Our objective is to determine the mechanisms involved in the selective regulation of gene expression during cartilage differentiation in the developing chick limb. As limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of type II collagen, cartilage-specific chondroitin sulfate proteoglycan, several minor collagen species including types IX and X, and a matrix link protein. Concomitantly, the differentiating cells cease synthesizing type I collagen and large amounts of fibronectin. Studies on regulation of cartilage-specific gene activity will be initiated by examining changes in the steady-state levels of mRNAs for each of the above matrix proteins during the progression of chondrogenesis in vitro and in vivo. Changes in mRNA levels will be correlated with the rates of synthesis of the proteins, and the morphological phases of cartilage differentiation. The effect of cAMP, cell shape changes, and other putative regulatory factors on cartilage-specific mRNA accumulation will be determined. The molecular levels at which the type I collagen, type II collagen, and proteoglycan core protein genes are regulated will be analyzed by examining the rate of transcription of the genes during various phases of chondrogenesis, and determining the extent to which post-transcriptional events such as nuclear RNA processing, mRNA stability, and translational regulatory mechanisms contribute to steady state mRNA levels. The 5'-promoter region of the type II collagen gene will be isolated, characterized, and used in DNA transfection experiments to study tissue specific regulation of the gene. The relationships between changes in chromatin structure (DNase I hypersensitivity) and regulation of cartilage-specific gene activity will be studied. The possibility that the low level activity of the type II collagen gene in limb mesenchymal cells prior to overt chondrogenesis may represent a molecular manifestation of the state of determination of the cells will be investigated. These studies will advance basic knowledge of the regulation of limb chondrogenesis, and hopefully, contribute to the prevention of developmentally-caused limb malformations.
