Dissertations / Theses on the topic 'Labyrinth (ear)'

Sox2, a HMG box transcription factor, is well known for its role in stem cell maintenance, iPS (induced pluripotent stem cell) induction, and development of neural tissues such as central nervous system and sensory organs. Sox2 has been demonstrated to be essential for the development of inner ear sensory patches. It has been shown that Sox2 is under the regulation of multiple regulatory elements to obtain a tissue specific manner. Two allelic mouse mutants, yellow submarine (Ysb) and Light coat and circling (Lcc) show hearing and balance impairments with different severity. They were made by random insertions of a transgene (pAA2) and X-ray irradiation respectively. Ysb and Lcc are both localized to chromosome 3 and involve complex chromosomal rearrangements. The Sox2 coding region is intact in the mutants, while the Sox2 expression in the otocyst is greatly reduced in Ysb and totally lost in Lcc, which indicates the tissue specific reduction of Sox2 may be due to the rearrangement of Sox2 regulatory element(s). Since Sox2 null mutants die before implantation, the two Sox2 inner ear mutants are valuable models for studying Sox2 knock down (Ysb) and Sox2 knock out (Lcc) condition in the inner ear. To understand the molecular basis behind Sox2 regulation in the inner ear, this project aims to identify the Sox2 otic regulatory elements, and potential Sox2 downstream targets involved in the development of inner ear. Previous work has indicated that Nop1 and Nop2 are the otic specific regulatory elements of Sox2 in chicken ear. In this project, transgenic mice were generated using Nop1-Nop2, and the result showed Nop1-Nop2 could drive Sox2 expression to the dorsal side of the otiv vesicle, which is different from the endogenous Sox2 expression pattern. Therefore, Nop1 and Nop2 may require other regulatory element(s) to gain a correct regulatory pattern. BAC(RP23-335P23), which contained the DNA sequences close to Ysb integration site 1 was also been tested in transgenic mice. Interestingly, the result showed that BAC(RP23-335P23) could drive Sox2 expression to the ventral side of the otic vesicle, indicating that this BAC may contain the Sox2 otic regulatory element(s). In this project, the binding relationship between Sox2 protein and Math1 enhancer has also been identified using chromatin immunoprecipitation (Ch-IP). Results showed that Sox2 could bind to Math1 enhancer A in the inner ear cochlea. So Sox2 may regulate Math1 through binding to Math1 enhancer A in inner ear development. Using a bioinformatics approach, potential Sox2 target genes in inner ear development have been identified from public microarray data on E9 to E15 inner ear tissue by the presence of conserved Sox2 binding sites. Among these potential targets, 4 genes (Itga6, Erbb3, Sox10 and Mycn) have been selected based on their known functions. Their expression patterns in the cochlea of wild type, Ysb and Lcc were verified. The identification of Sox2 downstream target genes using a bioinformatics approach will help us to understand the molecular basis of Sox2 regulation, and also understand the role of Sox2 in the inner ear development.
published_or_final_version
Biochemistry
Master
Master of Philosophy

Iroquois genes encode a family of highly conserved TALE homeodomain transcription factors that are involved in multiple developmental processes. Physiological tests indicated that Irx3 and Irx5 mutant mice displayed hearing impairment. However, the functions of these two genes during inner ear development are not known. The aim of this study is to characterize the roles of Irx3 and Irx5 during mammalian inner ear development using mouse models, in order to reveal the underlying mechanism for the hearing abnormality in the mutants. Two mouse mutants, Irx3tauLacZ and Irx3flox5EGFP with β-gal and EGFP reporters, were analyzed to examine the expression of these two genes in the otic vesicle and cochlear epithelium. In the otocyst, both Irx3 and Irx5 were expressed in the ventral-medial region. Irx5 expression was restricted to the non-sensory domain of the cochlear epithelia, while Irx3 was widely expressed, including the auditory sensory organ, the organ of Corti. The overlapping expression patterns of Irx3 and Irx5 suggest that they may share redundant functions. To investigate the roles of Irx3 and Irx5 during inner ear development, phenotypic analysis was performed on Irx3-/-, Irx5-/- and Irx3/5-/- mutant embryos. As shown by paint-filling analysis, Irx3/5-/- displayed shortened cochlear duct, enlarged cochlear lumen with fused sensory organ. Whole-mount phalloidin staining of hair cell bundles showed that Irx3-/- displayed occasional ectopic inner hair cells. Moreover, only supernumerary vestibular hair cell-like cells were developed in Irx3/5-/- mutant. These results suggest that Irx3 and Irx5 are required for inner ear morphogenesis and the formation of organ of Corti. To understand the effect of Irx3 and Irx5 in the cellular patterning of the cochlea, mutant cochleae were analyzed with markers for different regions of the cochlear epithelia. Altered expression domain of MyoVIIa, Sox2 and Gata2 in Irx3/5-/- cochlea revealed that the boundary between the Kolliker’s organ and the organ of Corti was lost and the location of sensory and non-sensory region was shifted. These results imply that Irx3 and Irx5 function in the establishment of the sensory/non-sensory boundary. It is known that p27kip1 regulates the wave of cell cycle exit in the developing organ of Corti and Sox2 takes part in prosensory specification. To explore the underlying reason for the patterning defects in Irx3/5-/- mutant, cochlear duct from prosensory stages were analyzed. Irx3/5-/- showed altered Sox2 and p27kip1 expression, with expanded prosensory domain and disrupted cell cycle exit. Ectopic prosensory proliferation was detected in the middle turn of the cochlear duct at E13.5 by BrdU incorporation assay. Therefore, Irx3 and Irx5 may participate in the subdivision of sensory territory in developing cochlea by controlling prosensory proliferation. In summary, this study demonstrates that Irx3 and Irx5 cooperate in multiple aspects of inner ear development: an early role to regulate prosensory proliferation and cell cycle exit; a second role to regulate cellular patterning of the cochlear duct by controlling the setting of sensory/non-sensory boundaries in the cochlea; a later role to regulate inner ear morphogenesis. This study supports the idea that Irx3 and Irx5 act as patterning genes during vertebrate evolution.
published_or_final_version
Biochemistry
Master
Master of Philosophy

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains vii, 114 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

A practical consideration in the development of cellular therapy technology for the inner ear is the development of an in vitro model for assessing the optimal conditions for successful application of cells. The first part of this thesis describes the adaptation of the cochleovestibular structure harvested from P1 mouse pups for analysis of factors critical for the optimal implantation of stem cells in the inner ear. Results of these studies establish that the c17.2 neural stem cell line can be introduced into the cochleovestibular structure in vitro. Using this model, c17.2 cells demonstrated survival predominantly within the vestibule and basal spiral ganglion regions. Furthermore, the addition of the ototoxin, cisplatin and the neurotrophin, Brain Derived Neurotrophic Growth Factor (BDNF) enhanced the survival and migration/dispersion of c17.2 cells within the cochleovestibular explant. The second part of this thesis examines the hypothesis that olfactory neurosphere (ONS) and progenitor cells harvested from the olfactory epithelium represent a viable source of graft material for potential therapeutic applications in the inner ear. Olfactory epithelium represents a unique source of pluripotent cells that may serve as either homografts or autografts. The feasibility of ONSs to survive and integrate into a mammalian cochlea in vivo was assessed. The ONSs were isolated as a crude fraction from the olfactory epithelium of P1 to P3 day old swiss webster mouse pups, ubiquitously expressing the Green Fluorescent Protein (GFP) marker. The ONSs were microinjected into the cochleae of adult CD1 male mice. Four weeks following their implantation, ONS cells expressing the GFP marker and stained by Nestin were identified in all areas of the cochlea and vestibule, including the spiral ganglion. Robust survival and growth of the implanted ONS and ONS derived cells in the cochlea also included the development of ???tumor-like??? clusters, a phenomenon not observed in control animals implanted with c17.2 neural stem cells. Collectively, the results of this thesis illustrate the potential of olfactory neurosphere and progenitor cells to survive in the inner ear and expose a potential harmful effect of their transplantation.

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.

Loss of sensory input from one vestibular labyrinth by unilateral vestibular deafferentation (UVD) results in a severe ocular motor (i.e., spontaneous nystagmus (SN)) and postural syndrome (i.e., yaw head tilt, (YHT) and roll head tilt (RHT)) which compensates over time in a behavioural recovery process known as vestibular compensation. It is generally accepted that the UVD-induced neuronal imbalance in the resting activity between the two vestibular nuclear complexes (VNCs) generates the ocular motor and postural syndrome and that the restoration of the resting activity in the ipsilateral VNC plays a causal role in the compensation of the static symptoms. γ-Aminobutyric acid (GABA) and the GABAA and GABAB receptors within the VNC are involved in normal vestibulo-ocular and --spinal pathways and it has been suggested that modification of GABAergic inhibition may be a mechanism responsible for the recovery of resting activity in the ipsilateral VNC. Behavioural, western blotting, and immunoassay techniques were used to address the role of the GABAA receptor in the VNC during vestibular compensation. The first study involved the characterization of SN, YHT, and RHT compensation in guinea pigs that had been anaesthetized with isoflurane during the UVD. These animals compensated rapidly (i.e., 30 hrs) and the time to compensate was independent of the duration of the anaesthesia. Using the 30 hrs time frame, the effects of the chronic infusion of the GABAA receptor agonist (muscimol) / antagonist (gabazine) into either the ipsilateral or the contralateral VNC on the compensation of SN, YHT, and RHT, were determined. Infusion of muscimol (250, 500, and 750 ng) into the contralateral VNC and gabazine (31.25, 62.5 and 125 ng) into the ipsilateral VNC significantly affected YHT and RHT (p < 0.05), but not their rate of compensation (p > 0.05). Interestingly, the effects of muscimol and gabazine on YHT and RHT were consistent throughout the first 30 hrs post-UVD. At 30 hrs post-UVD, the pumps were disconnected. In both experimental groups, the value and direction of the YHT and RHT returned to vehicle levels. Infusion of muscimol (62.5, 125, and 250 ng) into the ipsilateral VNC and gabazine (125, 375, and 750 ng) into the contralateral VNC had little effect on YHT and RHT, or their rate of compensation. At 30 hrs post-UVD, the pumps were disconnected. In both experimental groups, the value and direction of the YHT and RHT returned to vehicle levels. These results suggest that the ipsilateral gabazine and contralateral muscimol infusions were modifying the expression of the symptoms without altering the mechanism of compensation. Furthermore, the mechanism responsible for vestibular compensation can cope with the both the GABAA receptor-mediated and the UVD-induced decrease in resting activity. Results from the western blotting study indicated that compensation of SN, YHT, and RHT is not associated with changes in the protein levels of the GABAA receptor α₁, β₂, or γ₂ subunits. Compensation of SN, YHT, and RHT is associated with an elevation in cortisol salivary levels. Overall, the results suggest that the GABAA receptors are involved in the expression of YHT and RHT, but not in the mechanism that is responsible for their compensation.

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.

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

Bone morphogenetic proteins (BMPs) are diffusible molecules involved in a variety of cellular interactions during development. In particular, Bmp4 expression accompanies the development of the ear sensory organs during patterning and specification of sensory cell fates, and it has been shown to play a role in inner ear development and morphogenesis. However, there is no understanding of the cellular effects of BMP4 in prosensory progenitors, and about its role in the process of sensory fate specification. The present thesis project was aimed at exploring the effects of BMP-signaling on the development of hair-cells, using the chick inner ear as a model.
The specific aims proposed were:
1- Analyze the cellular effects caused by addition of BMP4 in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.
2- Analyze the cellular effects caused by inhibition of BMP4 signaling in a model of isolated chick otic vesicles in culture, measuring parameters of cell proliferation, cell death and sensory cell fate specification.
3- Analyze the expression in the innear ear of downstream targets of BMP signalling, in particular, analyse the members of Id gene family.
4- Analyze the regulation of Id genes by BMP signalling in the inner ear.
5- Analyze the expression of genes involved in the process of terminal differentiation, in particular, Btg1 and Btg2 genes
6- Analyze the regulation of Btg1 and Btg2 gene by BMP signalling in the inner ear

Thesis (Ph. D.)--West Virginia University, 2007.
Title from document title page. Document formatted into pages; contains xi, 82 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references.

A multitude of processes cooperate to produce the sensation of sound. The key initial step, the transformation from mechanical motion into an electrical signal, takes place in highly specialized mechanosensitive organelles that are called hair bundles due to their characteristic appearance. Each hair bundle comprises many apposed cylindrical stereocilia that are located in a liquid-filled compartment of the inner ear. The viscous liquid surrounding the hair bundle dissipates energy and dampens oscillations, which poses a fundamental physical challenge to the high sensitivity and sharp frequency selectivity of hearing. To understand the structure-function relationship in this complex system, a realistic physical model of the hair bundle with an appropriate representation of the fluid-structure interactions is needed to identify the relevant physical effects. In this work a novel approach is introduced to analyze the mechanics of the fluid-structure interaction problem in the inner ear. Because the motions during normal mechanotransduction are much smaller than the geometrical scales, a unified linear system of equations describes with sufficient accuracy the behavior of the liquid and solid in terms of a displacement variable. The finite-element method is employed to solve this system of partial differential equations. Based on data from the hair bundle of the bullfrog's sacculus, a detailed model is constructed that resolves simultaneously the interaction with the surrounding liquid as well as the coupling liquid in the narrow gaps between the individual stereocilia. The experimental data are from high-resolution interferometric measurements at physiologically relevant amplitudes in the range from a fraction of a nanometer to several tens of nanometers and over a broad range of frequencies from one millihertz to hundred kilohertz. Different modes of motion are analyzed and their induced viscous drag is calculated. The investigation reveals that grouping stereocilia in a bundle dramatically reduces the total drag as compared to the sum of the drags on individual stereocilia moving in isolation. The stereocilia in a hair bundle are interconnected by oblique tip links that transmit the energy in a sound to the mechanotransduction channels and by horizontal top connectors that provide elastic coupling between adjacent stereocilia. During hair-bundle deflections, the tip links induce additional drag by causing small but very dissipative relative motions between stereocilia; this effect is offset by the horizontal top connectors that restrain such relative movements, assuring that the hair bundle moves as a unit and keeping the total drag low. In the model the stiffness of the links, the stiffness of the stereocilia, and the geometry are carefully adjusted to match experimental observations. The references are stiffness and drag measurements, as well as the coherence measurements for the bundle's opposite edges, both with and without the tip links. The results are further validated by a comparison with the relative motions measured in a sinusoidally stimulated bundle for the distortion frequencies at which movements are induced by the nonlinearity imposed by channel gating. The model of the fluid-structure interactions described here provides insight into the key step in the perception of sound and the method presented provides an efficient and reliable approach to fluid-structure interaction problems at small amplitudes
Bei der Hörwahrnehmung eines Klangs spielen viele komplexe Prozesse zusammen. Der Schlüsselprozess, die Umwandlung mechanischer Schwingungsbewegung in elektrische Signale, findet in den Haarbündeln im Innenohr statt. Diese Haarbündel sind hoch entwickelte mechanosensitive Organellen, bestehend aus vielen nahe beieinander stehenden Stereozilien umgeben von Flüssigkeit. Die beträchtliche Viskosität dieser Flüssigkeit führt zur Energiedissipation und zur Schwingungsdämpfung, was im Gegensatz zur bekannten hohen Empfindlichkeit und der ausgezeichneten Frequenzselektivität der Hörwahrnehmung steht. Um die Komponenten des Haarbündelsystems in ihrem funktionalen Zusammenspiel besser zu verstehen, bedarf es eines wirklichkeitsgetreuen Modells unter Einbeziehung der Wechselwirkung zwischen Flüssigkeit und Struktur. Mit dieser Arbeit wird ein neuer Ansatz vorgestellt, um die Mechanik der Fluid-Struktur-Wechselwirkung im Innenohr zu analysieren. Da die Bewegungen bei der normalen Mechanotransduktion wesentlich kleiner als die geometrischen Abmessungen sind, ist es möglich, das Verhalten von Fluid und Struktur in Form der Verschiebungsvariable in einem linearen einheitlichen System von Gleichungen ausreichend genau zu beschreiben. Dieses System von partiellen Differentialgleichungen wird mit der Finite-Elemente-Methode gelöst. Basierend auf experimentell ermittelten Daten vom Haarbündel des Ochsenfrosches wird ein detailliertes Modell erstellt, welches sowohl die Interaktion mit der umgebenden Flüssigkeit als auch die koppelnde Flüssigkeit in den engen Spalten zwischen den einzelnen Stereozilien erfasst. Die experimentellen Daten sind Ergebnisse von hochauflösenden interferometrischen Messungen bei physiologisch relevanten Bewegungsamplituden im Bereich von unter einem Nanometer bis zu mehreren Dutzend Nanometern, sowie über einen breiten Frequenzbereich von einem Millihertz bis hundert Kilohertz. Das Modell erlaubt die Berechnung der auftretenden viskosen Widerstände aus der numerischen Analyse der verschiedenen beobachteten Bewegungsmoden. Es kann gezeigt werden, dass durch die Gruppierung zu einem Bündel der Gesamtwiderstand drastisch reduziert ist, im Vergleich zur Summe der Widerstände einzelner Stereozilien, die sich individuell und unabhängig voneinander bewegen. Die einzelnen Stereozilien in einem Haarbündel sind durch elastische Strukturen mechanisch miteinander verbunden: Die Energie des Schalls wird durch schräg angeordnete sogenannte Tiplinks auf die mechanotransduktiven Kanäle übertragen, wohingegen horizontale Querverbindungen die Stereozilien direkt koppeln. Während der Haarbündelauslenkung verursachen die Tiplinks zusätzlichen Widerstand durch stark dissipative Relativbewegungen zwischen den Stereozilien. Die horizontalen Querverbindungen unterdrücken diese Bewegungen und sind dafür verantwortlich, dass sich das Haarbündel als Einheit bewegt und der Gesamtwiderstand gering bleibt. Die Steifigkeit der Stereozilien und der Verbindungselemente sowie deren Geometrie sind in dem Modell sorgfältig angepasst, um eine Übereinstimmung mit den Beobachtungen aus verschiedenen Experimenten zu erzielen. Als Referenz dienen Steifigkeits- und Widerstandsmessungen, sowie Kohärenzmessungen für die gegenüberliegenden Außenkanten des Bündels, die jeweils mit und ohne Tiplinks durchgeführt wurden. Darüberhinaus sind die Ergebnisse durch den Vergleich mit experimentell beobachteten Relativbewegungen validiert, die das Haarbündel infolge von sinusförmiger Anregung bei Distorsionsfrequenzen zeigt. Diese haben ihren Ursprung in dem nichtlinearen Prozess des öffnens von Ionenkanälen. Das entwickelte Modell eines Haarbündels liefert neue Einblicke in den Schlüsselprozess der auditiven Wahrnehmung. Zur Behandlung von Problemen der Fluid-Struktur-Wechselwirkungen bei kleinen Amplituden hat sich der hier ausgearbeitete Ansatz als effizient und zuverlässig erwiesen

L’espèce humaine est caractérisée par une forme de locomotion bipède qui lui est propre. Toumaï, l’holotype de l’espèce Sahelanthropus tchadensis découverte au Tchad, est le plus ancien représentant connu du rameau humain. Daté de 7 millions d’années, il est très proche de la dichotomie entre ce rameau et celui des chimpanzés. C’est donc un témoin précieux des premières phases de l’évolution de la locomotion dans ce groupe. Le but de ce travail est de reconstituer le comportement locomoteur de Sahelanthropus tchadensis à partir de l’étude de la partie osseuse de son labyrinthe, organe de l’équilibre.Pour cela, les relations entre locomotion et morphologie du labyrinthe osseux sont recherchées chez les hominoïdes actuels. Les covariations entre le labyrinthe et le crâne sont étudiées chez Homo sapiens afin de déterminer l’influence sur la morphologie du labyrinthe de son intégration dans le crâne. Ces résultats sont enfin appliqués au labyrinthe de Toumaï, comparé à d’autres hominines fossiles.La morphométrie géométrique discrimine l’ensemble des espèces actuelles par l’orientation, la taille et la forme relatives de chacune des parties du labyrinthe. Cependant, la torsion et l’orientation relative des canaux semi-circulaires sont majoritairement liées à l’intégration du labyrinthe dans le crâne (orientation des pyramides pétreuses et de la face par rapport à la ligne médio-sagittale du basicrâne). Au contraire, l’excentricité des canaux et leur taille sont uniquement liées à la locomotion.Chez Sahelanthropus tchadensis, la taille des canaux semi-circulaires indique une espèce relativement plus agile que les grands singes. En revanche, il ne présente pas les rotations de canaux caractéristiques du genre Homo. La forme de ses canaux constitue une combinaison unique de caractères : canal postérieur rond et canal antérieur légèrement étiré, comme les humains et les australopithèques, mais canal latéral légèrement projeté postéro-latéralement, comme les gorilles et les babouins. Ceci indiquerait un comportement locomoteur associant le grimper vertical dans les arbres à des déplacements au sol bipèdes mais aussi quadrupèdes
Humans are characterised by a specific form of bipedal locomotion. Toumaï, holotype specimen of Sahelanthropus tchadensis discovered in Chad, is the oldest known member of the human branch. Dated from 7 million years ago, it is very close to the dichotomy between this branch and the branch of chimpanzees. Therefore, it gives valuable information on the first stages of the evolution of locomotion is this group. The purpose of this work is to reconstruct the locomotor behaviour of Sahelanthropus tchadensis based on the study of the osseous part of its labyrinth, the balance organ.To this aim, the relationships between the locomotion and the bony labyrinth morphology are investigated in extent hominoids. The covariations between the labyrinth and the skull are studied in Homo sapiens in order to determine the influence of cranial integration on labyrinth morphology. Finally, these results are applied to the bony labyrinth of Toumaï, compared to other hominin fossils.Geometric morphometrics discriminates all extant species by the relative orientation, size and shape of each part of the labyrinth. However, the torsion and the relative orientation of the semicircular canals are mainly due to the integration of the labyrinth in the skull (petrous pyramids and face orientations relative to the midline of the basicranium). On the contrary, canal eccentricity and size are linked only to locomotion.In Sahelanthropus tchadensis, the canal size indicates that this species was more agile than great apes. However, it does not have the semicircular canal rotations characterising the genus Homo. The shape of its canals constitutes a unique combination of features: a round posterior canal and a slightly stretched anterior canal, as in humans and australopithecines, but a slightly posterolaterally projected lateral canal, as in gorillas and baboons. This is the sign of a locomotor behaviour associating vertical climbing in trees to both bipedalism and quadrupedalism on the ground

The inner ear, the sensory organ responsible for hearing and balance, contains specialized sensory and non-sensory epithelia arranged in a highly complex threedimensional structure. To achieve this complexity, a tight coordination between morphogenesis and cell fate specification is essential during otic development. Tisúes surrounding the otic primordium, and more particularly the adjacent segmented hindbrain, have been implicated in specifying structures along the anteroposterior and dorsoventral axes of the inner ear. In this work we have first characterized the generation and axial specification of the otic neurogenic domain, and second, we have investigated the effects of the mutation of kreisler/MafB -a gene transiently expressed in the rhombomeres 5 and 6 of the developing hindbrain- in early otic patterning and cell specification. We show that kr/kr embryos display an expansion of the otic neurogenic domain, due to defects in otic patterning. Although many reports have pointed to the role of FGF3 in otic regionalization, we provide evidence that FGF3 is not sufficient to govern this process. Neither Krox20 nor Fgf3 null mutant embryos, in which Fgf3 is either downregulated or absent in r5 and r6, present ectopic otic neuroblasts in the otic primordium. However, Fgf3-/-Fgf10-/- double mutants show a phenotype very similar to kr/kr embryos: they present ectopic neuroblasts along the AP and DV otic axes. Finally, and remarkably, partial rescue of the kr/kr phenotype is obtained when Fgf3 or Fgf10 are ectopically expressed in the hindbrain of kr/kr embryos. These results highlight a compensatory mechanism between FGFs, and the importance of hindbrain-derived signals in instructing otic patterning and the establishment of the neurogenic domain.

La mise en évidence par la biologie moléculaire et par les données paléontologiques de l'appartenance des cétacés au groupe des artiodactyles constitue une des avancées majeures de ces 30 dernières années en mammalogie. Il n'y a cependant pas à l'heure actuelle de consensus quant aux relations phylogénétiques basales des artiodactyles fondées sur des caractères morphologiques et l'histoire évolutive du groupe est de fait, ponctuée de nombreux points d'interrogation. Cette thèse explore une source de caractères phylogénétiques prometteuse : la région auditive (os pétreux, bulle auditive, osselets de l'oreille moyenne, oreille interne) à partir notamment des nouvelles perspectives offertes par l'imagerie µCT Scan. Les principaux objectifs de cette thèse sont (1) de déterminer le signal phylogénétique porté par la région auditive chez les artiodactyles afin d’apporter une nouvelle source de caractères aux analyses et (2) d’explorer le signal écologique porté par les différents éléments de cette région sensorielle dédiée à l’audition (oreille externe, moyenne et canal cochléaire du labyrinthe osseux) et à l’équilibrioception (vestibule et canaux semi-circulaires du labyrinthe osseux). La première partie de cette thèse (I) nous emmène au Togo, où de nombreux restes inédits de la région auditive de « baleines à pattes » (Protocetidae Stromer, 1908) ont été récoltés. D’un point de vu anatomique, ces restes fossiles ont permis de documenter et de décrire pour la première fois le stapes, l’incus et le labyrinthe osseux d’un protocète ; des éléments indispensables pour comprendre leur audition. L’analyse morpho-fonctionnelle indique qu’une audition optimale était probablement possible dans l’air et dans l’eau pour ces cétacés semi-aquatiques. De plus, la morphologie de leur cochlée indique que leur capacité auditive était proche de celle de leurs cousins terrestres et que les spécialisations relatives aux capacités auditives remarquables des cétacés modernes (i.e. sensibilité aux infra- ou ultrasons) se sont opérées après la séparation historique entre les mysticètes et les odontocètes.La deuxième partie de ce travail (II) se concentre sur les origines de l’amphibiose au sein des Cetancodonta, à travers l’étude de plusieurs familles fossiles, connues pour leurs liens étroits au milieu aquatique. L’étude de la région auditive des hippopotamoïdes (Anthracotheriidae + Hippopotamidae), révèle que l’adaptation à un mode de vie semi-aquatique est apparue plusieurs fois, de façon convergente, dans son histoire évolutive et semble d’ailleurs indiquer une origine terrestre pour ce groupe. Quant au raoellidé Indohyus, son complexe pétro-tympanique présente une combinaison de caractères suggérant un certain degré d’adaptation au milieu aquatique, mais l’étude fonctionnelle de sa cochlée indique que ce taxon ne pouvait très probablement pas entendre de façon efficace sous l’eau. Pour finir, le dernier point de cette thèse explore également le potentiel phylogénétique de la région auditive à travers une analyse construite sur des caractères morphologiques du pétreux et du labyrinthe osseux à l’échelle des artiodactyles. Pour la première fois, les résultats de notre analyse concordent avec ceux des analyses moléculaires. Parmi les points les plus notables, le clade des Cetancodonta est bien soutenu par la morphologie du pétreux et la position d’Indohyus suggère fortement que les raoellidés sont des cétacés.Ainsi, la région auditive s’avère être un élément essentiel d’un point de vu phylogénétique et morphofonctionnel. En effet, comme nous avons pu le voir tout au long de cette thèse, lorsque la nature complexe et variée de la région auditive est appréhendée dans son ensemble, elle permet d’inférer l’écologie d’un taxon donné et d’en apprendre davantage sur ses relations de parenté. Par conséquent, la région auditive est encore loin d’avoir dit ses derniers mots... et nous n’avons pas encore fini d’en entendre parler
The discovery by both molecular biology and palaeontological data that cetaceans are artiodactyls constitutes one of the major breakthroughs in mammal’s evolutionary history of the past 30 years. However, no consensus has yet been reached regarding the basal relationship within the enlarged Artiodactyla clade and major questions of its evolutionary history remain to be solved. This thesis explores a promising source of phylogenetic characters: the auditory region (petrosal bone, tympanic bulla, middle ear ossicles, inner ear) from the new perspectives offered by µCT Scan imaging.The main objectives of this thesis are (1) to determine the phylogenetic signal carried by the auditory region in artiodactyls in order to provide a new source of characters to the analyses and (2) to explore the ecological signal carried by the different elements of this sensory region dedicated to hearing (outer ear, middle ear and cochlear canal of the bonny labyrinth) and to equilibrioception (vestibule and semicircular canals of the bony labyrinth).The first part of this thesis (I) brings us to Togo, where many fossil remains of the auditory region of ancient “legged whales” (Protocetida Stromer 1908) have been collected. From an anatomical viewpoint, these fossil remains document a nearly complete petrotympanic complex and allowed us to describe for the first time, the stapes, incus and bony labyrinth of a protocetid whale, which are crucial elements to understand their hearing. Morphofunctional analysis indicates that optimal hearing was probably possible both in air and underwater for these semi-aquatic whales. In addition, the morphology of their cochlea indicates that their hearing ability was close to that of their terrestrial kin and that the specializations related to the remarkable hearing abilities of modern cetaceans (i.e. sensitivity to infra- or ultrasound) occurred after the historical separation between mysticetes and odontocetes.The second part of this work (II) focuses on the origins of amphibiosis in Cetancodonta, through the study of several fossil families, known for their potamophilous tendencies. The study of the auditory region of hippopotamoids (Anthracotheriidae + Hippopotamidae) reveals that adaptation to a semi-aquatic lifestyle has emerged several times (i.e. in a convergent way) in its evolutionary history and seems to indicate a terrestrial origin for this group. As for the raoellid Indohyus, its petro-tympanic complex presents a combination of features suggesting some degree of adaptation to the aquatic environment, but the functional study of its cochlea indicates that this taxon probably could not hear efficiently underwater.The last point of this thesis explores the phylogenetic potential of the auditory region through an analysis built upon morphological characters of the petrosal and bony labyrinth at Artiodactyla scale. For the first time, the results of our analysis are consistent with that of molecular analyses. Among the most notable points, the Cetancodonta clade is well supported by the morphology of the petrosal and Indohyus’ position strongly suggests that raoellids are cetaceans.Thus, the auditory region turns out to be an essential element from a phylogenetic and morphofunctional viewpoint. Indeed, as we have seen throughout this thesis, when the complex and multifaceted nature of the auditory region is apprehended as a whole, it allows to infer the ecology of a given taxon and to clarify its phylogenetic relationships. Thus, the auditory region is still far from having said its last words… and we are not done hearing about it yet

This thesis cover two separate topics related to the function of the mammalian inner ear. Acoustic sensitivity of the vestibular system. Data are presented showing facilitation of the auditory startle response by tones outside the range of the mouse cochlea. The sensation of these low frequency tones is demonstrated to be mediated via the acoustically sensitive sacculus of the vestibular system by data collected from Nox3-/- mice. These mice lack the otoconia of the vestibular system and, unlike the wild-type mice, only show facilitation to tones within the range of the mouse cochlea, and not in response to tones <4 kHz. The mechanical properties of the tectorial membrane (TM). The mechanical properties of the TM are investigated using a laser interferometer-based method for tracking the longitudinal propagation of a radially shearing travelling wave in segments of TM isolated from the basal and apical regions of the wild-type cochlea. The properties of these travelling waves (wave propagation velocity and wave amplitude decay) are tracked over a range of stimulus frequencies (1-20 kHz). The viscoelastic properties, shear storage modulus (G') and shear viscosity (η), are estimated over this frequency range and are found to be lower in the apical TM segments compared to the basal TM segments, indicating the apical region of the TM is less stiff than the basal region. These data are compared to data collected from TM segments isolated from the basal cochlear region of three mutant groups, each lacking expression of TM-specific proteins; α-tectorin (TectaY1870C/+), β-tectorin (Tectb-/-) and otoancorin (OtoaEGFP/EGFP), using the same laser interferometer-based method. The viscoelastic properties are estimated for each of the mutants and indicate varying degrees of loss of structural integrity in their respective TM segments. Reflective difference between the wild-types and mutants are also observed and compared.

Alpha- and beta-tectorin (Tecta and Tectb) are major non-collagenous components of the tectorial membrane (TM). The presence of a zona pellucida (ZP) domain in both tectorins suggests that Tecta and Tectb can form hetero- or homopolymers. It is unclear, however, how these proteins assemble to form the TM matrix. The mechanisms of apical targeting, secretion and processing of the tectorins are also unexplored. I used fluorescently-tagged tectorin constructs for stable transfection into polarised epithelial MDCK cells or transient expression in mouse cochlear cultures to develop an in vitro model of TM matrix assembly. Significant amounts of matrix were not observed with stable tectorin expression in monolayer cultures of MDCK cells. In contrast, I observed substantial amounts of dense extracellular matrix on the apical surfaces of outgrowth zone cells when cochlear cultures were transiently transfected with either Tecta or Tectb. When ectopically expressed in hair cells, Tecta and Tectb locate to the distal tips of the hair bundle. To study the role of the inner-ear protein Ceacam16 in hearing, we generated a Ceacam16 functional null mouse model. The Ceacam16 gene was inactivated by targeted replacement of exons 2-5 with the bacterial lacZ gene. β-gal staining I performed reveals that Ceacam16 is expressed in the epithelial cells of the spiral limbus and inner sulcus, and in both the pillar cells and Deiter's cells. I first detected the presence of Ceacam16 in the TM at P12, four days before the defined striated-sheet matrix is observed. Transmission electron microscopy reveals a complete loss of striated-sheet matrix in Ceacam16 null mice in comparison to the wild-type. The results of this thesis suggest neonatal mouse cochlear cultures as a model for studying tectorin-based extracellular matrix production and also reveal that Ceacam16 is required for normal formation and/or maintenance of striated-sheet matrix.

La radiation adaptative des Equoidea est encore mal comprise en raison notamment de la méconnaissance de la phylogénie de cette super-famille. La principale irrésolution de ces relations de parenté porte sur les pachynolophes, Equoidea européens rapprochés des Equidae ou des Palaeotheriidae. Pendant une grande part de l'Éocène, l'Europe est isolée et subit à la fin de cette période de profonds changements climatiques. Lors de la Grande Coupure son isolement s'achève, tandis que l'aridité du climat s'installe, et des faunes migrantes provoquent l'extinction de nombreux groupes endémiques. Un Equoidea européen basal, richement représenté par un matériel bien préservé permet d'appuyer une des hypothèses phylogénétiques les plus récentes. Cependant, les caractères couramment utilisés pour débattre de cette question n'apportent pas de réponse claire et définitive. Aussi, cette étude se propose de mener des investigations sur des régions encore peu explorées de ces animaux comme le neurocrâne, grâce à la microtomographie (CT scan), qui permet un accès non destructif aux structures (encéphale, pétreux, labyrinthe osseux, sinus). Outre l'intérêt phylogénétique, ces organes peuvent, de par leurs fonctions, receler un intérêt paléoécologique. Jusqu'à présent, peu d'études à large échelle ont porté sur ces structures chez les Perissodactyla, s'agissant pour la plupart de mentions anecdotiques. Comme préalable indispensable, une étude modèle a été réalisée sur un Equidae sauvage actuel afin de mieux appréhender la variabilité de ces structures méconnues. Pour la première fois, un large échantillon d'Equoidea européens a été scanné et leurs structures neurocrâniennes reconstruites en trois dimensions virtuelles. Ce sont au total 20 espèces qui ont été échantillonnées, couvrant l'évolution de ces animaux de leur origine à leur extinction, pendant plus de 20 millions d'années. Leurs crânes ont été scannés, leurs structures internes reconstruites, comparées et analysées au moyen de la cladistique. Une nouvelle hypothèse phylogénétique propose des relations de parenté intra-Equoidea et montre la pertinence des caractères neurocrâniens, tout en conduisant à envisager une étude plus vaste. Les Palaeotheriidae apparaissent comme un groupe très diversifié, notamment au regard des Equidae éocènes d'Amérique du Nord, et caractérisé par une évolution en mosaïque. Ils connaissent une évolution cérébrale précoce par rapport aux faunes contemporaines (Equidae, Cetartiodactyla, Carnivora), ce qui, via le développement de nouvelles stratégies adaptatives, pourrait expliquer partiellement cette grande diversification familiale. Un parallèle est envisagé avec l'évolution endémique des Notoungulata, qui semblent eux aussi montrer une complexification cérébrale précoce. Cependant, face à un environnement biotique et abiotique bouleversé (fin de l'Éocène et lors de la Grande Coupure), ces structures complexes impliquant un coût métabolique important et une trop grande spécialisation, avec en conséquence, moins de potentiel adaptatif, auraient pu les désavantager et les conduire à l'extinction
The Equoidea adaptive radiation still remains badly known, especially due to the ignorance of their phylogeny. The main indecision of these relationships concerns the pachynolophs, European Equoidea either approached to the Equidae or to the Palaeotheriidae. During a great part of the Eocene times, Europe was isolated, and, at the end of this period, has undergone strong climatic changes. That isolation ended at the « Grande Coupure » event, whereas an arid climate moved, and migrant faunas caused the extinction of many endemic groups. A basal European Equoidea, richly represented by well-preserved material, can support one of the latest phylogenetic hypotheses. However, commonly used characters to discuss this issue do not provide a clear and definitive answer.Therefore, this study aims to investigate on unexplored regions of these animals as the neurocranium through microtomography (CT), which allows access to non-destructive structures (brain, petrosal, bony labyrinth, and sinus).Furthermore phylogenetic interest these bodies may, through their functions, harbor paleoecological interest. Until now, few large-scale studies have focused on those structures in the Perissodactyla, with regard to most were anecdotal reports. As a prerequisite, a model study was performed on a wild current Equidae to better understand the variability of these unknown structures. For the first time, a large sample of European Equoidea has been scanned and their neurocranium structures virtually reconstructed in three-dimensions. A total of 20 species were sampled, covering the evolution of these animals from their origin to their extinction, for over 20 million years. Their skulls were scanned; their internal structures reconstructed compared and analyzed using cladistics. A new phylogenetic hypothesis provides intra Equoidea relationships and shows the relevance of neurocranium characters, while driving to consider a larger study. The Palaeotheriidae appears as a highly diverse group, particularly with regard to Eocene Equidae in North America, and characterized by a mosaic evolution. Their brain evolved earlier than that of contemporary faunas (Equidae, Cetartiodactyla, Carnivora); which may partially explain the strong diversification of that family, through the development of new adaptive strategies

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.

The morphological diversity of the external and internal surfaces of the petrosal bone, which contains the structures of the inner ear, across a broad range of therian mammals is documented, and patterns of variation across taxa are identified. One pattern of variation is the result of ontogenetic changes in the ear region, as described for the external surface morphology of a sample of isolated petrosal bones referred to Proboscidea from Pleistocene deposits in central Texas. The morphology of the aquaeductus Fallopii for passage of the greater petrosal branch of the facial nerve supports an ontogenetic explanation for some variation within the proboscidean sample, and a sequence of ossification surrounding the aquaeductus Fallopii is hypothesized. Further ontogenetic patterns are investigated using digital endocasts of the bony labyrinth (preserved on the internal surfaces of the petrosal) constructed from CT data across a growth series of the opossum Monodelphis domestica. Strong correlation between skull length and age is found, but from 27 days after birth onward, there is no correlation with age among most dimensions of the inner ear. Adult dimensions of several of the inner ear structures are achieved before the inner ear is functional in M. domestica. Morphological variation within the inner ear of several eutherian mammals from the Cretaceous of Asia, including zhelestids from the Bissekty Formation of Uzbekistan, is described. The variation within the fossil sample is compared to that observed within extant species of placental mammals, and it is determined that the amount of variation within the Bissekty zhelestid population is within the range of that measured for extant species. Additional evolutionary and physiological patterns preserved within the walls of the bony labyrinth are identified through a high level anatomical comparison of the inner ear cavities across Placentalia as a whole. In particular, features of the inner ear support monophyly of Cetacea, Carnivora, Primatomorpha, and caviomorph Rodentia. The volumetric percentage of the vestibular apparatus (vestibule plus semicircular canals) of aquatic mammals is smaller than that calculated for terrestrial relatives of comparable body size. Thus, aspects of the bony labyrinth are both phylogenetically and physiologically informative.
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