Date of Award
Doctor of Philosophy (PhD)
The inner ear consists of two groups of organs, the auditory and vestibular organs. The auditory organ is cochlea and it is responsible for sensing sound. The vestibular organs are the sacculus, utricle, three semi-circular canals, and the ampulla. All of these organs contain mechanoreceptors called hair cells. Hair cells are epithelial cells with a bundle of stereocilia that protrudes from their apical surface. Hair cells are surrounded by another type of epithelial cell called a support cell. These two cell types, hair and support cells, together make up the sensory epithelia, which is a two-cell thick layer of cells that is the tissue of choice for this dissertation. Damage to the sensory hair cells is the major cause of hearing and balance impairments. In mammals, once the sensory hair cells are killed they are not replaced. This is not the case in lower-vertebrates such as birds. Birds maintain the ability to regenerate hair cells within the auditory and vestibular organs throughout their life.
To assess and compare the regenerative capabilities of the cochlea and utricle, the respective tissues were cultured with an aminoglycoside antibiotic to kill the hair cells within the sensory epithelia. The cultures were then allowed to recover in the absence of the antibiotic for up to 168 hours. The transcriptomes were generated with second-generation sequence (mRNA-seq) and compared. Of interest were the differentially expressed (and represented) transcription factors within these tissues as well as the differential usage of the Notch components. Hairy/enhancer-of-split 5 (HES5) is expressed in support cells and prevents acquisition of the hair cell fate (hair cells express atonal 1 [ATOH1] which inhibits HES5). HES5 is very abundant in regenerating utricles, but is not existent in regenerating cochlea. The most likely candidate to repress ATOH1 in the cochlea support cells is hairy/enhancer-of-split related with YRPW motif 1.
A second study examined dissociated sensory epithelia in culture. In the regeneration assay described in the first project, the sensory epithelia are kept intact on the stroma during culture whereas the dissociated sensory epithelial culture separates the sensory epithelia from the stroma and dissociates the sensory epithelia prior to plating. The dissociated sensory epithelia were cultured for up to 144 hours and their transcriptomes analyzed. Based on markers of epithelial and mesenchymal states, it appears as though the dissociated sensory epithelia undergo a epithelial-to-mesenchyme transition between 48 and 96 hours. A second surprising finding is the increase in the mesenchyme-specific splicing factor RNA binding protein fox-1 homolog 2 (RBFOX2) and the decrease in epithelial-specific splicing factors epithelial splicing regulatory proteins 1 and 2 (ESRP1 and ESRP2). This, along with differential isoform detection, suggests alternative splicing is occuring during the time in culture. It also gives another avenue for the creation of a high-throughput hair cell differentiation assay if the dedifferentiated tissues can be transitioned back into a differentiated state through the knock-down of RBFOX2.
The third study examined the regulatory elements associated with hair cell regeneration. This was carried out by performing chromatin immunoprecipation with the enhancer associated protein p300 on utricular sensory epithelia from the regeneration assay described in study one. P300 bound regions of DNA were enriched near differentially expressed genes identified in the utricular mRNA-seq dataset. The integration of the ChIP-seq data with the utricular mRNA-seq data allowed for better candidate selection. From the pool of high quality candidates, five were tested and all five validated in reporter assays. The p300 bounds regions at each time point were collectively analyzed for transcription factor binding sites; both known and predicted transcription factor binding sites were identified expanding the knowledge to potential networks of regulation.
Chair and Committee
Patrick Jay, John Rice, Elisha Roberson, Cristina de Guzman Strong, Mark Warchol
Renaud, Nicole Ann, "The Genomic Regulatory Architecture of Avian Inner Ear Hair Cell Regeneration" (2013). Arts & Sciences Electronic Theses and Dissertations. 1036.
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