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

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Title: Expression Analysis of the Regenerating Utricle Sensory Epithelia : From Macroarrays to Parsing Pathways
Author: Hawkins, Raymond David
Abstract: 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 .
URI: http : / /hdl .handle .net /2152 .5 /663
Date: 2005-05-03

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Expression Analysis of the Regenerating Utricle Sensory Epithelia : From Macroarrays to Parsing Pathways. Graduate School of Biomedical Sciences. Available electronically from http : / /hdl .handle .net /2152 .5 /663 .

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