VALIDATION OF CANDIDATE GENES IN RESPONSE TO VERSICAN MANIPULATION IN DEVELOPING SYNOVIAL JOINTS

Abstract

Little is understood about the complex process of synovial joint formation in early limb development. It has been shown that versican is highly expressed in the extracellular matrix of these joints (Snow et al., 2005; Shepard et al., 2007) and the knockdown of versican leads to malformation of the interzone, tissues that form the articular cartilage and synovial cavity (Nagchowdhuri et al., 2012). It is also thought that these effects impact gene expression in cells that are involved in the developing joint. Versican is chondroitin sulfate proteoglycan. It has four functional-modules: the N-terminal domain, the C-terminal domain, and GAG-[alpha], and GAG-[beta] chondroitin sulfate attachment regions. The N-terminal domain is also known as the G1 domain and the C-terminal domain is also known as the G3 domain (Kimata et al., 1986 and Zimmerman et al., 1989). In previous studies versican protein expression has been reduced and the G1 domain has been over-expressed to observe how these changes in the developing joint tissue effect gene expression. This study is a validation of microarray data specifically focusing on hyaluronan and Wnt pathway genes as they pertain to the misexpression of versican. RT-PCR and real-time PCR were used in this study to validate expression of the genes chosen. Through the use of these techniques the degree of expression has been quantified and compared to the fold changes observed with the microarray data. Overall, G1 versican mediated gene expression in the developing joint with regard to hyaluronan and Wnt pathway transcript regulation were in agreement with results obtained by RNA array.