Transcriptome Characterization of Adult Zebrafish Inner Ear Hair Cells

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Authors
Barta, Cody Lincoln
Issue Date
2016-02-16
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Thesis
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en_US
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Abstract
Hair cells are the vital mechanotransducer cells found in both the mammalian auditory and vestibular systems. Mammalian hair cells are non-regenerative, and thus their death leads to irreversible losses in hearing. Hair cells in zebrafish and other non-mammalian organisms such as chickens have regenerative properties, which makes them important to study. In addition to the inner ear sensory epithelia of the zebrafish, hair cells exist on the zebrafish exterior in lateral-line neuromasts. This convenient and accessible anatomy has been exploited by researchers to screen drugs for possible ototoxicity and hair cell regeneration studies. Our study focuses on the transcriptomes of the adult inner ear hair cells. Using a pou4f3 promoter-driven GAP-GFP line of transgenic adult zebrafish, hair cells were isolated from the three inner sensory epithelia: the utricle, saccule, and lagena. Two thousand GFP+ cells (hair cells) and 2000 GFP- cells (non-sensory cells in the inner ear) were individually collected by suction pipetting. RNA-seq was performed on GFP+ hair cells, and GFP- surrounding cells. The transcriptomes of the GFP+ and GFP- cells were then analyzed and compared. Using a 1 FPKM threshold, 12,444 total genes were found to be expressed in GFP+ hair cells, with 2,291 genes being differentially upregulated when being compared to the GFP- cells. A total of 13,119 total genes were found in the GFP- cells, with 1,757 genes being differentially upregulated compared to hair cells. The top ten differentially expressed genes in hair cells are anxa5a, s100s, cd164l2, pvalb9, s100t, pvalb8, si:ch73-199k24.2, atp1b2b, cabp2b, and atp1a3b. In addition, 73 zebrafish orthologs of human deafness-related genes were examined; 57 of these genes are present in adult zebrafish hair cells. The transcriptome was characterized by examining stereocilia related genes, cell cycle control genes, and ion channel and neurotransmission genes. Validation of our RNA-seq data was accomplished by RT-qPCR and literature review of published data. Our RNA-seq data of purified adult zebrafish hair cells provides an extremely valuable resource for understanding the molecular mechanisms underlying hair cell morphology, function, and pathology. This dataset also establishes a framework for future characterization of all genes expressed in hair cells.
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Creighton University
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Copyright is retained by the Author. A non-exclusive distribution right is granted to Creighton University and to ProQuest following the publishing model selected above.
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