Even at very early stages of development (i.e., stages 22–24), the embryonic chick limb bud consists of a heterogeneous mixture of chondrogenic, myogenic, and fibrogenic progenitor cells in various stages of differentiation. In contrast, the distal subridge region of a stage 25 wing bud appears to be a relatively homogeneous population of undifferentiated chondrogenic progenitor cells. In the present study, we confirm the results of a number of investigators that when the heterogeneous cells of whole stage 23/24 limb buds are subjected to high density cell culture (micromass culture) the cells in various discrete regions of the culture aggregate with one another and differentiate into cartilage nodules. The cartilage nodules in such cultures, however, are separated from one another by a considerable amount of nonchondrogenic tissue. In fact, nonchondrogenic tissue constitutes a relatively high proportion of the micromass cultures of whole stage 23/24 limb bud cells. In contrast, when stage 25 subridge mesenchymal cells are subjected to micromass culture, a virtually uniform sheet of cartilage is formed throughout the culture, and little, if any, nonchondrogenic tissue is detectable. The micromass culture of stage 25 subridge mesenchymal cells thus provides an excellent system in which a relatively homogeneous group of chondrogenic progenitor cells progressively and uniformly undergo chondrogenesis in a readily manipulable culture situation. Such a system is ideally suited for studies on the molecular regulation of cartilage differentiation.

In the present study, we have also demonstrated that exogenous prostaglandin E₂ (PGE₂) elicits a dose‐dependent stimulation of chondrogenesis in micromass cultures of both stage 25 subridge mesenchymal cells and the cells of whole stage 23/24 limb buds. The stimulatory effect of PGE₂ is greatly potentiated by the phosphodiesterase inhibitor theophylline, suggesting that its influence on chondrogenesis is mediated by its ability to increase cAMP levels. PGE₁ is just as effective as PGE₂ in stimulating chondrogenesis, whereas PGF_1α, 6‐keto PGF_1α and thromboxane B₂ have no effect. On the basis of these observations and previous studies, the possibility that endogenous prostaglandins might regulate limb cartilage differentiation by acting as local mediators of cAMP accumulation is discussed.