Genomic approaches for pathway identification in regenerating sensory epithelia of the inner ear

The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these sensory hair cells lack the capacity for regeneration and if damaged lead to hearing or balance disorders. However, non-mammalian vertebrates such as birds maintain their regenerative abilities throughout their life. We completed a gene expression profiling time course of regenerating sensory epithelia (SE) in avian cochlea and utricle on a custom transcription factor microarray following damage by laser and chemical ablation and identified genes from known signaling cascades differentially expressed during SE regeneration. In the second study, we selected 27 of these genes for knockdown by siRNA or small molecule inhibition to determine their requirement for SE regeneration and identified downstream targets. We assessed affects on proliferation using a 96 well high throughput assay and profiled gene expression changes that resulted from each knockdown. Using these techniques we have determined genes required for SE proliferation and identified novel epistatic relationships between many of these genes.;In a third study we used 3 complimentary approaches to identify downstream targets of GATA3 in the avian utricle. The zinc finger transcription factor GATA3 is required for inner ear development and mutations cause sensory neural deafness in humans. In a previous study we had observed that GATA3 is expressed throughout the SE in the cochlea; however, expression is limited to the striola of the utricle. The striola corresponds to an abrupt change in morphologically distinct hair cell types and a 180° shift in hair cell orientation. We used microarray expression profiling of direct comparisons between cells micro-dissected from the striola vs. extra-striola, GATA3 knockdown by siRNA in utricle sensory epithelia and GATA3 over-expression to identify genes potentially regulated by GATA3 in the inner ear. We confirmed the direct in vivo interaction of GATA3 with two of these targets (LMO4 and MBNL2) by chromatin immunoprecipitation (ChIP) using GATA3 antibodies and also demonstrated by RNA in situs that both these genes exhibit patterns of expression consistent with their direct regulation by GATA3.