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The formation of cartilage elements in the developing vertebrate limb, where they serve as primordia for the appendicular skeleton, is preceded by the appearance of discrete cellular condensations. Control of the size and spacing of these condensations is a key aspect of skeletal pattern formation. Limb bud cell cultures grown in the absence of ectoderm formed continuous sheet-like masses of cartilage. With the inclusion of ectoderm, these cultures produced one or more cartilage nodules surrounded by zones of noncartilaginous mesenchyme. Ectodermal fibroblast growth factors (FGF2 and FGF8), but not a mesodermal FGF (FGF7), substituted for ectoderm in inhibiting chondrogenic gene expression, with some combinations of the two ectodermal factors leading to well-spaced cartilage nodules of relatively uniform size. Treatment of cultures with SU5402, an inhibitor FGF receptor tyrosine kinase activity, rendered FGFs ineffective in inducing perinodular inhibition. Inhibition of production of FGF receptor 2 (FGFR2) by transfection of wing and leg cell cultures with antisense oligodeoxynucleotides blocked appearance of ectoderm- or FGF-induced zones of perinodular inhibition of chondrogenesis and, when introduced into the limb buds of developing embryos, led to shorter, thicker, and fused cartilage elements. Because FGFR2 is expressed mainly at sites of precartilage condensation during limb development in vivo and in vitro, these results suggest that activation of FGFR2 by FGFs during development elicits a lateral inhibitor of chondrogenesis that limits the expansion of developing skeletal elements. Key Words: limb development; pattern formation; fibroblast growth factor; FGF receptor; lateral inhibition.

Fibronectin 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).sup.+] 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-[beta] 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-[beta] 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 aminoterminal 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 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.

Morphogenetic differences between fore and hind limb cartilage mesenchymal cells obtained from stages 21-26 chicken embryos have been studied using a serum-free culture system. The quantity and spatial assembly of cartilage generated varied for wing and leg cultures in all stages. Differences were evident in relative cohesivity.