The proposed investigation tests two hypotheses: 1) that transforming growth factor beta (TGF-B) plays an active role in mediating epithelial- mesenchymal tissue interactions during otic capsule formation and 2) that the target of action for TGF-B in mediating otic capsule chondrogenesis is the regulation of extracellular matrix. To test these hypotheses, the proposal consists of a series of a series of 6 experiments: 1A) Modulation of chondrogenesis by TGF-B; 1B) Specificity of effects of TGF-B on cartilage differentiation; 2) Initiation of chondrogenesis by TGF-B in early mesenchyme; 3) Localization of endogenous TGF-B; 4) Effects of anti- TGF-B on the action of otic epithelium; 5) Modulation of extracellular matrix (ECM) production in response to TGF-B; 6) Effects of TGF-B on gene expression of matrix proteins in the cells of the periotic mesenchyme. All six experiments utilize high density cultures to model otic capsule chondrogenesis. Expts. 1 & 2 compare the chondrogenic capacities of periotic mesenchyme cultured in the presence or absence of exogenous TGF-B to identify induction, enhancement, or suppression of this process in response to this growth factor during otic capsule formation. Expt. 3 utilizes indirect immuno-fluorescence to localize endogenous TGF-B in cultured epithelial and mesenchymal tissues of the inner ear. Expt. 4 seeks to define whether endogenous TGF-B mediates the action of otic epithelium on chondrogenesis utilizing antibodies specific for TGF-B. Expts. 5 & 6 test the second hypothesis and will further define the parameters of the hypothesis that suggest a mechanism of action for TGF-B involving the ECM. These experiments utilize the techniques of RNA isolation, gel electrophoresis, and Western blot, Northern blot and slot blot hybridization. Testing of the two hypotheses stated above will produce information to further our understanding of the influence of growth factors such as TGF-B on the interactions between epithelial and mesenchymal tissues during otic capsule formation. This information will also advance the general understanding epithelial induced differentiation in other chondrifying tissues. There is additional application of these studies in that many common congenital anomalies occur in tissues in which TGF-B appears to have morphogenetic action. These findings may therefore have application to further our understanding of basic mechanisms that when perturbed can result in patients with congenital defects of the inner ear.
