Biology Theses and Dissertations

Permanent URI for this collectionhttp://hdl.handle.net/1903/2749

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    IDENTIFICATION OF THE MOLECULAR MECHANISMS OF ZEBRAFISH INNER EAR HAIR CELL REGENERATION USING HIGHTHROUGHPUT GENE EXPRESSION PROFILING
    (2010) Liang, Jin; Popper, Arthur N.; Neuroscience and Cognitive Science; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    All nonmammalian vertebrates studied can regenerate inner ear mechanosensory receptors, i.e. hair cells, but mammals only possess a very limited capacity for regeneration after birth. As a result, mammals suffer from permanent deficiencies in hearing and balance once their inner ear hair cells are lost. The mechanisms of hair cell regeneration are poorly understood. Because the inner ear sensory epithelium is highly conserved in all vertebrates, we chose to study the hair cell regeneration mechanism in adult zebrafish, hoping the results would be transferrable to inducing hair cell regeneration in mammals. We defined the comprehensive network of genes involved in hair cell regeneration in the inner ear of adult zebrafish with the powerful transcriptional profiling technique, Digital Gene Expression (DGE), which leverages the power of next-generation sequencing. We also identified a key pathway, stat3/socs3, and demonstrated its role in promoting hair cell regeneration through stem cell activation, cell division, and differentiation. In addition, transient pharmacological up-regulation of stat3 signaling accelerated hair cell regeneration without over-producing cells. Taking other published datasets into account, we propose that the stat3/socs3 pathway is a key response in all tissue regeneration and thus an important therapeutic target not only for hair cell regeneration, but also for a much broader application in tissue repair and injury healing. The dissertation contains four supplemental files. Supplemental file 1 contains raw data of five expression profiles generated by DGE. It is a tab-delimited text file with six columns. The first column contains the sequences of the tags and the second to sixth columns contain the count of the corresponding tags in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 2 contains UniGene clusters identified from unambiguously mapped tags. It is a tab-delimited text file with six columns. The first column contains the UniGene IDs. The second to sixth columns contain the count of the corresponding UniGene clusters in control, 0-hpe, 24-hpe, 48-hpe, and 96-hpe profiles respectively. Supplemental file 3 contains candidate genes identified by comparison of the expression profiles during regeneration to the control profiles. It is a tab-delimited text file with 19 columns. The contents in each column are specified in the header. Supplemental file 4 contains a list of the candidate genes known to be expressed in the inner ear and/or the lateral line system during development. It is a tab-delimited text file with four columns which contain UniGene IDs, ZFIN IDs, Entrez Gene IDs, and gene symbols respectively.
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    Unconventional Myosins in Fish Ears
    (2005-04-21) Coffin, Allison; Popper, Arthur N; Kelley, Matthew W; Biology; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)
    Unconventional myosins are critical motor proteins in the vertebrate inner ear. Mutations in both myosins VI and VIIa cause multiple forms of human hereditary deafness but the precise function of these proteins is unknown. This dissertation uses a comparative approach to better understand the role of myosins VI and VIIa in vertebrate ears. Gene expression and protein distribution for these two myosins is examined in the ears of evolutionarily diverse fishes. RT-PCR data shows that myo7a is expressed in the ears of all taxonomically diverse fish species examined here, and immunofluorescence reveals that myo7a protein is distributed throughout the sensory hair bundles of all inner ear regions. Myosin VI expression and distribution is more complex. Studies in other laboratories show that zebrafish (Danio rerio) have two myo6 paralogs with differing gene expression patterns. This dissertation extends previous findings by showing that all teleost fishes have two myo6 genes while non-teleost fishes and tetrapods have one, suggesting that myo6 duplication occurred in an ancestral teleost, probably during a genome-wide duplication. RT-PCR experiments suggest that both myo6 paralogs are expressed in teleost ears. mRNA localization with in situ hybridization shows, however, that myo6a is not expressed in sensory epithelia. Immunocytochemical data shows that myo6 protein is distributed throughout hair bundles in all inner ear end organs of the sea lamprey (Petromyzon marinus) and the zebrafish but is not found in utricular hair bundles in other fishes. While protein expression studies find that the myo6 antibody used in this dissertation binds to both myo6 proteins in the zebrafish, the gene expression studies suggest that only myo6b is expressed in hair cells, and therefore that this is differential distribution of a single protein. This dissertation adds depth to current studies of myo6-associated hereditary deafness and suggests that comparative studies between zebrafish and other fishes such as shad (Alosa sapidissima) that differ in myo6 protein distribution will help elucidate the function of this critical hair cell protein. Comparisons between the two myo6 paralogs will further aid in functional studies and shed light on evolutionary processes during the teleost radiation.