A molecular approach to the analysis of pre-chondrogenic condensation in the avian limb

Limb bud chondrogenesis in vivo and in vitro appears to depend upon extensive cell contacts leading to localized increases in relative cell density. When cultured in suspension, dissociated early limb bud cells segregate into aggregating and non-aggregating populations. While the non-aggregating cells die, resulting aggregates differentiate exclusively as cartilage. We are investigating the role of the cell surface in prechondrogenic condensation employing aggregation in suspension culture. Immediately after their dissociation limb bud cells readily undergo Ca -independent, temperature-dependent aggregation. This 'early' mechanism is completely and reversibly sensitive to cycloheximide, although this drug does not inhibit recovery of all cell surface proteins. Addition of type I collagen and/or fibronectin, isolated fibronectin cell binding fragment, or anti-fibronectin Fab' fragments do not affect this process. Fab' fragments prepared from antisera directed against the surface of these cells also fail to inhibit their aggregation. Aggregation is partially inhibited by synthetic lecithin vessicles and stimulated by mixed ganglioside micelles. In contrast, after 16 h recovery equivalent cells exhibit a different, 'late', aggregation mechanism which is totally Ca-dependent and demonstrates a distinct sensitivity to cycloheximide. Immunohistochemistry with an antiserum prepared against these recovered cells preferentially stains limb bud pre-chondrogenic core cells. Fab fragments prepared from this same antiserum inhibit the 'late', but not 'early', aggregation mechanism. We conclude that two independent mechanisms of limb bud cell aggregation exist. The 'early' mechanism is most probably mediated through the lipid portion of the cell surface and leads to non-specific cell association. This initial phase is subsequently replaced by the 'late', specific mechanism mediated primarily through cell surface protein(s). We are now attempting to isolate the surface components involved in the 'late' aggregation mechanism and characterize their relevance to chondrogenic expression.