The chicken inner ear is unique in that adult hair cells regenerate after noise or gentamicin-induced damage. However, the background of this proposal shows that limited viability and very little regeneration has been achieved in culture. Thus, it is reasonable to suspect that the behavior of these cells is guided by their micro-environment. The experiments set forth in this proposal will test the hypothesis that viable and regenerating cultures of the avian sensory epithelium can be developed by mimicking the features of the in vivo system that promote hair cell viability and regeneration. I believe that two important but unexplored features of the in vivo system are the composition of the basement membrane and the stimulation of hair cells. By anchoring epithelium on a matrix that mimics the epitheliums' matrix and stimulating the cells, the hair cells will remain viable in culture. The macromolecular patterning of the chick supporting cell/basement membrane junction will be assessed with Mass Spectometry and antibody-based methods. The sensory epithelium will be cultured on an artificial membrane that mimic the soft tissue structure and macromolecule content of the chick basement membrane. The importance of macromolecules on preserving hair cell and supporting cells will be assessed through their selective removal from the culture system. The importance of stimulating the cells through an ionic and electrical gradient and the application of tension across the cells will also be assessed. We will characterize cell density, identity and functionality. Finally, in vitro scrapes will be made across the width of the sensory epithelium, and we will characterize the proliferation and differentiation events that occur. The events will then be assessed as a function of the microenvironment. These experiments will enable us to develop a theory of the elements of the microenvironment that are necessary and sufficient for chick hair cell viability and regeneration. Because the microenvironment obviously plays an important role in guiding cell behavior, it is feasible to believe that adult mouse hair cells and supporting cells will behave like the chick counterparts in the appropriate microenvironment. ? ? ?
