Expression Analysis of the Regenerating Utricle Sensory Epithelia : From Macroarrays to Parsing Pathways




Hawkins, Raymond David

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I have used a human transcription factor microarray developed in the laboratory of Dr. Michael Lovett to study gene expression differences in the chicken inner ear sensory epithelia. In the initial study, the sensory epithelium from the utricle was compared to that of the cochlea. The purpose of this study was to identify gene expression differences between these two organs. The sensory epithelium from each organ is made up of hair cells and supporting cells. These hair cells are necessary for the detection of sound in the cochlea and for the detection of movement and acceleration in the utricle. The chicken sensory epithelia is of great research interest as it possesses the ability to fully regenerate hair cells that are damaged, whereas mammalian epithelia, once damaged, cannot regenerate. These two organs were compared because the utricle is in a constant state of hair cell turnover, and the cochlea remains quiescent, unless damaged. In order to carry out such microarray expression studies on a small number of cells, between 30,000-50,000 cells, a micro-cDNA amplification method, developed in the lab, was implemented and is described here as well. The experiments were carried out via cross-species hybridizations, and subsequently, a number of genes were validated by quantitative PCR and in situ analysis. Additionally, a library subtraction was used to identify additional genes expressed in the utricle sensory epithelia. In a second microarray expression study, the utricle sensory epithelia was damaged by two independent methods and allowed to recover for various time points for expression profiling on the same transcription factor array. The first method of damage was by laser microbeam to ablate the hair cells such that they die almost instantly. The second method of damage was using ototoxic antibiotics. In each time course, the time points were compared to time matched control epithelia (undamaged). The analysis of this data reveals some very important signaling cascades and developmental pathways involved in hair cell regeneration. Finally, in an effort to functionally validate many of the genes identified during regeneration, gene transcripts were targeted by RNA interference to reduce the expression level and determine the effect on hair cell proliferation. Through this method, several genes were identified to reduce proliferation. Additionally, these experiments were profiled as a means for networking genes into pathways by identifying putative downstream targets in the expression data. An intersection of genes downregulated following inhibitory experiments reveals how several genes potentially lie downstream of one another and form a pathway containing some common regulatory elements.

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