The objective of the proposed study is to test the hypothesis that embryonic chick limb mesenchymal cells respond to fibroblast growth factors (FGFs) in a position-related manner by synthesizing and responding to prostaglandins (PGs) prior to entering the chondrogenic pathway for skeletal formation. The investigator hypothesizes that cells taken from each of three different limb fields (pre-axial, post-axial, and distal tip) and placed in culture under conditions conducive for chondrogensis will respond differently to pretreatments with FGFs. Tests of this hypothesis will include determinations of cell numbers, total protein, DNA, and cartilage matrix accumulations, cAMP accumulation in response to PG exposure, and PG synthesis after exposure of the cultures to concentration-ranges of both aFGF and bFGF. Assays will be performed at days 0,1,3, and 6 of culture in all experiments.
The specific aims of the study are 1) to characterize the basic features of the cultures after exposure to a-FGF and b-FGF (1, 10, and 100ng/ml) and PGE2 and PGI2 (0.05, 0.5, and 5.0 ug/ml), including measuring cartilage matrix accumulation; 2) to determine cAMP accumulation in the cultured cells in the presence and absence of the phosphodiesterase inhibitor MIX after exposure to FGFs and PGs; 3) to determine cAMP accumulation in the cultured cells in the presence and absence of MIX after pretreatment with either aFGF or bFGF and exposure to physiologic concentrations of either PGE2 or PGI2; and 4) to determine by RIA the amount of PGE2 and PGI2 synthesized by the cultured cells after exposure to the FGFs. By comparing the growth, cAMP accumulation, and PG synthesis responses of distal tip, pre-axial, and post-axial limb cells in response to the FGFs, the investigator hopes to gain insight into the relationship between FGFs and PGs in stimulating chondrogenesis, and the role of FGFs in limb development.
