Different Roles for Fibronectin in the Generation of Fore and Hind Limb Precartilage Condensations

AbstractFibronectin expression and spatiotemporal distribution were examined in relation to the distinctive patterns of mesenchymal condensation and chondrogenesis seen in high-density serum-free cultures of chicken wing and leg bud precartilage cells. More fibronectin protein was produced on a per cell basis by leg than by wing mesenchyme, both in freshly isolated tissue and during the prechondrogenic condensation period in culture, where the difference was twofold. The quantitative difference in fibronectin expression in freshly isolated wing and leg mesenchyme was also seen at the level of total and poly(A)+RNA. During the condensation phase, fibronectin was distributed in the wing and leg mesenchymal cultures in a way that prefigured the eventual distribution of cartilage in these cultures: in wing cultures condensations were broad and flat, and rich in diffusely organized fibronectin; in leg cultures, condensations were compact and spheroidal, and contained abundant deposits of fibronectin. In addition, the leg condensations were connected by long fibronectin-rich fibers. Transient treatment with TGF-β early during the culture period led to increase in fibronectin production and expansion of condensations in both wing and leg cultures. Leg mesenchyme was more responsive to transforming growth factor-β than wing mesenchyme with respect to fibronectin production, and this was reflected in a greater enhancement of cartilage formation in later cultures. Treatment of cultures with monoclonal antibody 304 directed against the amino-terminal heparin-binding domain of fibronectin inhibited condensation formation and reduced chondrogenesis in wing mesenchyme, but left these two processes unchanged in leg mesenchyme, despite disruption by the antibody of the leg-specific fibronectin fibers. These studies indicate that for both wing and leg mesenchyme the morphology, extent, and spatiotemporal regulation of precartilage condensation and subsequent chondrogenesis closely parallels the deposition of fibronectin. But whereas the interaction between cells and fibronectin in wing bud mesenchyme is mediated in part by the protein's amino-terminal domain, this domain does not appear to be involved in analogous interactions in leg bud mesenchyme