OREGON HEALTH & SCIENCE UNIVERSITY
The long term goals of our laboratory are to understand the molecular mechanisms responsible for cell fate specification and sensory patch formation in the developing mammalian inner ear. Progress toward understanding these essential aspects of inner ear development have been hampered by the inaccessibility of the mouse embryo in utero. Equally confounding is the lack of molecular tools to dynamically manipulate developmental gene expression in situ. We apply experimental embryology to study the mouse inner ear in vitro and in vivo. We aim: 1) to fate map mouse otic cup closure in wild type and mutant mice; 2) to determine the lineage relationships among constituent cells in the inner ear; and 3) to probe the molecular mechanisms underlying mammalian sensory patch formation. Mouse whole embryo culture supports inner ear development from the placode through early otocyst stages. The fate map will be conducted by iontophoretic injection of fluorescent tracer dye into designated positions in the rim and concavity of the cup. Fate mapping will teach us the morphogenetic movements of otic epithelial progenitors during cup closure and permit their correlation with know domains of gene expression. Tranuterine microinjection of bioactive reagents into the early otocyst stage mouse embryo in vivo enables a broad range of studies in the developing and postnatal inner ear. Lineage analysis will be performed by transuterine microinjection of a retroviral construct encoding alkaline phosphatase and a complex 24 base pair library into the otocyst with subsequent clonal analysis in the mature, postnatal inner ear. Lineage analysis will show us the types and timing of cell fate choices made by otic epithelial progenitors that give rise to sensory and nonsensory cells. Sensory patch formation will be investigated by transuterine microinjection of expression plasmid into the early otocyst followed by in vivo electroporation for gain-of-function studies. We will misexpress transcription factors know to be involved in sensory patch formation in wild type and mutant mouse inner ears to gain insight into their mechanistic roles in establishing the auditory sensory epithelium. A clear understanding of otic vesicle morphogenesis, lineage relationships, and sensory patch formation is essential for the definition of regenerative medical approaches to ameliorate hearing loss and balance disorders in humans.