Thesis (Ph. D.)--University of Hong Kong, 2006. / Also available online.

Sox10 is a high mobility group (HMG) domain transcription factor which is an important regulator for neural crest development. SOX10 mutations have been identified in Waardenburg-Shah syndrome type 4 (WS4) patients who suffer from sensorineural deafness. However, the mechanisms underlying the hearing defect of SOX10-mediated WS4 are unclear. The aim of this study is to elucidate the function of Sox10 during mouse inner ear development using a mutant mouse model, in order to reveal the underlying basis for SOX10 mutation associated sensorineural deafness in WS4 patients. The mammalian inner ear originates from the otic placode epithelium as well as neural crest cells (NCCs). To understand the role of Sox10 in inner development, I investigated the contribution of cranial NCCs to the cochleovestibular ganglion (CVG) by lineage tracing analysis, using Wnt1-cre;ZEG mice in which all NCCs were marked by GFP. Co-expression of GFP-positive cells with the glial marker BFABP suggested that glial cells in the CVG were derived from NCCs. Furthermore, Sox10-expressing NCCs were found to invade the CVG at 30-somite stage. These results suggest a role of Sox10 in regulating cranial NCCs contribution to CVG glia. In our laboratory we have generated a mouse mutant Sox10EGFP in which the Sox10 N-terminal domain was fused to the EGFP reporter. To investigate the function of Sox10 in NCCs invasion and gliogenesis of CVG, phenotypic analysis of Sox10NGFP mutant mouse were performed. EGFP expression in the CVG and inner ear epithelium of Sox10NGFP/+ embryos recapitulated the dynamic expression pattern of Sox10. Sox10NGFP/NGFP mutants displayed a reduced number of migrating NCCs and lacked NCCs or glia in their CVG. Moreover, loss of glial cell in the developing spiral ganglia of Sox10NGFP/NGFP mice led to disorganized fasciculation and degeneration of axonal filaments. These data suggest that Sox10 is required for maintaining the cranial NC stem cell pool, and is also essential for CVG gliogenesis and normal growth and innervation of spiral ganglion neurons. To study the function of Sox10 in regulating cochlear morphogenesis, morphological and histological analysis of mutant cochlear were performed. As illustrated by paint-filling analysis, Sox10NGFP/NGFP mice developed a shortened cochlear duct, reduced cochlear turning and enlarged endolymph lumen. Sensory hair cell patterning in the organ of Corti was normal in the Sox10 mutant as shown by immunohistochemistry analysis, suggesting that cochlear lumen enlargement was not due to disrupted planar cell polarity (PCP) pathway. To explore the molecular basis of Sox10-mediated cochlear morphogenic defect, expression of genes related to cochlear development were examined by qRT-PCR. Candidate genes included those involved in fluid homeostasis, which are known to affect the size of cochlear lumen. Up-regulated expression of Aquaporin 3, a water channel protein in the cochlear epithelium that facilitates water transport across the cell membrane, was observed in Sox10NGFP/NGFP cochlear. These results suggest that Sox10 may regulate cochlear morphogenesis by controlling endolymph homeostasis. In conclusion, Sox10 is required in multiple processes during inner ear development including NCC invasion, gliogenesis and cochlear morphogenesis, and their abnormal development can lead to sensorineural deafness in WS4 syndrome. / published_or_final_version / Biochemistry / Doctoral / Doctor of Philosophy

No description available.

Damage to one vestibular apparatus (unilateral vestibular deafferentation, UVD) results in severe postural and ocular motor disturbances (such as spontaneous nystagmus, SN) that recover over time in a process known as vestibular compensation. However, the underlying neurochemical mechanisms of vestibular compensation are poorly understood. While UVD affects many areas in the CNS, attention has focused upon the partially deafferented second order neurons in the vestibular nuclei complex (VNC). Several converging lines of evidence suggest that Ca�⁺-permeable ion channels (N-methyl-D-aspartate receptors and L-type voltage-gated Ca�⁺-channels) and intracellular Ca�⁺-dependent protein kinases play an important role in vestibular compensation. However, the nature of this involvement and the locus of these changes are unknown. The aim of this thesis was to investigate the role of Ca�⁺ signalling pathways in the VNC during vestibular compensation in guinea pig. These issues were investigated in three separate experiments that utilised two methodological approaches: i) in vitro assays were used to determine the nature and extent of protein phosphorylation within the VNC at various stages of compensation; and ii) ion channel blockers or cell-permeable kinase inhibitors were injected directly into the VNC immediately before UVD to determine whether or not these systems were causally involved in compensation. The results of experiment 1 (Chapter 5) showed that a bolus intra-VNC injection of an uncompetitive NMDA receptor antagonist, but not an L-type voltage-gated Ca�⁺ channel antagonist, temporarily reduced SN frequency at the earliest measurement time (6 hours post-UVD). These results suggested that the initial expression of SN required, in part, the activation of NMDA receptors in the VNC on the side of the UVD, and by inference, Ca�⁺ entry through the ion channel. The results of experiment 2 (Chapter 6) revealed that the medial VNC contains abundant Ca�⁺/calmodulin-dependent and Ca�⁺/phospholipid-dependent protein kinase activities. The same VNC tissue removed from animals at various times after UVD, showed that vestibular compensation is accompanied by specific changes in the phosphorylation of several major protein kinase C substrates. These included an unidentified 46-kDa band, and a 75-kDa band with similar characteristics to the myristoylated alanine-rich C kinase substrate (MARCKS). These results suggest that protein kinase C signalling pathways may be involved in vestibular compensation. The results of experiment 3 (Chapter 7) are consistent with these results showing that intra-VNC infusion of a protein kinase C inhibitor, but not a Ca�⁺/calmodulin-dependent protein kinase II inhibitor, significantly increased SN at the earliest measurement times (6 and 8 hours), but had no effect upon the time taken to achieve compensation or on postural compensation. These results suggest that the induction of SN compensation involves protein kinase C activity in the VNC. Taken together, these findings suggest that the mechanisms underlying the expression of SN (e.g., Ca�⁺ influx via NMDA receptors) are possibly distinct from those that initiate its compensation (e.g., PKC activation). The downstream effects of raised intracellular Ca�⁺ may involve protein kinase C-dependent phosphorylation of key intracellular proteins that initiate long-lasting changes in cellular function within the VNC.

Thesis (Ph. D.)--University of Oregon, 2008. / Typescript. Includes vita and abstract. Includes bibliographical references (leaves 43-47). Also available online in ProQuest, free to University of Oregon users.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / The inner ear contains specialized sensory epithelia that detect head movements, gravity and sound. Hearing loss and imbalance are primarily caused by degeneration of the mechanosensitive hair cells in sensory epithelia or the sensory neurons that connect the inner ear to the brain. The controlled derivation of inner ear sensory epithelia and neurons from pluripotent stem cells will be essential for generating in vitro models of inner ear disorders or developing cell-based therapies. Despite some recent success in deriving hair cells from mouse embryonic stem (ES) cells, it is currently unclear how to derive inner ear sensory cells in a fully defined and reproducible manner. Progress has likely been hindered by what is known about induction of the nonneural and preplacodal ectoderm, two critical precursors during inner ear development. The studies presented here report the step-wise differentiation of inner ear sensory epithelia from mouse ES cells in three-dimensional culture. We show that nonneural, preplacodal and pre-otic epithelia can be generated from ES cell aggregates by precise temporal control of BMP, TGFβ and FGF signaling, mimicking in vivo development. Later, in a self-guided process, vesicles containing supporting cells emerge from the presumptive otic epithelium and give rise to hair cells with stereocilia bundles and kinocilium. Remarkably, the vesicles developed into large cysts with sensory epithelia reminiscent of vestibular sense organs (i.e. the utricle, saccule and crista), which sense head movements and gravity in the animal. We have designated these stem cell-derived structures inner ear organoids. In addition, we discovered that sensory-like neurons develop alongside the organoids and form putative synapses with hair cells in a similar fashion to the hair cell-to-neuron circuit that forms in the developing embryo. Our data thus establish a novel in vitro model of inner ear organogenesis that can be used to gain deeper insight into inner ear development and disorder.

Accessing the inner ear presents a significant challenge for the diagnosis and treatment of inner ear diseases. Many existing techniques to access the inner ear are invasive and can cause permanent damage to the cochlea. Recently, a novel microneedle has been fabricated to perforate the round window membrane (RWM) – a membrane sealing one of the two openings in the cochlea. These perforations enhance drug delivery into the inner ear, potentially improving the efficacy of therapeutics. Furthermore, they allow for the aspiration of perilymph samples, which is essential for diagnosing inner ear diseases. However, owing to limited knowledge about the mechanical properties of the RWM, certain technical aspects remain unexplored. Specifically, the interaction between the RWM and the microneedle during perforation is yet to be examined. This investigation is pivotal for the optimal design of microneedles — those robust enough to perforate RWMs yet delicate enough to minimize damage. In this thesis, we conduct a thorough examination of the guinea pig RWM, encompassing its geometry and its mechanical responses to pressures from the middle ear and inner ear. Additionally, we also formulate a comprehensive constitutive law for the guinea pig RWM. Our exploration begins with the creation of a U-Net model tailored to automatically segment the RWM. Despite the presence of other structures in the same image—such as bone, the basilar membrane, and ambient noise—the model proved invaluable for efficiently and automatically segmenting the RWM. To enhance accuracy, post-processing techniques like connected component analysis and majority voting were incorporated. Using this 3D model, we proceeded to study the RWM’s geometry. Recognizing the shrinkage observed in fixed RWMs, we integrated fresh RWM data to estimate the shrinkage ratio. Subsequently, we analyzed both the overall RWM thickness and that of the middle connective tissue layer—crucial metrics for future RWM modeling. Next, we proposed a method to evaluate the in-plane deformation of the RWM due to applied pressure. This involved using a bulge test system to pressurize and deform the RWM, combined with confocal microscopy to track stained nuclei or pre-introduced fluorescent beads on the RWM. We then utilized the coherent point drift (CPD) algorithm to measure the displacement of beads and nuclei. Results indicated that both markers could be successfully used to measure the RWM’s displacement. Further analysis revealed the in-plane Lagrangian strain of the RWM, with a significant observation being that the direction of maximum in-plane Lagrangian strain is perpendicular to the fiber direction. This underscores the crucial role of collagen fibers in determining the RWM’s mechanical properties. To conclude our study, we devised a constitutive law for the RWM, conceptualizing it as a combination of the ground substance and a family of dispersed fibers. This model was integrated into a FEBioStudio plugin, facilitating simulations of the RWM’s mechanical reactions to different pressures. Although our simulations closely aligned with experimental findings, some discrepancies were noted, likely stemming from an incomplete understanding of fiber dispersions. Nevertheless, our constitutive law reinforces the notion that fibers primarily govern the RWM’s mechanical characteristics.

Inner ear hair cells have been identified as the sites of mechanoelectrical transduction from a mechanical event (e.g. hearing, motion) to an electrical event (e.g. neural response). Deflection of bundles of hair-like stereocilia extending from these cells has been associated with the transduction process. Stereocilia bundle structure and stiffness controls deflection and thus the fundamental sensitivity of the transduction process. The finite-element method was used along with analytical techniques to characterize individual stereocilium and stereocilia bundle stiffnesses. A three ‘stack’ bundle with a Young’s modulus of 3 GPa (F-actin protein) and Poisson’s ratio of 0.4 (nearly incompressible) resulted in a stiffness of K = 2.1 x 10⁻³ N/m. This value is within the range of experimentally determined stiffmesses. Tip-link and subapical band interconnecting structures each contribute significantly to bundle stiffness and each could act as the gating-spring in transduction models, which propose gating structures as a means of regulating ionic activity and therefore neural activity. Stiffness depends most strongly on individual stereocilium geometry and material description, tip-link orientation and material description, and stereocilia bundle width. Stiffness depends least on stereocilia height variations and subapical bands configuration. Linear analysis was reliable up to deflections of 3.5 um, the upper limit of physical response. Preliminary dynamic response indicates a natural frequency of 382 kHz for the vibration mode resembling physical deformation behavior. Future models should include hexagonal bundle arrangements, transversely isotropic stereocilia material descriptions, and viscoelastic tip-link behavior. / Master of Science

published_or_final_version / abstract / Biochemistry / Doctoral / Doctor of Philosophy