THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. This research project will define the role of growth factors and extracellular matrix (ECM) molecules in the signaling of the epithelial- mesenchymal interactions of the developing inner ear by studying the consequences of disruption of these tissue interactions by the teratogen all-trans retinoic acid (RA). Two hypotheses will be investigated by this study: 1) Exposure to exogenous RA alters TGFbeta1, FGF2 and FGF3 signaling of the epithelial-mesenchymal interactions of the developing mouse inner ear, and 2) Alteration of TGFbeta1, FGF2, and FGF3 signaling by exogenous RA is associated with subsequent changes in the levels of ECM factors. To test these hypotheses, the proposal consists of a series of 9 interrelated experiments: 1) Modulation of endogenous TGFbeta1, FGF2, and FGF3 expression by RA in periotic mesenchyme and otic epithelium in vivo and in vitro; 2) Effects of excess growth factor(s) on the inhibitory action of RA in vitro; 3) Modulation of growth factor gene expression by RA in inner ear tissues in vivo and in vitro; 4) Effects of in vivo exposure of otic epithelium/periotic mesenchyme to RA on the interactions between these RA-exposed tissues and normal inner ear tissues in vitro; 5) Distribution pattern of TGFbeta and FGF receptor mRNAs in RA-exposed and control mouse inner ears; 6) The role of RA receptors (RAR-alpha1 & RAR-beta) in RA induced teratogenicity of the inner ear; 7) Effects of RA exposure on the expression of ECM molecules in periotic mesenchyme in vivo and in vitro; 8) Effects of alpha TGFbeta1, alphaFGF2, and/or alphaFGF3 antibodies on ECM accumulation in cultured periotic mesenchyme + otic epithelium; and 9) Effects of the in vivo RA exposure of otic epithelium/periotic mesenchyme on ECM accumulation when these RA exposed tissues are interacted with normal inner ear tissues in vitro. These experiments will employ the techniques of high-density culture, immunohistochemistry, enzyme-linked immunosorbent assay (ELISA), Northern blot/RNAase protection assays, Southern blot analysis, and in situ hybridization. In summary, the proposed experiments will utilize an in vivo and in vitro RA model to probe endogenous mechanisms of inner ear development, determining the role that growth factors play in signaling the tissue interactions of the developing inner ear, and in regulating the accumulation of morphogenetic molecules in the mesenchymal ECM. The information provided by this study will have additional application by furthering our understanding of the fundamental relationship between tissue interactions, growth factors, and ECM molecules that, when altered by teratogenic means, can result in congenital disorders of the inner ear.
