Long-term in Vitro Analysis of Limb Cartilage Development: Involvement of Wnt Signaling

J Cell Biochem. 2004 Oct 15;93(3):526-41. doi: 10.1002/jcb.20190.


Endochondral skeletal development involves the condensation of mesenchymal cells, their differentiation into chondrocytes, followed by chondrocyte maturation, hypertrophy, and matrix mineralization, and replacement by osteoblasts. The Wnt family of secreted proteins have been shown to play important roles in vertebrate limb formation. To examine the role(s) of Wnt members and their transmembrane-spanning receptor(s), Frizzled (fz), we retrovirally misexpressed Wnt-5a, Wnt-7a, chicken frizzled-1 (Chfz-1), and frizzled-7 (Chfz-7) in long-term (21 day) high density, micromass cultures of stage 23/24 chick embryonic limb mesenchyme. This culture system recapitulates in vitro the entire differentiation (days 1-10), growth (days 5-12), and maturation and hypertrophy (from day 12 on) program of cartilage development. Wnt-7a misexpression severely inhibited chondrogenesis from day 7 onward. Wnt-5a misexpression resulted in a poor hypertrophic phenotype by day 14. Chfz-7 misexpression caused a slight delay of chondrocyte maturation based on histology, whereas Chfz-1 misexpression did not affect the chondrogenic phenotype. Misexpression of all Wnt members decreased collagen type X expression and alkaline phosphatase activity at day 21. Our findings implicate functional role(s) for Wnt signaling throughout embryonic cartilage development, and show the utility of the long-term in vitro limb mesenchyme culture system for such studies.

Publication types

  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Alkaline Phosphatase / metabolism
  • Animals
  • Cadherins / metabolism
  • Cartilage / embryology*
  • Cartilage / metabolism
  • Cell Culture Techniques
  • Cell Differentiation / physiology
  • Chick Embryo
  • Chondrogenesis / physiology*
  • Collagen Type X / metabolism
  • Cytoskeletal Proteins / metabolism
  • Embryonic Development / physiology
  • Intercellular Signaling Peptides and Proteins / metabolism*
  • Limb Buds / cytology
  • Limb Buds / embryology*
  • Limb Buds / metabolism
  • Receptors, Cell Surface / metabolism*
  • Trans-Activators / metabolism
  • Wnt Proteins
  • beta Catenin


  • Cadherins
  • Collagen Type X
  • Cytoskeletal Proteins
  • Intercellular Signaling Peptides and Proteins
  • Receptors, Cell Surface
  • Trans-Activators
  • Wnt Proteins
  • beta Catenin
  • Alkaline Phosphatase