Comprehensive Transcriptome Analysis of Utricle Hair Cell Regeneration in the Inner Ear.

摘要

Inner ear hair cells (HCs) are the main mechanoreceptors involved in the detection of sound, balance and orientation. In mammals, HCs develop during embryogenesis and can be lost in later life as a result of various forms of damage. Loss of HCs is a leading cause of deafness and balance disorders. Unlike mammals, lower vertebrates, such as birds, are able to regenerate sensory hair cells in response to injury. Avian HC death triggers one of two outcomes in the surrounding supporting cells (SCs) which are bipotent stem cells. They can either directly convert into new HCs or they can re-enter the cell cycle and generate new HCs and SCs by regenerative proliferation. Aminoglycoside antibiotic-ablation of the chicken utricle sensory epithelia has been widely used to study hair cell regeneration. In this thesis, I use this damage regimen, in conjunction with Next Generation Sequencing Technology (NGS) to investigate the regenerative transcriptomes of inner ear sensory epithelia in birds and mice. In chapters 2 and 3 I describe and analyze comprehensive transcriptome profiles of avian utricles, including mRNAs and miRNAs. For mRNA, important gene families and pathways, such as NOTCH, FGF and WNT signaling are extracted and new expression patterns and components are identified by Self-Organizing Maps. These patterns are further correlated to distinct biological processes during regeneration. A subset of ∼500 potential hair cell markers is discovered and 3 of these are validated by immunohistochemistry. In chapter 3, differentially expressed miRNAs are identified during hair cell regeneration. Five miRNAs are selected for functional validation by overexpression and inhibition. The phenotypic effects are measured by cell proliferation assays. Their expression profiles are correlated to the mRNA dataset to identify possible miRNA : mRNA interactions. In chapter 4, the transcriptome changes in chicken and mouse utricles in response to the forced phenotypic conversion of supporting cells to hair cells (by gamma-secretase inhibitor treatments) are profiled by NGS. Transcriptome signatures are discovered for cell proliferation and differentiation. By comparing and contrasting the similarities and differences between these datasets I identify potential regulators which appear to distinguish the differing regenerative capabilities of avian and mammalian sensory epithelia.