Text

theses

ETD doctoral

The integration of auditory and vestibular sensory inputs from our environment underlies our ability to hear, maintain spatial orientation, and balance. Otic mechanosensory cells (hair cells) and neurons are responsible for conveying and relaying sensory information to the brain. Inner ear disorders are often a result of mutations in transcription factors involved in the development of neurosensory progenitors that give rise to hair cells and otic neurons. The discovery and study of genetic factors that contribute to the development of the inner ear neurosensory cells will clarify the molecular etiology inner ear impairments and promote the advancement of strategies to treat inner ear disorders.

In this thesis, I have reviewed the key molecular mechanisms that drive inner ear development, morphology, and function. To study and identify transcription factors involved in otic neurosensory cell development, I utilized a fate restricted stem cell line of self-renewing immortalized multipotent otic progenitors (iMOPs). Under distinct culture conditions, iMOP cells can self-renew and maintain an otic progenitor state or differentiate into neurons that molecularly and morphologically resemble otic neurons. Furthermore, to holistically characterize the role of iMOP identified transcription factors during otic neurosensory development, I utilized the evolutionarily and functionally conserved inner ear of the embryonic zebrafish.

Using the iMOP cellular system, the role of SOX2 in otic neuron development was studied. A crucial milestone in otic neuronal development is the cell cycle exit of neuronal progenitor cells. Chromatin immunoprecipitation of SOX2 followed by sequencing of the pulled-down DNA fragments in proliferating iMOP (neurosensory progenitor-like cells) and iMOP-derived neurons showed SOX2 to be enriched in the promoter region of cell cycle inhibitors (e.g., Cdkn1b) in both cellular states. Furthermore, knockdown of SOX2 significantly reduced the expression of CDKN1B in iMOP derived neurons and prevented neuronal differentiation of iMOP cells. These experiments lead us to conclude that SOX2 is a critical regulator of otic neuron differentiation. SOX2 does this by regulating the expression of cell cycle inhibitors to promote a post-mitotic state, a critical stage in the progression of otic neuronal differentiation.

To further understand the molecular progression of otic neuronal differentiation, a pseudo time trajectory-based model of neuronal differentiation was generated using the iMOP cellular system. To build this model, TUBB3 labeled neurites from morphologically heterogeneous post-mitotic iMOP-derived neuron cultures were measured and clustered based on neurite length similarities. The neurite length of iMOP-derived neurons defined the differentiation state. The clustered neurites were arranged in an ascending manner. Fluorescence intensity of transcription factors critical for otic neuronal differentiation SOX2 and NEUROD1 was measured in pseudo time order iMOP-derived neurons. During early iMOP neuronal differentiation, SOX2 and NEUROD1 protein expression meet at an inflection point. Where SOX2 and NEUROD1 protein levels gradually decrease or increase, respectively. Quantifying subtle differences in protein expression during otic neuronal differentiation provides a deeper understanding of the molecular trajectory of neuronal differentiation. This is relevant for properly recapitulating otic neuronal differentiation in potential stem cell therapies.

To characterize iMOP- identified transcription factors involved in neurogenesis and neuron development, the homeobox transcription factor Shox2 was studied during zebrafish inner ear development. Several studies have identified SHOX2 expression during early inner ear neuronal development. However, its role in this process has not been studied before. To characterize the role of SHOX2 in a way that would better recapitulate human disease, the embryonic zebrafish inner ear model system was used. SHOX2 and its related gene family members (SHOX) are highly conserved from humans to zebrafish but are not present in mice. With a variety of transgenic reporter zebrafish lines, it was determined that Shox2 expression is restricted to a subpopulation of otic neuron progenitors. In this study, several shox2 loss-of-function alleles were generated. The ablation of shox2 gene resulted in a reduction of utricular neurons. Finally, single-cell RNA sequencing of the developing zebrafish embryo inner ear was performed to determine how the transcriptional landscape and cell populations were altered in the absence of Shox2. This experiment showed that the absence of Shox2 alters the pool of otic neuronal progenitors. These experiments allowed us to conclude that Shox2 is essential for the establishment of a subpopulation of neuronal and neurosensory progenitor cells in the inner ear.

ETD_11346

Ph.D.

Includes bibliographical references

doi:10.7282/t3-cdq3-y819

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