Etude du développement de la cochlée dans une perspective de régénération neurosensorielle

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 d’origine neurosensorielle ainsi que la mise au point de stratégies visant à restaurer la structure cellulaire de l’oreille 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 l’organogenèse 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 l’oreille interne, en l’absence 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 n’apparaît pas comme un facteur indispensable à l’induction 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 l’oreille interne. Cependant, à des stades ultérieurs du développement, la structure parfaitement conservée de l’organe de Corti nous laisse penser que l’action de Sox10 est compensée par d’autres gènes du groupe SoxE, par exemple Sox9, dont nous avons mis en évidence l’expression dans les cellules de soutien de l’organe de Corti (Cook et al., 2005;Sock et al., 2001;Stolt et al., 2004;Wegner, 1999).Par ailleurs, nous avons montré qu’au cours du développement de l’organe de Corti, la première cellule identifiable était la cellule pilier interne. Cette cellule échapperait au système d’inhibition latérale lié à Notch et pourrait avoir un rôle dans la différenciation des autres cellules de l’organe 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 l’origine 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. C’est dans ce modèle d’explants 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, l’organe de Corti (Lefebvre et al., 1992) et que cette dépendance persiste à l’âge adulte. Ce modèle de culture permet de conserver l’architecture du ganglion spiral, se rapprochant ainsi de la situation observée in vivo. Laissés seuls, en l’absence de facteurs trophiques exogènes, le nombre de neurones auditifs chute drastiquement après 24 heures de culture. L’organe de Corti, source de neurotrophines tant durant la période développementale qu’à l’âge adulte (Oestreicher et al., 2000;Ylikoski et al., 1993), prévient significativement cette mort neuronale lorsqu’il reste associé au ganglion spiral à la mise en culture. Dans ce modèle de dégénérescence rétrograde, nous avons étudié l’expression de la périphérine et constaté que cette protéine était réexprimée dans les neurones auditifs de type I après lésion. De plus, l’étude de l’expression de la périphérine dans le ganglion spiral du rat a permis d’observer qu’elle 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 d’intervenir à la fois lors du développement embryonnaire et réactivée lors des phénomènes de lésions tissulaires cochléaires

Sox9 Inhibits Cochlear Hair Cell Fate by Upregulating Hey1 and HeyL Antagonists of Atoh1.

peer reviewedIt is widely accepted that cell fate determination in the cochlea is tightly controlled by different transcription factors (TFs) that remain to be fully defined. Here, we show that Sox9, initially expressed in the entire sensory epithelium of the cochlea, progressively disappears from differentiating hair cells (HCs) and is finally restricted to supporting cells (SCs). By performing ex vivo electroporation of E13.5-E14.5 cochleae, we demonstrate that maintenance of Sox9 expression in the progenitors committed to HC fate blocks their differentiation, even if co-expressed with Atoh1, a transcription factor necessary and sufficient to form HC. Sox9 inhibits Atoh1 transcriptional activity by upregulating Hey1 and HeyL antagonists, and genetic ablation of these genes induces extra HCs along the cochlea. Although Sox9 suppression from sensory progenitors ex vivo leads to a modest increase in the number of HCs, it is not sufficient in vivo to induce supernumerary HC production in an inducible Sox9 knockout model. Taken together, these data show that Sox9 is downregulated from nascent HCs to allow the unfolding of their differentiation program. This may be critical for future strategies to promote fully mature HC formation in regeneration approaches

Hair cell progenitors: identification and regulatory genes

Hair cell loss in higher vertebrates appears to be permanent. Progenitors that are quiescent in the organ of Corti are the best candidates for the restoration of the different cell types in the organ of Corti. However, little is known about the presence of these progenitors and their capacity to differentiate into hair cells. This review will first highlight recent findings concerning the identification of progenitor cells that are able to proliferate and to differentiate into hair cells. Principal factors impinging on this process are then reviewed. Auditory hair cell progenitors have been identified and, under appropriate conditions, are capable of proliferating and differentiating into hair cells. Characterization of signals that maintain, expand and regulate these progenitors will be essential for the biomedical application of stem cell populations to restore hearing

Early identification of inner pillar cells during rat cochlear development.

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Here, we examine its spatio-temporal development in rats from embryonic day 16 (E16) to E19 by using cytochemical and immunocytochemical methods at the light- and electron-microscope levels. We demonstrate that the organ of Corti develops from a non-proliferating cell zone that is located in the junctional region between two edges of the dorsal epithelium of the cochlear duct. We also reveal that the first cells to develop in this zone are the inner pillar cells, a particular type of non-sensory supporting cell, which arise in the base of the cochlear duct at the boundary between the two ridges at E16. Cell differentiation in this prosensory region continues according to a base-to-apex gradient; the inner hair cells appear in the greater epithelial ridge at E17 and the outer hair cells in the lesser epithelial ridge at E18. At E19, the various cell types of the organ of Corti are in place. Finally, we show that unlike the development of all the supporting cell types of the organ of Corti, the development of inner pillar cells within the prosensory domain seems not to involve Notch1 activation. These results highlight the central role that the inner pillar cells probably play in the development of the organ of Corti

Autocrine/paracrine activation of the GABA(A) receptor inhibits the proliferation of neurogenic polysialylated neural cell adhesion molecule-positive (PSA-NCAM+) precursor cells from postnatal striatum.

GABA and its type A receptor (GABA(A)R) are present in the immature CNS and may function as growth-regulatory signals during the development of embryonic neural precursor cells. In the present study, on the basis of their isopycnic properties in a buoyant density gradient, we developed an isolation procedure that allowed us to purify proliferative neural precursor cells from early postnatal rat striatum, which expressed the polysialylated form of the neural cell adhesion molecule (PSA-NCAM). These postnatal striatal PSA-NCAM+ cells were shown to proliferate in the presence of epidermal growth factor (EGF) and formed spheres that preferentially generated neurons in vitro. We demonstrated that PSA-NCAM+ neuronal precursors from postnatal striatum expressed GABA(A)R subunits in vitro and in situ. GABA elicited chloride currents in PSA-NCAM+ cells by activation of functional GABA(A)R that displayed a typical pharmacological profile. GABA(A)R activation in PSA-NCAM+ cells triggered a complex intracellular signaling combining a tonic inhibition of the mitogen-activated protein kinase cascade and an increase of intracellular calcium concentration by opening of voltage-gated calcium channels. We observed that the activation of GABA(A)R in PSA-NCAM+ neuronal precursors from postnatal striatum inhibited cell cycle progression both in neurospheres and in organotypic slices. Furthermore, postnatal PSA-NCAM+ striatal cells synthesized and released GABA, thus creating an autocrine/paracrine mechanism that controls their proliferation. We showed that EGF modulated this autocrine/paracrine loop by decreasing GABA production in PSA-NCAM+ cells. This demonstration of GABA synthesis and GABA(A)R function in striatal PSA-NCAM+ cells may shed new light on the understanding of key extrinsic cues that regulate the developmental potential of postnatal neuronal precursors in the CNS

New insights into peripherin expression in cochlear neurons

Peripherin is an intermediate filament protein that is expressed in peripheral and enteric neurons. In the cochlear nervous system, peripherin expression has been extensively used as a differentiation marker by preferentially labeling the type II neuronal population at adulthood, but yet without knowing its function. Since the expression of peripherin has been associated in time with the process of axonal extension and during regeneration of nerve fibers in other systems, it was of interest to determine whether peripherin expression in cochlear neurons was a static phenotypic trait or rather prone to modifications following nerve injury. In the present study, we first compared the expression pattern of peripherin and beta III-tubulin from late embryonic stages to the adult in rat cochlea. The staining for both proteins was seen before birth within all cochlear neurons. By birth, and for 2 or 3 days, peripherin expression was gradually restricted to the type II neuronal population and their projections. In contrast, from postnatal day (P) 10 onwards, while the expression of beta III-tubulin was still found in projections of all cochlear neurons, only the type I population had beta III-tubulin immunoreactivity in their cell bodies. We next investigated the expression of peripherin in axotomized cochlear neurons using an organotypic explant model. Peripherin expression was surprisingly re-expressed in a vast majority of neurons after axotomy. In parallel, the expression and localization of beta III-tubulin and peripherin in dissociated cultures of cochlear neurons were studied. Both proteins were distributed along the entire neuronal length but exhibited complementary distribution, especially within the projections. Moreover, peripherin immunoreactivity was still abundant in the growth cone, whereas that of beta III-tubulin was decreasing at this compartment. Our findings are consistent with a model in which peripherin plays an important structural role in cochlear neurons and their projections during both development and regenerative processes and which is compatible with the assumption that frequently developmentally regulated factors are reactivated during neuronal regeneration

Strategies to Regenerate Hair Cells: Identification of Progenitors and Critical Genes

Deafness commonly results from a lesion of the sensory cells and/or of the neurons of the auditory part of the inner ear. There are currently no treatments designed to halt or reverse the progression of hearing loss. A key goal in developing therapy for sensorineural deafness is the identification of strategies to replace lost hair cells. In amphibians and birds, a spontaneous post-injury regeneration of all inner ear sensory hair cells occurs. In contrast, in the mammalian cochlea, hair cells are only produced during embryogenesis. Many studies have been carried out in order to demonstrate the persistence of endogenous progenitors. The present review is first focused on the occurrence of spontaneous supernumerary hair cells and on nestin positive precursors found in the organ of Corti. A second approach to regenerating hair cells would be to find genes essential for their differentiation. This review will also focus on critical genes for embryonic hair cell formation such as the cell cycle related proteins, the Atoh1 gene and the Notch signaling pathway. Understanding mechanisms that underlie hair cell production is an essential prerequisite to defining therapeutic strategies to regenerate hair cells in the mature inner ear

Supernumerary outer hair cells arise external to the last row of sensory cells in the organ of corti.

During the development of the mammalian inner ear, the number of hair cells produced is highly regulated and remains constant throughout life. The mechanism underlying this regulation is beginning to be understood although many aspects still remain obscure. When late embryonic or early postnatal rat organs of Corti were cultured, the production of supernumerary hair cells was observed. This overproduction of sensory cells could be modulated by the addition of several growth factors. In this study, we examined explants of rat organs of Corti that produced supernumerary hair cells. In the supernumerary hair cell region, up to two rows of inner hair cells and five rows of outer hair cells were observed. Morphological evaluation of these specimens revealed that less mature hair cells were located in the most external rows of these sensory cells. When a supernumerary hair cell was produced, a supporting cell (i.e. Deiters' cell) was also produced, strongly suggesting that the conversion of a Deiters' cell into a hair cell was not the mechanism that produced these extra hair cells. Based on these results, we propose that prosensory cells located at the external edge of the organ of Corti retain a capacity to form hair cells and that it is these prosensory cells that differentiate into supernumerary hair cells and Deiters' cells