Indian hedgehog and syndecans‐3 coregulate chondrocyte proliferation and function during chick limb skeletogenesis

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Abstract
Hedgehog proteins exert critical roles in embryogenesis and require heparan sulfate proteoglycans (HS‐PGs) for action. Indian hedgehog (Ihh) is produced by prehypertrophic chondrocytes in developing long bones and regulates chondrocyte proliferation and other events, but it is not known whether it requires HS‐PGs for function. Because the HS‐PG syndecan‐3 is preferentially expressed by proliferating chondrocytes, we tested whether it mediates Ihh action. Primary chick chondrocyte cultures were treated with recombinant Ihh (rIhh‐N) in absence or presence of heparinase I or syndecan‐3 neutralizing antibodies. While rIhh‐N stimulated proliferation in control cultures, it failed to do so in heparinase‐ or antibody‐treated cultures. In reciprocal gain‐of‐function studies, chondrocytes were made to overexpress syndecan‐3 by an RCAS viral vector. Cells became more responsive to rIhh‐N, but even this response was counteracted by heparinase or antibody treatment. To complement the in vitro data, RCAS viral particles were microinjected in day 4–5 chick wing buds and effects of syndecan‐3 misexpression were monitored over time. Syndecan‐3 misexpression led to widespread chondrocyte proliferation and, interestingly, broader expression and distribution of Ihh. In addition, the syndecan‐3 misexpressing skeletal elements were short, remained cartilaginous, lacked osteogenesis, and exhibited a markedly reduced expression of collagen X and osteopontin, products characteristic of hypertrophic chondrocytes and bone cells. The data are the first to indicate that Ihh action in chondrocyte proliferation involves syndecan‐3 and to identify a specific member of the syndecan family as mediator of hedgehog function. Developmental Dynamics 229:607–617, 2004.