CHARACTERIZATION OF CHONDROGENESIS IN CELLS ISOLATED FROM LIMB BUDS IN MOUSE

Micromass cultures of mesenchymal cells isolated from limb buds of 11. 5-day-old mouse fetuses were used to study chondrogenesis. After 3 day s of culture, dense cell aggregates were observed. They then were conv erted into macroscopically visible cartilage foci during the following 2-4 days. Comparison of 2-, 4- and 7-day-old cultures has shown that the cells first expressed collagen type I, then switched to collagen t ype II expression as shown by immunohistochemistry and in situ hybridi zation. At day 7, proteoglycans were synthesized centrally in the foci . At the same time, most cells expressed collagen type II, with the hi ghest expression in the periphery of the aggregates. The oncogene c-fo s and homeodomain protein FS-1 were found in the cells expressing coll agen type II, indicating that these transcription factors may be invol ved in the regulation of cell differentiation. The expression of alkal ine phosphatase was detected first in mature cartilage foci (day 4) an d increased during culture. Early in culture, DNA-replicating cells we re uniformly distributed. With differentiation, the proliferating cell s were present predominantly between the aggregates and their total nu mber became significantly reduced. Our results indicate that the proce ss of chondrogenesis in micromass cultures of mesenchymal cells mimics the differentiation process occurring during fetal development in viv o and can be directly studied by in situ hybridization, immunohistoche mical and histochemical methods.