Sensory hair cell (HC) loss is a major cause of human deafness, tinnitis and balance disorders. However, despite great progress in many laboratories, the molecular bases for both hair cell development and regeneration are still not understood. We propose to test the novel hypothesis that mammalian hair cell regeneration in vivo will be possible if we can induce HCs to re-express the critically important growth factor Bone Morphogenetic Protein 4 (BMP4). In the experiments proposed here, we will make a transgenic """"""""knock-in"""""""" mouse, in which BIVIP4 can be expressed in mammalian hair cells under a dual control system. We will engineer constructs that place a full-length BMP4 under the control of the hair-cell specific brn3.1 promoter, in a tet-on inducible system, so that providing the transgenic mice with doxycycline (DOX) will turn on BMP4 expression specifically in terminally differentiated hair cells of the inner ear that normally do not express BIVIP4. BIVIP4 is an axial patterning morphogen, but more significantly, it is a major """"""""plasticity"""""""" gene that keeps sensory and neuronal cells in a proliferative state, resembling stem cells. We will first express this tet-on construct in immortalized inner ear cell lines derived from day 9 Immortomouse otocysts and in mouse embryonic stem cells (ES) in culture. We have made constructs that drive luciferase, to test the proof of principle. We can also compare the genetic expression repertoire of the cells induced with DOX to those of cells that are not induced by RT-PCR and by gene array techniques. Next we will make a transgenic knock-in mouse that is capable of re-expressing BMP4 in all of the hair cells of the inner ear. We will study the cellular morphology and HC repair and regenerative capacity of these mice in the presence and absence of DOX; that is, when the hair cells are either capable of re-expressing BIVIP4 or not. We are testing the hypothesis that if BIVIP4 is re-expressed in hair cells of the inner ear, returning these cells to an earlier more 11 stem cell-like"""""""" state, we will be able to regenerate a sensory epithelium after either noise-induced trauma or ototoxic aminoglycoside exposure. By using focal noise exposure or precisely dosed and delivered aminoglycosides, we can vary the site and extent of the lesions, and determine the degree to which repair, regeneration and/or preservation play a role in restoring a functional sensory epithelium.
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