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Le développement de la cochlée des mammifères est un phénomène complexe qui implique la coordination de nombreux gènes. La compréhension de la physiopathologie des surdités dorigine neurosensorielle ainsi que la mise au point de stratégies visant à restaurer la structure cellulaire de loreille interne ne nous paraît possible que grâce à la compréhension fine des processus qui régulent et sous-tendent son développement. Les connaissances actuelles, nous permettre en effet de constater que de nombreuses molécules qui contrôlent lorganogenèse au cours du développement sont souvent activées ou impliquées dans les phénomènes de régénération tissulaire après un traumatisme y compris à l'échelle de l'oreille interne (Levic et al., 2007). Lors de notre travail, nous avons montré que dans loreille interne, en labsence de Sox10, les cellules dérivées des crêtes neurales, à savoir les mélanocytes cochléaires et les cellules de Schwann du ganglion spiral étaient absentes, soulignant le caractère dépendant de ces cellules au gène Sox10. Au niveau du ganglion spiral, nous avons montré que contrairement aux neurones des ganglions rachidiens (Honore et al., 2003;Sonnenberg-Riethmacher et al., 2001), le développement et la survie embryonnaire des neurones auditifs étaient indépendants des cellules gliales et de Sox10. Par ailleurs, nous avons également montré que Sox10 napparaît pas comme un facteur indispensable à linduction et au développement de la placode otique, mais que son absence conduit à une diminution de la population de cellules progénitrices du canal cochléaire. Cette diminution aboutit à une réduction de la longueur de la cochlée, et suggère que Sox10 joue un rôle primordial dans le déterminisme du pool des progéniteurs de la portion auditive de loreille interne. Cependant, à des stades ultérieurs du développement, la structure parfaitement conservée de lorgane de Corti nous laisse penser que laction de Sox10 est compensée par dautres gènes du groupe SoxE, par exemple Sox9, dont nous avons mis en évidence lexpression dans les cellules de soutien de lorgane de Corti (Cook et al., 2005;Sock et al., 2001;Stolt et al., 2004;Wegner, 1999). Par ailleurs, nous avons montré quau cours du développement de lorgane de Corti, la première cellule identifiable était la cellule pilier interne. Cette cellule échapperait au système dinhibition latérale lié à Notch et pourrait avoir un rôle dans la différenciation des autres cellules de lorgane de Corti. La destruction des cellules ciliées provoque à plus ou moins long terme une dégénérescence rétrograde des neurones du ganglion spiral, un phénomène à lorigine de nombreuses surdités neurosensorielles (Bichler et al., 1983;Koitchev et al., 1982). Afin de mieux comprendre les signaux qui régulent la réponse neuronale suite à un traumatisme au cours de leur régénération, nous avons mis au point un modèle de culture organotypique de neurones déafférentés de rats postnataux. Cest dans ce modèle dexplants de ganglions spiraux en culture que nous avons évalué la mort neuronale lors des processus de déafférentation des neurones auditifs. Il a été démontré que les neurones auditifs survivent en partie grâce aux facteurs trophiques produits par leur cible périphérique, lorgane de Corti (Lefebvre et al., 1992) et que cette dépendance persiste à lâge adulte. Ce modèle de culture permet de conserver larchitecture du ganglion spiral, se rapprochant ainsi de la situation observée in vivo. Laissés seuls, en labsence de facteurs trophiques exogènes, le nombre de neurones auditifs chute drastiquement après 24 heures de culture. Lorgane de Corti, source de neurotrophines tant durant la période développementale quà lâge adulte (Oestreicher et al., 2000;Ylikoski et al., 1993), prévient significativement cette mort neuronale lorsquil reste associé au ganglion spiral à la mise en culture. Dans ce modèle de dégénérescence rétrograde, nous avons étudié lexpression de la périphérine et constaté que cette protéine était réexprimée dans les neurones auditifs de type I après lésion. De plus, létude de lexpression de la périphérine dans le ganglion spiral du rat a permis dobserver quelle était ubiquitaire dans la population neuronale en développement puis réduite seulement aux neurones de type II lors de la période postnatale. La périphérine serait donc une molécule susceptible dintervenir à la fois lors du développement embryonnaire et réactivée lors des phénomènes de lésions tissulaires cochléaires.

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

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第20255号 / 医博第4214号 / 新制||医||1020(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 萩原 正敏, 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

Indiana University-Purdue University Indianapolis (IUPUI) / Inner ear development requires the complex interaction of numerous cell types arising from multiple embryologic origins. Current knowledge of inner ear organogenesis is limited primarily to animal models. Although most mechanisms of cellular development show conservation between vertebrate species, there are uniquely human aspects of inner ear development which remain unknown. Our group recently described a model of in vitro human inner ear organogenesis using pluripotent stem cells in a 3D organoid culture system. This method promotes the formation of an entire sensorineural circuit, including hair cells, inner ear neurons, and Schwann cells. Our past work has characterized certain aspects of this culture system, however we have yet to fully define all the cell types which contribute to inner ear organoid assembly. Here, our goal was to reconstruct a time-based map of in vitro development during inner ear organoid induction to understand the developmental elements captured in this system. We analyzed inner ear organoid development using single-cell RNA sequencing at ten time points during the first 36 days of induction. We reconstructed the on-target progression of undifferentiated pluripotent stem cells to surface ectoderm, pre-placodal, and otic epithelial cells, including supporting cells, hair cells, and neurons, following treatment with FGF, BMP, and WNT signaling modulators. Our data revealed endogenous signaling pathwayrelated gene expression that may influence the course of on-target differentiation. In addition, we classified a diverse array of off-target ectodermal cell types encompassing the neuroectoderm, neural crest, and mesenchymal lineages. Our work establishes the Inner ear Organoid Developmental Atlas (IODA), which can provide insights needed for understanding human biology and refining the guided differentiation of in vitro inner ear tissue. / 2024-08-02

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Doctor of Philosophy / Biochemistry Interdepartmental Program / Antje Philine Wangemann / Mutations of SLC26A4 are the most common cause of the hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in the cochlea, the vestibular labyrinth, and the endolymphatic sac of the inner ear. Slc26a4Δ/Δ mice, devoid of pendrin expression, develop an enlarged membranous labyrinth which leads to the failure to develop hearing, thereby recapitulating the human disease. Identifying the ionic composition of the endolymph and evaluating the importance of pendrin expression at various sites are initial steps towards developing strategies for preventing enlargement of the endolymph volume and subsequently restoring the inner ear functions. The major aims of the present study are 1) To determine the ionic composition of inner ear fluids during the developmental phase in which the enlargement of the endolymph volume occurs; 2) To test the hypothesis that pendrin expression in the endolymphatic sac is more important than its expression in the cochlea and the vestibular labyrinth. Here, we determined the Na+ and K⁺ concentrations in the cochlea and the endolymphatic sac with double-barreled ion-selective electrodes and generated a mouse model that restores pendrin expression in the endolymphatic sac while lacking expression in the cochlea and the vestibular labyrinth. High Na⁺ and low K⁺ concentrations were found in the cochlear endolymph during the embryonic stage. A rise of the K⁺ concentration along with a decline of the Na⁺ concentration occurred shortly before birth. The site-specific restoration of pendrin to the endolymphatic sac prevented enlargement and rescued hearing and balance. In conclusion, these data demonstrate that endolymph, in the phase of luminal enlargement during the embryonic development, is a Na⁺-rich fluid that is modified into a K⁺-rich fluid just before birth; restoration of pendrin in the endolymphatic sac is sufficient for developing normal inner ear function. Furthermore, these data suggest enlargement of endolymph volume caused by the loss of Slc26a4 is a consequence of disrupted Na⁺ absorption. Moreover, pharmacological strategies that correct fluid transport, as well as spatially and temporally limited restorations of pendrin, might restore normal inner ear functions in humans carrying mutations of SLC26A4.

Introduction: Hearing loss affects over 500 million people worldwide and results from irreversible damage to inner ear hair cells. The only available treatment is cochlear implants, which may be unable to provide sensory input if neuronal connections are lost, as they are in mouse models. Thus, regeneration of hair cells offers the only permanent cure; however, such therapeutic intervention requires a detailed molecular understanding inner ear development and hair cell maintenance. During mouse development, there is a balance between proliferation and differentiation that not only determines the size of the ear, but also is needed to form a functional sensory unit. The fulcrum to this balance is N-Myc, a key transcription factor that acts as a node incorporating many upstream growth signaling pathways and funnels them to directly alter the cell cycle and at the same time inhibits differentiation. The loss of N-Myc results in major morphogenetic abnormalities, including a progressive loss of cochlear, despite their initial formation. Interestingly, N-Myc is present in inner ear hair cells after birth, long after proliferation in the inner ear ceased. In addition to N-Myc, L-Myc is co-expressed throughout development in the inner ear. This data suggests that N-Myc and L-Myc may play partially redundant roles both early during development and later in hair cells. Elucidating the relative importance of the Mycs and their interdependent roles in maintaining the balance between proliferation and differentiation may shed light on future hair cell regeneration avenues. Methods: We generated two Cre-LoxP lines, knocking out both N-Myc and L-Myc before (Pax2-Cre) and after (Atoh1-Cre) hair cell formation. We assessed the possibility of Myc redundancy through 3D reconstructions generated from confocal image stacks from E10.5-E18.5 and the effects of early Myc loss on the balance between proliferation and differentiation through a quantitative PCR study that assessed relative changes in gene expression, using the Pax2-Cre N-Myc f/f L-Myc f/f mice. We assessed organ of Corti development and functionality at P21 and four months of age in the Atoh1-Cre N-Myc f/f L-Myc f/f mice. Results: The development of the Pax2-Cre N-Myc f/f L-Myc f/f mutant ear was more severely impacted than the Pax2-Cre N-Myc f/f alone, as shown by an additional 50% reduction in size. Genes important to cell cycle maintenance were downregulated whereas differentiation transcription factors were initially downregulated but subsequently later upregulated to normal levels. In Atoh1-Cre N-Myc f/f L-Myc f/f mice, there were no defects in hair cell development. Discussion: There appears to be redundancy between N-Myc and L-Myc with N-Myc playing a more important role in inner ear formation. The late-onset defects seen in the Pax2-Cre N-Myc f/f mice appear to be a result of abnormal formation of hair cells due to the disruption in the balance between proliferation and differentiation much earlier on. This is the first time such a late-onset hair cell loss has been shown to be due to a defect sustained much earlier and is an important finding as the majority of people suffer from late-onset hearing loss. Additionally, these findings highlight the continued therapeutic importance in elucidating the molecular interactions controlling the delicate shift from a proliferating precursor to a differentiating cell.