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The sensory cells of the inner ear, hair cells, provide vertebrates with the ability to detect auditory stimuli and balance. In mammals, cochlear hair cells, those responsible for hearing, do not regenerate. Zebrafish hair cells do regenerate. Gaining an understanding of the role and regulation of the genes involved in the formation and regeneration of these cells may provide information important for the development of genetic therapies. We show that zebrafish atoh1 acts as the proneural gene responsible for defining the equivalence group from which hair cells form. Expression of atoh1 is dependent upon Fgf and Pax. Atoh1 induces expression of delta, resulting in activation of Notch and subsequent lateral inhibition. Another factor known to be important for hair cell formation in mice is Sox2. In zebrafish, sox2 expression is downstream of Atoh1, Notch and Fgf. Zebrafish Sox2 is not required for hair cell formation, but rather Sox2 is important for hair cell maintenance. In zebrafish, otic hair cell regeneration has not yet been characterized. We show that, following laser ablation, hair cells regenerate by way of transdifferentiation. We further show that this regeneration requires Sox2 activity. These data suggest that Sox2 acts to maintain support cell plasticity. This role is likely conserved because Sox2 is also important for stem cell plasticity in mammals. This new understanding of the role and regulation of both Atoh1 and Sox2 provides essential information that can be used to further efforts to provide genetic therapies for hair cell regeneration in mammals.

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京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18168号 / 医博第3888号 / 新制||医||1003(附属図書館) / 31026 / 京都大学大学院医学研究科医学専攻 / (主査)教授 伊藤 壽一, 教授 大森 治紀, 教授 影山 龍一郎 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

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The hair cell, a specialized cell in the inner ear, is responsible for hearing and balance. The hair cell is an exquisite sensor that captures mechanical stimuli and generates neurosensory signals. A theory called gating theory has been developed and widely used to analyze the experimental data of hair cell transduction. Despite increasing knowledge about molecular structures of hair cells, the mechanical model in the gating theory remained simple. Efforts to make the most of the recent findings regarding the hair cell structures led to the development of hair cell finite element (FE) model (Cotton & Grant, 2000, 2004a, b). I have extended this approach by adding channel kinetics and structural dynamics to the FE structural model of the hair cell. I have expanded the previous static and passive model to a dynamic and active model. It is the most detailed hair cell structural model and includes up-to-date knowledge of the hair cell structure such as the stereocilia and various extracellular links. In order to observe the dynamic response of hair bundles in the endolymph fluid, I have included fluid drag in the model. Link nonlinearity has been added to reflect recent observations (Tsuprun 2003). The lateral links stiffen as they stretch and prevent contact between stereocilia when they compress. In addition to these structural features, I added channel kinetics such as the fast adaptation. In my study, the Ca²⁺ diffusion kinetics plays a key role in the hair cell adaptations. The Ca²⁺ association rate to the fast adaptation modulator is postulated to govern the fast adaptation. I assumed that two factors--the tip link tension and the Ca²⁺ concentration at the tip of stereocilia govern the hair cell mechanoelectric transduction. My dissertation comprises three parts--structure, dynamics and mechanotransduction of hair cells. First, the mechanical properties of hair bundle were sought by comparing my FE model with other experiments. The quantified Young's modulus of stereocilia and the stiffness of tip link agree well with other recent estimates. The stiffness of other structural elements (upper lateral and shaft links) was newly estimated through this effort. Second, I established equations of motion for the hair bundle in the fluid. Two possible loading conditions to the hair bundle were simulated. Two different hair bundles were subjected to a point load and a load induced by fluid flow. The results showed that some vestibular hair cells' transduction might be dominated by the fluid-induced force. Finally, I observed the hair cell transduction in various stimulus conditions. The results showed that the hair cell's sensitivity highly depends on the stimulus method. The fluid-jet stimulus activated fewer channels than the glass fiber and made the hair cell less sensitive. A faster stimulus opened more channels and made the hair cell more sensitive. The resting tension in the tip link, which is believed to be controlled by the Ca²⁺ concentration, also affected the hair cell sensitivity. A higher resting tension, equivalent to a lower Ca²⁺ concentration, tended to make the hair cell more sensitive. In conclusion, I developed a new tool to study the hair cell mechanoelectric transduction. My hair cell computational model enables us (1) to study how the hair cells' morphological variations are related to their function; (2) to investigate the hair cell mechanoelectric transduction at the single channel level, in silico, as opposed to the statistical approach; (3) to test the response of hair cells under in situ force boundary conditions. / Ph. D.

The vestibular system is one of our main organs responsible for the sense of balance. This system is located within the inner ear and contains cells with ciliary bundles. These hair cells are transducers that convert a mechanical movement, detected by the bundle of cilia extending from their top surface, into an electrochemical signal to be sent to the brain. The bundles vary structurally within the organs of the inner ear, and this structural difference may play a role in the mechanical properties of each bundle. Analyzing the mechanical properties of the cells will provide information necessary for understanding the transduction process. In an effort to evaluate one of these properties, cell bundle stiffness, a system was designed to mechanically stimulate the bundles within their physiological range and then measure the resulting displacement. The mechanical stimulation was the result of a force applied to the tip of a bundle with the end of a glass whisker. The distance the base of the whisker moves is measured by an extrinsic Fabry-Perot interferometer (EFPI). The magnitude of this movement is compared with the amount the bundle is deflected, detected by a photoelectronic motion transducer (PMT). Knowing these displacements and the stiffness of the glass whisker, simple kinematics is used to determine the bundle stiffness. System tests were conducted on imitation bundles (whiskers of known stiffness) and the experimental stiffness differed from the known value by less than 4.5% for every test. These results lead us to conclude the system was in good working order and could be used to conduct tests on cell bundles. For tissue tests, this work focused on the hair cells located within the utricle, which senses linear accelerations of the head. Within the utricle, we examined two types of hair cells: non-striolar (medial type II) and striolar. Tests on twelve medial type II cells found bundles ranging in stiffness from 0.26 to 2.62 x 10⁻⁵ N/m. Results with striolar bundles provided a range from 2.83 to 27.10 x 10⁻⁵ N/m. The results of the preliminary tissue tests lead us to conclude that the average stiffness of the striolar and non-striolar bundles seems to vary by an order of magnitude. This is consistent with the relative relationship produced through a computer model. However, the model predicted larger stiffness values for both types of cells. / Master of Science

The vestibular system of vertebrates consists of the utricle, saccule, and the semicircular canals. Head movement causes deformation of hair cell bundles in these organs, which translate this mechanical stimulus into an electrical response sent to the nervous system. This study consisted of two sections, both utilizing a Fortran-based finite element program to study hair cell bundle response. In the first part, the effects of variations in geometry and material properties on bundle mechanical response were studied. Six real cells from the red eared slider turtle utricle were modeled and their response to a gradually increased point load was analyzed. Bundle stiffness and tip link tension distributions were the primary data examined. The cells fell into two groups based on stiffness. All cells exhibited an increase in stiffness as the applied load was increased, but cells in the stiffer group showed a greater increase. Tip link tensions in the compliant group were approximately 3 times as high as those in the stiffer group. Cells in the stiffer group were larger, with more cilia, and also had a higher stereocilia/kinocilium height ratio than the cells in the other group. The stereocilia/kinocilium height ratio was the most important geometric factor in influencing bundle stiffness. Modeling a bundle as just its middle row of stereocilia resulted in some decrease in stiffness, but more significantly, a stiffness that was virtually constant as applied load increased. Tip link tension distributions showed serial behavior in the core rows of stereocilia and parallel behavior in the outer rows; this trend intensified if the tip link elastic modulus was increased. It was demonstrated that full three-dimensional modeling of bundles is critical for obtaining complete and accurate results. In the second part of the study, tip link ion gates were modeled. Sufficient tension in a tip link caused that link's ion gate to open, increasing the length of the link and causing its tension to decrease or the link to go slack. The two parameters that were varied were tip link elastic modulus and tip link gating distance d (change in length of the link). Bundle stiffness drops of up to 25% were obtained, but only when tip links went slack after gate opening; tip link slackening was dependent on tip link gating distance. Higher tip link modulus resulted in higher stiffness drops. Variable tip link modulus and tip link pre-tensioning were modeled. Variable tip link modulus resulted in increased bundle stiffness, especially under high applied loads, and in some cases, resulted in greater bundle stiffness drops when ion gates opened. Tip link pre-tensioning had no noticeable effect on bundle response. No evidence against inclusion of pre-tensioning or variable tip link elastic modulus was found. / Master of Science

The ear is the organ used for hearing and maintaining equilibrium. In the inner ear, the vestibular system is responsible for the sense of balance. The main organs of the vestibular system are the semicircular canals, the saccule, and the utricle. Within each of the vestibular organs, sensory receptors in the form of hair cells detect motion and send a message to the brain for interpretation. Hair cells found in different parts of the inner ear are structurally different and are mechanically specialized to perform different functions. In this study, the linear and torsional stiffnesses were measured for hair cells located in the red ear slider turtle utricle. The system used to measure the stiffnesses was composed of a glass whisker (attached to a pipette) used to produce a force on the tip of the bundle, an extrinsic Fabry-Perot interferometer (EFPI) to measure the displacement of the pipette, and a photoelectronic motion transducer (PMT) to measure the displacement of the bundle. Using the measured values of whisker stiffness, whisker displacement, and bundle displacement, the stiffness of the bundle was calculated using statics. For each bundle tested, the location of the bundle was determined by measuring its position from a landmark in the utricle, the line of polarity reversal, characterized by a 180o change in direction of the hair bundles. Stiffness results showed that the linear stiffness of a bundle increased in the area surrounding the line of polarity reversal, otherwise referred to as the striolar region (average linear stiffness of 2.27 E-04 N/m). The average linear stiffness value of bundles found lateral to the striolar region was 6.30 E-05 N/m and in the region medial to the striolar region was 1.16 E-04 N/m. A wide range of linear stiffnesses were found in hair cells medial to the striolar region. There was no correlation found between the torsional stiffness of a bundle and its position and the height of a bundle and its linear or torsional stiffness. As the force applied to a hair bundle was increased, the measured linear stiffness of the bundle also increased. / Master of Science