European Spine Journal

, Volume 22, Issue 8, pp 1774–1784 | Cite as

Comparative immunolocalisation of perlecan, heparan sulphate, fibroblast growth factor-18, and fibroblast growth factor receptor-3 and their prospective roles in chondrogenic and osteogenic development of the human foetal spine

  • Cindy Shu
  • Susan S. Smith
  • Christopher B. Little
  • James MelroseEmail author
Original Article



A comparative immunolocalisation study of perlecan, HS, FGF-18 and FGFR-3 in the 12–20-week gestational age human foetal spine was undertaken to identify spatiotemporal associations between these components to provide insights into prospective roles in spinal development.


Comparative immunolocalisations of matrix and cell associated components in Histochoice-fixed paraffin-embedded human foetal spinal tissues.


The 12–14-week-old human foetal spine was a predominantly cartilaginous structure with the discs displaying a relative paucity of proteoglycan compared to the adjacent cartilaginous vertebral rudiments, notochordal remnants were also observed. HS and perlecan had a widespread distribution throughout the spine at 12 weeks, however, FGF-18 was only localised to the outer AF margins and hypertrophic cell condensations in the vertebral bodies. This contrasted with HS distributions at 14–20 weeks, which were prominent in the developing intervertebral disc (IVD). Ossification centres were also evident centrally within the vertebral rudiments surrounded by small columns of hypertrophic chondrocytes which expressed FGFR-3 and FGF-18 and upregulated levels of perlecan. FGF-18 also had a prominent localisation pattern in the developing IVD and the cartilaginous endplate while FGFR-3 was expressed throughout the disc interspace. This suggested roles for perlecan, FGF-18 and FGFR-3 in chondrogenic and osteogenic events which drive discal development and ossification of the vertebral bodies.


The above data supported a role for FGF-18 in discal development and in the terminal osteogenic differentiation of chondroprogenitor cell populations, which promote vertebral ossification during spinal development.


Fibroblast growth factor (FGF)-18 Fibroblast growth factor receptor (FGFR)-3 Perlecan Heparin sulphate (HS) 


Conflict of interest



  1. 1.
    Glenn OA, Barkovich AJ (2006) Magnetic resonance imaging of the fetal brain and spine: an increasingly important tool in prenatal diagnosis, part 1. AJNR Am J Neuroradiol 27:1604–1611PubMedGoogle Scholar
  2. 2.
    Cheng PJ, Huang SY, Shaw SW, Chueh HY, Soong YK (2010) Evaluation of fetal spine biometry between 11 and 14 weeks of gestation. Ultrasound Med Biol 36:1060–1065. doi: 10.1016/j.ultrasmedbio.2010.04.007 PubMedCrossRefGoogle Scholar
  3. 3.
    Babic MS (1991) Development of the notochord in normal and malformed human embryos and fetuses. Int J Dev Biol 35:345–352PubMedGoogle Scholar
  4. 4.
    Bagnall KM, Harris PF, Jones PR (1982) A radiographic study of the growth in width of the human fetal vertebral column. Anat Rec 204:265–270. doi: 10.1002/ar.1092040311 PubMedCrossRefGoogle Scholar
  5. 5.
    Nerlich AG, Boos N, Wiest I, Aebi M (1998) Immunolocalization of major interstitial collagen types in human lumbar intervertebral discs of various ages. Virchows Arch 432:67–76PubMedCrossRefGoogle Scholar
  6. 6.
    Zhu Y, McAlinden A, Sandell LJ (2001) Type IIA procollagen in development of the human intervertebral disc: regulated expression of the NH(2)-propeptide by enzymic processing reveals a unique developmental pathway. Dev Dyn 220:350–362. doi: 10.1002/dvdy.1115 PubMedCrossRefGoogle Scholar
  7. 7.
    Hayes AJ, Smith SM, Gibson MA, Melrose J (2011) Comparative immunolocalization of the elastin fiber-associated proteins fibrillin-1, LTBP-2, and MAGP-1 with components of the collagenous and proteoglycan matrix of the fetal human intervertebral disc. Spine (Phila Pa, 1976) 36:E1365–E1372. doi: 10.1097/BRS.0b013e31821fd23e CrossRefGoogle Scholar
  8. 8.
    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 132:491–503PubMedCrossRefGoogle Scholar
  9. 9.
    Melrose J, Smith S, Ghosh P, Whitelock J (2003) Perlecan, the multidomain heparan sulfate proteoglycan of basement membranes, is also a prominent component of the cartilaginous primordia in the developing human fetal spine. J Histochem Cytochem 51:1331–1341PubMedCrossRefGoogle Scholar
  10. 10.
    Sinowatz F (2010) Musculo-skeletal system. In: Hyttel P, Sinowatz F, Vejlsted M (eds) Essentials of domestic animal embryology, 1st edn. Saunders Elsevier, Philadelphia, pp 286–316Google Scholar
  11. 11.
    Risbud MV, Schaer TP, Shapiro IM (2010) Toward an understanding of the role of notochordal cells in the adult intervertebral disc: from discord to accord. Dev Dyn 239:2141–2148. doi: 10.1002/dvdy.22350 PubMedCrossRefGoogle Scholar
  12. 12.
    McGeady TA, Quinn PJ, Fitzpatrick ES (2006) Muscular and skeletal systems. In: McGeady TA, Quinn PJ, Fitzpatrick ES (eds) Veterinary embryology, 1st edn. Blackwell Publishing, Oxford, pp 184–204Google Scholar
  13. 13.
    Smith CA, Tuan RS (1994) Human PAX gene expression and development of the vertebral column. Clin Orthop Relat Res 302:241–250PubMedGoogle Scholar
  14. 14.
    Ornitz DM, Xu J, Colvin JS, McEwen DG, MacArthur CA, Coulier F, Gao G, Goldfarb M (1996) Receptor specificity of the fibroblast growth factor family. J Biol Chem 271:15292–15297PubMedCrossRefGoogle Scholar
  15. 15.
    Chuang CY, Lord MS, Melrose J, Rees MD, Knox SM, Freeman C, Iozzo RV, Whitelock JM (2010) Heparan sulfate dependent signaling of fibroblast growth factor (FGF) 18 by chondrocyte-derived perlecan. Biochemistry. doi: 10.1021/bi1005199 Google Scholar
  16. 16.
    Smith SM, Shu C, Melrose J (2010) Comparative immunolocalisation of perlecan with collagen II and aggrecan in human foetal, newborn and adult ovine joint tissues demonstrates perlecan as an early developmental chondrogenic marker. Histochem Cell Biol 134:251–263. doi: 10.1007/s00418-010-0730-x PubMedCrossRefGoogle Scholar
  17. 17.
    Arikawa-Hirasawa E, Watanabe H, Takami H, Hassell JR, Yamada Y (1999) Perlecan is essential for cartilage and cephalic development. Nat Genet 23:354–358. doi: 10.1038/15537 PubMedCrossRefGoogle Scholar
  18. 18.
    Ornitz DM (2000) FGFs, heparan sulfate and FGFRs: complex interactions essential for development. Bioessays 22:108–112. doi: 10.1002/(SICI)1521-1878(200002)22 PubMedCrossRefGoogle Scholar
  19. 19.
    Gibson G, Francki K, Caterson B, Foster B (1996) Type X collagen is colocalized with a proteoglycan epitope to form distinct morphological structures in bovine growth cartilage. Bone 19:307–315PubMedCrossRefGoogle Scholar
  20. 20.
    Drury RAB, Wallington EA (1967) Carleton's histological technique, 4th edn. Oxford University Press, pp 150–151Google Scholar
  21. 21.
    Peters K, Ornitz D, Werner S, Williams L (1993) Unique expression pattern of the FGF receptor 3 gene during mouse organogenesis. Dev Biol 155:423–430. doi: 10.1006/dbio.1993.1040 PubMedCrossRefGoogle Scholar
  22. 22.
    Knox S, Merry C, Stringer S, Melrose J, Whitelock J (2002) Not all perlecans are created equal: interactions with fibroblast growth factor (FGF) 2 and FGF receptors. J Biol Chem 277:14657–14665PubMedGoogle Scholar
  23. 23.
    Liu Z, Xu J, Colvin JS, Ornitz DM (2002) Coordination of chondrogenesis and osteogenesis by fibroblast growth factor 18. Genes Dev 16:859–869. doi: 10.1101/gad.965602 PubMedCrossRefGoogle Scholar
  24. 24.
    Ohbayashi N, Shibayama M, Kurotaki Y, Imanishi M, Fujimori T, Itoh N, Takada S (2002) FGF18 is required for normal cell proliferation and differentiation during osteogenesis and chondrogenesis. Genes Dev 16:870–879. doi: 10.1101/gad.965702 PubMedCrossRefGoogle Scholar
  25. 25.
    Liu Z, Lavine KJ, Hung IH, Ornitz DM (2007) FGF18 is required for early chondrocyte proliferation, hypertrophy and vascular invasion of the growth plate. Dev Biol 302:80–91. doi: 10.1016/j.ydbio.2006.08.071 PubMedCrossRefGoogle Scholar
  26. 26.
    Ellman MB, An HS, Muddasani P, Im HJ (2008) Biological impact of the fibroblast growth factor family on articular cartilage and intervertebral disc homeostasis. Gene 420:82–89. doi: 10.1016/j.gene.2008.04.019 PubMedCrossRefGoogle Scholar
  27. 27.
    Ellsworth JL, Berry J, Bukowski T, Claus J, Feldhaus A, Holderman S, Holdren MS, Lum KD, Moore EE, Raymond F, Ren H, Shea P, Sprecher C, Storey H, Thompson DL, Waggie K, Yao L, Fernandes RJ, Eyre DR, Hughes SD (2002) Fibroblast growth factor-18 is a trophic factor for mature chondrocytes and their progenitors. Osteoarthr Cartil 10:308–320. doi: 10.1053/joca.2002.0514 PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Cindy Shu
    • 1
  • Susan S. Smith
    • 1
  • Christopher B. Little
    • 1
  • James Melrose
    • 1
    Email author
  1. 1.Raymond Purves Bone and Joint Research Laboratories, Institute of Bone and Joint Research, The Kolling Institute of Medical ResearchUniversity of Sydney at the Royal North Shore HospitalSt. LeonardsAustralia

Personalised recommendations