INTRODUCTION Cell-cell adhesion and the events that subsequently take place between interacting cells, play important functional roles in biological processes (Grunwald, 1991). Cell adhesion molecules are recognized as being important mediators of both embryonic developmental and morphogenetic events, as well as stabilizers of adult tissue structures. The two main families of cell adhesion molecules are the Ca 2+ -dependent cadherin superfamily, and the Ca 2+ -independent immunoglobulin super- family (CAMs; Edelman and Crossin, 1991; Takeichi, 1990, 1991; Geiger and Ayalon, 1992). Members of both of these families of cell adhesion molecules mediate cell-cell adhesion in a homophilic manner. The classical cadherins, such as N- cadherin (Hatta et al., 1988) and E-cadherin (Nagafuchi et al., 1987), are transmembrane glycoproteins of about 120×10 3 M r that require interaction with the cytoskeleton in order to mediate extracellular binding. Such interactions are likely to involve the catenins, a group of at least three proteins (α, β, and γ) which both co-localize and co-immunoprecipitate with solubilized cadherins (Ozawa et al., 1989; Peifer et al., 1992). The three catenins, α, β, and γ, are closely related to vinculin, armadillo, and plakoglobin, respectively. The expression of a cell adhesion molecule during develop- ment usually coincides with substantial morphogenetic events. Chondrogenesis in the embryonic chick limb bud represents an interesting model system, in view of the early cellular con- densation phase (Fell and Canti, 1934; Thorogood and Hinch- liffe, 1975; Ede, 1983; Newman et al., 1985). At around stage 23/24 of development (Hamburger and Hamilton, 1951), mes- enchymal cells of the limb bud core come into close apposi- tion with one another (i.e. condensation) and subsequently dif- ferentiate into the first cartilaginous structure in the limb. This is accompanied by the synthesis and secretion of collagen type II and the protein core of the large chondroitin-rich proteogly- can known as aggrecan. The close apposition of the mes- enchymal cells could result from or facilitate the formation of cell-cell interactions necessary for chondrogenesis. Interest- ingly, Widelitz et al. (1993) recently reported that N-CAM, a neural Ca 2+ -independent cell adhesion molecule, was 177 Development 120, 177-187 (1994) Printed in Great Britain  The Company of Biologists Limited 1994 Cell adhesion molecules have been shown to be important mediators of morphogenesis and pattern formation. In this study, we have shown that N-cadherin is expressed in a specific spatiotemporal manner in the developing limb bud during chondrogenesis in vivo and in cultured limb mes- enchyme in vitro. The time period of maximal expression of N-cadherin corresponds to the period of active cellular condensation, an event believed to be a necessary prereq- uisite for chondrogenic differentiation. To directly assess the functional involvement of N-cadherin in cellular con- densation, we have examined the effects of perturbing N- cadherin activity on both cell aggregation and chondroge- nesis using NCD-2, a rat monoclonal antibody directed against the binding region of N-cadherin. Non-immune rat IgG was used as a control. Our results show that functional N-cadherin is necessary for chondrogenesis to proceed both in vivo and in vitro. Limb mesenchymal cells exhibited characteristic Ca 2+ -dependent cell aggregation in suspen- sion, which was inhibited in the presence of exogenous NCD-2. In micromass cultures of limb mesenchymal cells, NCD-2 inhibited overt chondrogenesis in a dose-dependent manner. Furthermore, NCD-2 inhibition of chondrogene- sis in micromass cultures was time-dependent, suggesting that N-cadherin is crucially involved during the latter half of the first 24 hours of culture, a time period most likely corresponding to active cellular condensation. NCD-2 also significantly influenced limb development when injected into embryonic limb buds in vivo. In addition to significant inhibition of chondrogenesis and developmental delays, gross developmental deformities and perturbation of overall pattern formation were also observed. Taken together, these results demonstrate that N-cadherin is func- tionally required in mediating the cell-cell interactions among mesenchymal cells important for chondrogenesis in micromass culture in vitro and in the intact limb bud in vivo. Key words: cell-cell interaction, morphogenesis, extracellular matrix, limb mesenchyme, skeletogenesis, cellular condensation SUMMARY Expression and functional involvement of N-cadherin in embryonic limb chondrogenesis Steven A. Oberlender and Rocky S. Tuan* Department of Orthopaedic Surgery and Department of Biochemistry and Molecular Biology, Thomas Jefferson University, Philadelphia, PA, 19107, USA *Author for correspondence