PBS

Phosphate-buffered saline

PG

Proteoglycan

qPCR

Quantitative polymerase chain reaction

RT

Reverse transcription

runx2

Runt-related transcription factor 2

Sox9

(Sex determining region Y) box 9

SP

Substance P

Zn12

Zebrafish neuron marker 12

Supplementary material

441_2010_1069_MOESM1_ESM.jpg (5.8 mb)
Fig. S1 Western blot of (a) Zn12 (standard in lane 1 Novex Sharp Pre-Stained Protein Standards, Invitrogen), (b) Aggrecan, and (c) Perlecan. (JPEG 5925 kb)

References

  1. Adams DS, Keller R, Koehl MAR (1990) The mechanics of notochord elongation, straightening and stiffening in the embryo of Xenopus laevis. Development 110:115–130PubMedGoogle Scholar
  2. Adamus MA, Dabrowski ZJ (2001) Effect of the neuropeptide substance P on the rat bone marrow-derived osteogenic cells in vitro. J Cell Biochem 81:499–506CrossRefPubMedGoogle Scholar
  3. Amenta PS, Clark CC, Martinezhernandez A (1983) Deposition of fibronectin and laminin in the basement-membrane of the rat parietal yolk-sac—immunohistochemical and biosynthetic-studies. J Cell Biol 96:104–111CrossRefPubMedGoogle Scholar
  4. Anderson MJ (1993) Differences in growth of neurons from normal and regenerated teleost spinal-cord in vitro. In Vitro Cell Dev Biol Anim 29:145–152CrossRefGoogle Scholar
  5. Antoniou J, Steffen T, Nelson F, Winterbottom N, Hollander AP, Poole RA, Aebi M, Alini M (1996) The human lumbar intervertebral disc—evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, ageing, and degeneration. J Clin Invest 98:996–1003CrossRefPubMedGoogle Scholar
  6. Aviezer D, Hecht D, Safran M, Eisinger M, David G, Yayon A (1994) Perlecan, basal lamina proteoglycan, promotes basic fibroblast growth factor-receptor binding, mitogenesis, and angiogenesis. Cell 79:1005–1013CrossRefPubMedGoogle Scholar
  7. Baluk P, Bowden JJ, Lefevre PM, McDonald DM (1997) Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats. Am J Physiol Lung Cell Mol Physiol 17:L565–L571Google Scholar
  8. Barnes PJ (2001) Neurogenic inflammation in the airways. Respir Physiol 125:145–154CrossRefPubMedGoogle Scholar
  9. Bartels H, Potter IC (1998) Membrane structure of the cells of the lamprey notochord. J Electron Microsc 47:627–636Google Scholar
  10. Bjurholm A (1991) Neuroendocrine peptides in bone. Int Orthop 15:325–329CrossRefPubMedGoogle Scholar
  11. Brown JC, Sasaki T, Gohring W, Yamada Y, Timpl R (1997) The C-terminal domain V of perlecan promotes beta 1 integrin-mediated cell adhesion, binds heparin, nidogen and fibulin-2 and can be modified by glycosaminoglycans. Eur J Biochem 250:39–46CrossRefPubMedGoogle Scholar
  12. Caterson B, Flannery CR, Hughes CE, Little CB (2000) Mechanisms involved in cartilage proteoglycan catabolism. Matrix Biol 19:333–344CrossRefPubMedGoogle Scholar
  13. Cleaver O, Krieg PA (2001) Notochord patterning of the endoderm. Dev Biol 234:1–12CrossRefPubMedGoogle Scholar
  14. Couchman JR, Ljubimov AV (1989) Mammalian tissue distribution of a large heparin sulphate proteoglycan detected by monoclonal antibodies. Matrix 9:311–321PubMedGoogle Scholar
  15. Deng WM, Ruohola-Baker H (2000) Laminin A is required for follicle cell-oocyte signaling that leads to establishment of the anterior-posterior axis in Drosophila. Curr Biol 10:683–686CrossRefPubMedGoogle Scholar
  16. Dodge GR, Jimenez SA (2003) Glucosamine sulfate modulates the levels of aggrecan and matrix metalloproteinase-3 synthesized by cultured human osteoarthritis articular chondrocytes. Osteoarthritis Cartilage 11:424–432CrossRefPubMedGoogle Scholar
  17. Domowicz M, Li H, Hennig A, Henry J, Vertel BM, Schwartz NB (1995) The biochemically and immunologically distinct CSPG of notochord is a product of the aggrecan gene. Dev Biol 171:655–664CrossRefPubMedGoogle Scholar
  18. Fleming A, Keynes R, Tannahill D (2004) A central role for the notochord in vertebral patterning. Development 131:873–880CrossRefPubMedGoogle Scholar
  19. Gjerde EAB, Karlsen TV, Reed RK (2003) Lowering of interstitial fluid pressure in rat trachea after substance P alone and in combination with calcitonin gene-related peptide. Acta Physiol Scand 178:123–127CrossRefPubMedGoogle Scholar
  20. Glickman NS, Kimmel CB, Jones MA, Adams RJ (2003) Shaping the zebrafish notochord. Development 130:873–887CrossRefPubMedGoogle Scholar
  21. Gorman KF, Breden F (2007) Teleosts as models for human vertebral stability and deformity. Comp Biochem Physiol C Toxicol Pharmacol 145:28–38CrossRefPubMedGoogle Scholar
  22. Goto T, Yamaza T, Kido MA, Tanaka T (1998) Light- and electron-microscopic study of the distribution of axons containing substance P and the localization of neurokinin-1 receptor in bone. Cell Tissue Res 293:87–93CrossRefPubMedGoogle Scholar
  23. Goto T, Nakao K, Gunjigake KK, Kido MA, Kobayashi S, Tanaka T (2007) Substance P stimulates late-stage rat osteoblastic bone formation through neurokinin-1 receptors. Neuropeptides 41:25–31CrossRefPubMedGoogle Scholar
  24. Grotmol S, Nordvik K, Kryvi H, Totland GK (2005) A segmental pattern of alkaline phosphatase activity within the notochord coincides with the initial formation of the vertebral bodies. J Anat 206:427–436CrossRefPubMedGoogle Scholar
  25. Grotmol S, Kryvi H, Keynes R, Krossoy C, Nordvik K, Totland GK (2006) Stepwise enforcement of the notochord and its intersection with the myoseptum: an evolutionary path leading to development of the vertebra? J Anat 209:339–357CrossRefPubMedGoogle Scholar
  26. Hassell JR, Robey PG, Barrach HJ, Wilczek J, Rennard SI, Martin GR (1980) Isolation of a heparan sulfate-containing proteoglycan from basement-membrane. Proc Natl Acad Sci USA 77:4494–4498CrossRefPubMedGoogle Scholar
  27. Hayashi K, Madri JA, Yurchenco PD (1992) Endothelial-cells interact with the core protein of basement-membrane perlecan through beta-1 and beta-3 integrins—an adhesion modulated by glycosaminoglycan. J Cell Biol 119:945–959CrossRefPubMedGoogle Scholar
  28. Hayes AJ, Benjamin M, Ralphs JR (2001) Extracellular matrix in development of the intervertebral disc. Matrix Biol 20:107–121CrossRefPubMedGoogle Scholar
  29. Hogan BLM, Cooper AR, Kurkinen M (1980) Incorporation into Reicherts membrane of laminin-like extracellular proteins synthesized by parietal endoderm cells of the mouse embryo. Dev Biol 80:289–300CrossRefPubMedGoogle Scholar
  30. Hökfelt T, Kellerth JO, Nilsson G, Pernow B (1975) Substance-P—localization in central nervous-system and in some primary sensory neurons. Science 190:889–890CrossRefPubMedGoogle Scholar
  31. Holmqvist BI, Ekstrom P (1991) Galanin-like immunoreactivity in the brain of teleosts—distribution and relation to substance-P, vasotocin, and isotocin in the Atlantic salmon (Salmo salar). J Comp Neurol 306:361–381CrossRefPubMedGoogle Scholar
  32. Hukkanen M, Konttinen YT, Rees RG, Gibson SJ, Santavirta S, Polak JM (1992) Innervation of bone from healthy and arthritic rats by substance-P and calcitonin gene related peptide containing sensory fibers. J Rheumatol 19:1252–1259PubMedGoogle Scholar
  33. Hunter CJ, Matyas JR, Duncan NA (2003) The three-dimensional architecture of the notochordal nucleus pulposus: novel observations on cell structures in the canine intervertebral disc. J Anat 202:279–291CrossRefPubMedGoogle Scholar
  34. Ida-Yonemochi H, Ohshiro K, Swelam W, Metwaly H, Saku T (2005) Perlecan, a basement membrane-type heparan sulfate proteoglycan, in the enamel organ: its intraepithelial localization in the stellate reticulum. J Histochem Cytochem 53:763–772CrossRefPubMedGoogle Scholar
  35. Kanemoto M, Hukuda S, Komiya Y, Katsuura A, Nishioka J (1996) Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 in human intervertebral discs. Spine 21:1–8CrossRefPubMedGoogle Scholar
  36. Kauppila LI (1995) Ingrowth of blood-vessels in disc degeneration—angiographic and histological studies of cadaveric spines. J Bone Joint Surg Am 77A:26–31Google Scholar
  37. Kim JH, Deasy BM, Seo HY, Studer RK, Vo NV, Georgescu HI, Sowa GA, Kang JD (2009) Differentiation of intervertebral notochordal cells through live automated cell imaging system in vitro. Spine 34:2486–2493CrossRefPubMedGoogle Scholar
  38. Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF (1995) Stages of embryonic-development of the zebrafish. Dev Dyn 203:253–310PubMedGoogle Scholar
  39. Koehl MAR, Quillin KJ, Pell CA (2000) Mechanical design of fiber-wound hydraulic skeletons: the stiffening and straightening of embryonic notochords. Am Zool 40:28–41CrossRefGoogle Scholar
  40. Kuraishi Y, Hirota N, Sato Y, Hino Y, Satoh M, Takagi H (1985) Evidence that substance-P and somatostatin transmit separate information related to pain in the spinal dorsal horn. Brain Res 325:294–298CrossRefPubMedGoogle Scholar
  41. Kvellestad A, Hoie S, Thorud K, Torud B, Lyngoy A (2000) Platyspondyly and shortness of vertebral column in farmed Atlantic salmon Salmo salar in Norway—description and interpretation of pathologic changes. Dis Aquat Organ 39:97–108CrossRefPubMedGoogle Scholar
  42. Lee EC, Lotz MM, Steele GD, Mercurio AM (1992) The integrin alpha-6-beta-4 is a laminin receptor. J Cell Biol 117:671–678CrossRefPubMedGoogle Scholar
  43. Linsenma TF, Trelstad RL, Gross J (1973) Collagen of chick embryonic notochord. Biochem Biophys Res Commun 53:39–45CrossRefGoogle Scholar
  44. Lotz JC (2004) Animal models of intervertebral disc degeneration—lessons learned. Spine 29:2742–2750CrossRefPubMedGoogle Scholar
  45. Lundberg JM, Brodin E, Hua XY, Saria A (1984) Vascular-permeability changes and smooth-muscle contraction in relation to capsaicin-sensitive substance-P afferents in the guinea-pig. Acta Physiol Scand 120:217–227CrossRefPubMedGoogle Scholar
  46. McDonald DM (1988) Neurogenic inflammation in the rat trachea. 1. Changes in venules, leukocytes and epithelial-cells. J Neurocytol 17:583–603CrossRefPubMedGoogle Scholar
  47. Melrose J, Roberts S, Smith S, Menage J, Ghosh P (2002) Increased nerve and blood vessel ingrowth associated with proteoglycan depletion in an ovine anular lesion model of experimental disc degeneration. Spine 27:1278–1285CrossRefPubMedGoogle Scholar
  48. Metcalfe WK, Myers PZ, Trevarrow B, Bass MB, Kimmel CB (1990) Primary neurons that express the L2/Hnk-1 carbohydrate during early development in the zebrafish. Development 110:491–504PubMedGoogle Scholar
  49. Millward-Sadler SJ, Mackenzie A, Wright MO, Lee HS, Elliott K, Gerrard L, Fiskerstrand CE, Salter DM, Quinn JP (2003) Tachykinin expression in cartilage and function in human articular chondrocyte mechanotransduction. Arthritis Rheum 48:146–156CrossRefPubMedGoogle Scholar
  50. Miner JH, Li C, Mudd JL, Go G, Sutherland AE (2004) Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation. Development 131:2247–2256CrossRefPubMedGoogle Scholar
  51. Morita H, Yoshimura A, Inui K, Lodeura T, Watanabe H, Wang L, Soininen R, Tryggvason K (2005) Heparan sulfate of perlecan is involved in glomerular filtration. J Am Soc Nephrol 16:1703–1710CrossRefPubMedGoogle Scholar
  52. Nachemso A, Lewin T, Maroudas A, Freeman MAR (1970) In-vitro diffusion of dye through end-plates and annulus fibrosus of human lumbar inter-vertebral discs. Acta Orthop Scand 41:589–596CrossRefGoogle Scholar
  53. Nordvik K, Kryvi H, Totland GK, Grotmol S (2005) The salmon vertebral body develops through mineralization of two preformed tissues that are encompassed by two layers of bone. J Anat 206:103–114CrossRefPubMedGoogle Scholar
  54. Oegema TR (2002) The role of disc cell heterogeneity in determining disc biochemistry: a speculation. Biochem Soc Trans 30:839–844CrossRefPubMedGoogle Scholar
  55. Olsvik PA, Lie KK, Jordal AEO, Nilsen TO, Hordvik I (2005) Evaluation of potential reference genes in real-time RT-PCR studies of Atlantic salmon. BMC Mol Biol 6:21CrossRefPubMedGoogle Scholar
  56. Parsons MJ, Pollard SM, Saude L, Feldman B, Coutinho P, Hirst EMA, Stemple DL (2002) Zebrafish mutants identify an essential role for laminins in notochord formation. Development 129:3137–3146PubMedGoogle Scholar
  57. Pfaffl MW, Horgan GW, Dempfle L (2002) Relative expression software tool (REST) for group-wise comparison and statistical analysis of relative expression results in real-time PCR. Nucleic Acids Res 30:1CrossRefGoogle Scholar
  58. Roberts S, Menage J, Urban JPG (1989) Biochemical and structural properties of the cartilage endplate and its relation to the intervertebral disc. Spine 14:166–174CrossRefPubMedGoogle Scholar
  59. Sandell LJ (1994) In-situ expression of collagen and proteoglycan genes in notochord and during skeletal development and growth. Microsc Res Tech 28:470–482CrossRefPubMedGoogle Scholar
  60. Shively JE, Conrad HE (1976) Nearest neighbour analysis of heparin: identification and quantitation of the products formed by selective depolymerization procedures. Biochemistry 15:3943–3950CrossRefPubMedGoogle Scholar
  61. Sivan SS, Tsitron E, Wachtel E, Roughley PJ, Sakkee N, Ham F van der, DeGroot J, Roberts S, Maroudas A (2006) Aggrecan turnover in human intervertebral disc as determined by the racemization of aspartic acid. J Biol Chem 281:13009–13014CrossRefPubMedGoogle Scholar
  62. Smith SM, Whitelock JM, Iozzo RV, Little CB, Melrose J (2009) Topographical variation in the distributions of versican, aggrecan and perlecan in the foetal human spine reflects their diverse functional roles in spinal development. Histochem Cell Biol 132:491–503CrossRefPubMedGoogle Scholar
  63. Smyth N, Vatansever HS, Murray P, Meyer M, Frie C, Paulsson M, Edgar D (1999) Absence of basement membranes after targeting the LAMC1 gene results in embryonic lethality due to failure of endoderm differentiation. J Cell Biol 144:151–160CrossRefPubMedGoogle Scholar
  64. Sonnenberg A, Calafat J, Janssen H, Daams H, Vanderraaijhelmer LMH, Falcioni R, Kennel SJ, Aplin JD, Baker J, Loizidou M, Garrod D (1991) Integrin-alpha-6-beta-4 complex is located in hemidesmosomes, suggesting a major role in epidermal-cell basement-membrane adhesion. J Cell Biol 113:907–917CrossRefPubMedGoogle Scholar
  65. Streuli CH, Schmidhauser C, Bailey N, Yurchenco P, Skubitz APN, Roskelley C, Bissell MJ (1995) Laminin mediates tissue-specific gene-expression in mammary epithelia. J Cell Biol 129:591–603CrossRefPubMedGoogle Scholar
  66. Sun HB, Chen JC, Liu Q, Guo MF (2010) Substance P stimulates differentiation of mice osteoblast through up-regulating osterix expression. J Traumatol 13:46–50Google Scholar
  67. Sundarraj N, Fite D, Ledbetter S, Chakravarti S, Hassell JR (1995) Perlecan is a component of cartilage matrix and promotes chondrocyte attachment. J Cell Sci 108:2663–2672PubMedGoogle Scholar
  68. Takaishi H, Nemoto O, Shiota M, Kikuchi T, Yamada H, Yamagishi M, Yabe Y (1997) Type-II collagen gene expression is transiently upregulated in experimentally induced degeneration of rabbit intervertebral disc. J Orthop Res 15:528–538CrossRefPubMedGoogle Scholar
  69. Tarakçý BG, Köprücü SS (2002) Regulatory peptides in gastroenteropancreatic endocrine cells of the rainbow trout (Oncorhynchus mykiss Walbaum, 1792). J Fish Aquat Sci 19:157–162Google Scholar
  70. Timpl R (1996) Macromolecular organization of basement membranes. Curr Opin Cell Biol 8:618–624CrossRefPubMedGoogle Scholar
  71. Tingbø MG, Kolset SO, Ofstad R, Enersen G, Hannesson KO (2006) Identification and distribution of heparan sulfate proteoglycans in the white muscle of Atlantic cod (Gadus morhua) and spotted wolffish (Anarhichas minor). Comp Biochem Physiol [B] 143:441–452CrossRefGoogle Scholar
  72. Urban JPG, Mcmullin JF (1985) Swelling pressure of the intervertebral disk—influence of proteoglycan and collagen contents. Biorheology 22:145–157PubMedGoogle Scholar
  73. Urban JPG, Roberts S (2003) Degeneration of the intervertebral disc. Arthritis Res Ther 5:120–130CrossRefPubMedGoogle Scholar
  74. Urban JPG, Smith S, Fairbank JCT (2004) Nutrition of the intervertebral disc. Spine 29:2700–2709CrossRefPubMedGoogle Scholar
  75. Vincent M, Duband JL, Thiery JP (1983) A cell-surface determinant expressed early on migrating avian neural crest cells. Dev Brain Res 9:235–238CrossRefGoogle Scholar
  76. Witten PE, Rosenthal H, Hall BK (2002) The kype of male Atlantic salmon (Salmo salar): restart of bone development in adult animals. Integr Comp Biol 42:1337Google Scholar
  77. Witten PE, Gil-Martens L, Hall BK, Huysseune A, Obach A (2005) Compressed vertebrae in Atlantic salmon Salmo salar: evidence for metaplastic chondrogenesis as a skeletogenic response late in ontogeny. Dis Aquat Organ 64:237–246CrossRefPubMedGoogle Scholar
  78. Witten PE, Obach A, Huysseune A, Baeverfjord G (2006) Vertebrae fusion in Atlantic salmon (Salmo salar): development, aggravation and pathways of containment. Aquaculture 258:164–172CrossRefGoogle Scholar
  79. Yasuma T, Arai K, Yamauchi Y (1993) The histology of lumbar intervertebral disc herniation—the significance of small blood-vessels in the extruded tissue. Spine 18:1761–1765CrossRefPubMedGoogle Scholar
  80. Ytteborg E, Baeverfjord G, Hjelde K, Torgersen J, Takle H (2010a) Molecular pathology of vertebral deformities in hyperthermic Atlantic salmon (Salmo salar). BMC Physiol 10:12CrossRefPubMedGoogle Scholar
  81. Ytteborg E, Torgersen J, Baeverfjord G, Hjelde K, Takle H (2010b) Morphological and molecular characterization of developing vertebral fusions using a teleost model. BMC Physiol (in press)Google Scholar
  82. Yu J, Fairbank JCT, Roberts S, Urban JPG (2005) The elastic fiber network of the anulus fibrosus of the normal and scoliotic human intervertebral disc. Spine 30:1815–1820CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2010

Authors and Affiliations

  • Elisabeth Ytteborg
    • 1
    • 5
  • Jacob Seilø Torgersen
    • 1
  • Mona E. Pedersen
    • 2
  • Grete Baeverfjord
    • 3
  • Kirsten O. Hannesson
    • 2
  • Harald Takle
    • 1
    • 4
  1. 1.Nofima Marine AS, Fisheries and Aquaculture ResearchNorwegian Institute of FoodAasNorway
  2. 2.Nofima Food ASAasNorway
  3. 3.Nofima Marine ASSunndalsøraNorway
  4. 4.AVS ChilePuerto VarasChile
  5. 5.Department of Animal and Aquaculture SciencesNorwegian University of Life SciencesAasNorway

Personalised recommendations