Morphogenetic Differences between Fore and Hind Limb Precartilage Mesenchyme: Relation to Mechanisms of Skeletal Pattern Formation

Abstract

Using a serum-free culture system, we have investigated morphogenetic differences between fore and hind limb precartilage mesenchymal cells derived from stages 21-26 chicken embryos. Across all stages, wing and leg cultures were intrinsically different in the amount and spatial organization of cartilage that they produced. By stage 24, leg cells began to produce a nodular pattern of cartilage while wing cells continued to produce sheets of cartilage. This pattern difference persisted throughout the later stages and was not due to differences in cell survival as judged by DNA content or to the presence of distinctive cell subpopulations in either tissue, as determined by flow cytometry. Chondrogenesis in wing and leg precartilage cell cultures was affected differently by 10% fetal bovine serum, TGF-β1 (1 ng/ml for 5 hr on the day after plating), and retinoic acid (5 ng/ml). In wing cultures, the extent of chondrogenesis was significantly enhanced by serum or by a combination of TGF-β1 and retinoic acid, but the cartilage pattern was not altered with any treatment. In leg cultures, the extent of chondrogenesis was enhanced by TGF-β1 alone, inhibited by retinoic acid alone, and the cartilage pattern was changed from nodular to sheet-like by treatment with TGF-β1. Wing and leg cultures also differed from each other in relative cohesivity and in condensation morphology and organization of fibronectin during the early phase of differentiation. Wing cells produced broad, flat condensations containing diffusely organized fibronectin, whereas leg cells elaborated an extensive network of long fibronectin-rich fibrils connecting very compact, fibronectin-rich condensations. These intrinsic and induced differences in fore and hind limb mesenchyme provide insight into the mechanisms that are common to the formation of all limb skeletal elements and those that distinguish skeletal elements from different limb regions and limb types.