We shall employ micro-cDNA methods, gene chips and custom oligonucleotide microrarrays to investigate which genes are specifically up or down regulated in the early stages of mammalian inner ear development the progenitors of the inner ear appear as an ectodermal thickening at about day 8 of mouse development gradually this structure invaginates and the otocyst/otic vesicle is formed which by day 15 postcoitum holds most of the complex cellular machinery of the inner ear we intend to conduct a detailed gene chip analysis of this time course in the mouse we shall also employ a custom transcription factor microarray to measure expression changes in these important regulatory molecules early inductive mechanisms will be investigated by comparing gene expression in mesoderm, hind brain and the ectodermal thickening that precedes actual otic placode development in addition to these relatively conventional analyses, we shall explore innovative cdna target depletion strategies designed to measure changes in much lower abundance transcripts our third aim is to develop a custom oligonucleotide microarray of important genes identified using the gene chip data and by interrogation of a large microarray database we shall use this to reanalyze our mouse time courses and for a more limited parallel study of microdissected human embryonic otic structures to confirm the relevance of our observations to human ear development we shall conduct confirmatory assays to validate our observations from expression arrays finally, we intend to apply the knowledge gained from normal otic development to an investigation of expression profiles in mouse embryonic stem cell lines preliminary data indicate that is cells can be initiated into what may be a """"""""otic"""""""" pathway we shall investigate the gene expression profiles in these clones of cells and also apply the normal otic expression profiles to """"""""directed differentiation"""""""" experiments in the es system we expect that the type of gene expression profiles we shall build for the developing inner ear will highlight candidate genes for hearing loss, that they will pinpoint at least some of the players in a sequential genetic pathway leading to auditory hair cell differentiation and innervation (as just two examples), and that they will prove useful in the intelligent design of differentiation strategies for embryonic stem cells for future replacement therapies. ? ?
