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See detailDistribution of glycogen during the development of the organ of Corti
Thelen, Nicolas ULg; Cloes, Marie ULg; Johnen, Nicolas ULg et al

Poster (2011, January 31)

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Using cytochemical methods at the light and ... [more ▼]

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Using cytochemical methods at the light and electron microscope levels, we examined the spatiotemporal distribution of polysaccharides during the development of the organ of Corti in rats from embryonic day 16 (E16) to postnatal day 15 (P15). At E16, small polysaccharide inclusions were detected in the cytoplasm of the future inner pillar cells by electron microscope only. These inclusions became obvious at the light microscope level at E17. At E19, the polysaccharide deposits were important within the inner pillar cells and they arose in the Hensen cells cytoplasm. Polysaccharide accumulations also appeared in the outer pillar cells and the Deiters cells from P3-P4. As the organ of Corti developed, the amount of polysaccharide inclusions within the inner and outer pillar cells decreased. At P15, large amount of polysaccharide deposits were visible in the Deiters cells whereas they had almost disappeared from the inner and outer pillar cells. Finally, we showed that the polysaccharide deposits present in the developing organ of Corti are PAS-positive and can be digested with a salivary amylase, suggesting that they are essentially constituted of glycogen. [less ▲]

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See detailSpatio-temporal localisation of β-actin and γ-actin isoforms during the development of the organ of Corti in rat from the embryonic day 18 (E18) to the post-natal day 25 (P25).
Johnen, Nicolas ULg; Thelen, Nicolas ULg; Cloes, Marie ULg et al

Poster (2011, January 31)

The auditory organ, the organ of Corti (OC), is a highly specialized structure composed by specific cellular types. The sensory cells (HC) are characterized by stereocilia at their apex and are necessary ... [more ▼]

The auditory organ, the organ of Corti (OC), is a highly specialized structure composed by specific cellular types. The sensory cells (HC) are characterized by stereocilia at their apex and are necessary for the sound perception. Theses cells are supported by supporting cells. Based on their morphology and physiology, at least four types of supporting cells can be identified in the OC: inner and outer pillar cells (PC), phalangeal cell and Deiter’s cells. Sensory and supporting cells have cytoskeletons containing β-actin and γ-actin isoforms. In the adult mammalian cochlea, amounts of γ-actin increase and β-actin decrease in the order: outer pillar cells, inner pillar cells, Deiters’ cells and hair cells. In sensory cells, γ-actin appears to be the most prominent component with an apparent γ-actin/β-actin ratio of approximately 2:1 (Hofer et al., 1997). β-actin is present in the cuticular plate but is more concentrated in the stereocilia, especially at the base where the stereocilia insert into the cuticular plate. The amount of γ-actin differs less between these structures, the stereocilia and cuticular plate, although its expression is apparently higher towards the tip of stereocilia and it is the predominant isoform of the hair cell's lateral wall (Furness et al., 2005). The differential subcellular localization of two actin isoforms suggests they may play different functions in auditory organ. In the brain, β-actin is restricted to dynamic structures whereas γ-actin is more ubiquitously distributed and occurs in relatively quiescent regions (Micheva et al., 1998). In the present study, by using confocal microscopy, we investigated the spatio-temporal localisation of β-actin and γ-actin isoforms during the development of the OC in rat from the embryonic day 18 (E18) to the post-natal day 25 (P25). Our results indicated that the labelling for both actin isoforms changed during the OC development. Between E18 and P25, we observed a labelling for β-actin in the apical region of the HC. Between P8 and P25, the feet of PC are also β-actin-positive. Unlike β-actin, between E18 and P10, γ-actin is detected in the basal region of supporting cells. Between P12 and P25, the labelling for γ-actin is preferentially localized in the apical surface of the HC. Our results revealed that during development β-actin isoform preceded γ-actin isoform in the apical region of HC. They also suggest that γ-actin isoform might be involved in attachment of supporting cells with their basal membrane and that β-actin isoform might play a role in PC reorganization during the formation of Corti tunnel. [less ▲]

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See detailSPATIO-TEMPORAL LOCALIZATION OF BETA TUBULIN III IN THE ORGAN OF CORTI AND IN THE SPIRAL GANGLIA BETWEEN THE EMBRYONIC DAY (E18) AND THE POST-NATAL DAY (P25) IN RAT.
Johnen, Nicolas ULg; Thelen, Nicolas ULg; Cloes, Marie ULg et al

Poster (2010, October 22)

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, at least two types of ... [more ▼]

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, at least two types of sensory cells can be identified in the OC: inner and outer hair cells. The structure of this organ is well reported in adult but its development is still little-known. By using confocal microscopy, we studied the spatial-temporal distribution of beta tubulin III during the differentiation of the OC in rat from the embryonic day 18 (E18) to the postnatal day (P25). The beta tubulin III is typical for neural cells in the OC. We observed that beta III tubulin is present in the extensions innerving the row of inner hair cells at E18. At E19, the extensions innerving the inner hair cells and the two first rows of outer hair cells were immunolabelled. From E21 to P25, all of hair cells were connected to the spiral ganglion. In the latter, the intensity of immunolabelling decreased between E18 to P25 and the labelling only concerned some cells. These results reveal that beta III tubulin appears before birth in the nervous extensions connecting the sensory cells of the OC according to a modiolar-to-striolar gradient. In the spiral ganglia, the labelling progressively decreases during its development. [less ▲]

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See detailGlial but not neuronal development in the cochleo-vestibular ganglion requires Sox10.
Breuskin, Ingrid ULg; Bodson, Morgan ULg; Thelen, Nicolas ULg et al

in Journal of Neurochemistry (2010), 114(6), 1827-39

The cochleo-vestibular ganglion contains neural crest-derived glial cells and sensory neurons that are derived from the neurogenic otic placode. Little is known about the molecular mechanisms that ... [more ▼]

The cochleo-vestibular ganglion contains neural crest-derived glial cells and sensory neurons that are derived from the neurogenic otic placode. Little is known about the molecular mechanisms that regulate the tightly orchestrated development of this structure. Here, we report that Sox10, a high-mobility group DNA-binding domain transcription factor that is required for the proper development of neural crest cell derivatives, is specifically expressed in post-migratory neural crest cells in the cochleo-vestibular ganglion. Using Sox10-deficient mice, we demonstrate that this transcription factor is essential for the survival, but not the generation, of the post-migratory neural crest cells within the inner ear. In the absence of these neural crest-derived cells, we have investigated the survival of the otocyst-derived auditory neurons. Surprisingly, auditory neuron differentiation, sensory target innervation and survival are conserved despite the absence of glial cells. Moreover, brain-derived neurotrophic factor expression is increased in the hair cells of Sox10-deficient mice, a compensatory mechanism that may prevent spiral ganglion neuronal cell death. Taken together, these data suggest that in the absence of neural crest-derived glial cells, an increase trophic support from hair cells promotes the survival of spiral ganglion neurons in Sox10 mutant mice. [less ▲]

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See detailSPATIO-TEMPORAL LOCALIZATION OF INTERMEDIATE FILAMENTS IN THE ORGAN OF CORTI BETWEEN THE EMBRYONIC DAY 18 (E18) AND THE POST-NATAL DAY 15 (P15) IN RAT
Johnen, Nicolas ULg; Thelen, Nicolas ULg; Malgrange, Brigitte ULg et al

Poster (2009, October 17)

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, a least four types of ... [more ▼]

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, a least four types of supporting cells can be identified in the OC: inner pillar cell, outer pillar cell, phalangeal cell and Deiter’s cells. The structure of this organ is well reported in adult but its development is still little known. Using antibodies directed against different proteins of intermediate filaments cytoskeleton, we studied the spatial-temporal localization of cytokeratins (typical of epithelial cells) and vimentin (typical of mesenchymal cells) during the differentiation of the OC in rat from the embryonic day 18 (E18) to the postnatal day (P15). Whatever the antibody used, we observed an obvious labelling over the supporting cells after the birth. In particular, an intense labelling is observed in the pillar cells and in the Deiters’ cells at P8 and at P10. These results suggest that the epithelial-mesenchymal transition might be implicated in the opening of Corti’s tunnel between the pillar cells and the formation of the Nuel’s spaces between the Deiters’ cell and their outer hair cells, at P8 and at P10 respectively. [less ▲]

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See detailSupporting cell cytoskeleton during development of the organ of Corti in rat
Johnen, Nicolas ULg; Thelen, Nicolas ULg; Malgrange, Brigitte ULg et al

Poster (2009, May 11)

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, a least four types of ... [more ▼]

The mammalian auditory organ, the organ of Corti (OC), is composed of mechanosensory hair cells and nonsensory supporting cell types. Based on their morphology and physiology, a least four types of supporting cells can be identified in the OC: inner pillar cell, outer pillar cell, phalangeal cell and Deiter’s cells. All supporting cells are highly specialized cells that are characterized by the presence of bundled microtubules with 15 protofilaments instead of 13. Using antibobies against different proteins of cytoskeleton (tubulin, cutokeratin and vimentin), we investigated by confocal microscopy the setting up of supporting cells' cytoskeleton during the differentiation of the OC in art from the embryonic day 18 (E18) to the postnatal 15 (P15). We showed that the inner pillar cells are labelled with an anti-beta IV tubulin from P0. Using an antibody to cytokeratin, a labelling appeared in Deiters' cells from E22. We also revealed that during the development of the OC, supporting cells were labelled with an anti-vimentin antibody from P0. [less ▲]

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See detailLocalization of Nopp140 within mammalian cells during interphase and mitosis
Thiry, Marc ULg; Cheutin, Thierry; Lamaye, Françoise et al

Poster (2009)

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See detailSox10 promotes the survival of cochlear progenitors during the establishment of the organ of Corti
Breuskin, Ingrid ULg; Bodson, Morgan ULg; Thelen, Nicolas ULg et al

in Developmental Biology (2009), 15(335), 327-339

Transcription factors of the SoxE family are critical players that underlie various embryological processes. However, little is known about their function during inner ear development. Here, we show that ... [more ▼]

Transcription factors of the SoxE family are critical players that underlie various embryological processes. However, little is known about their function during inner ear development. Here, we show that Sox10 is initially expressed throughout the otic vesicle epithelium and becomes later restricted to supporting cells as cell differentiation proceeds in the organ of Corti. Morphological analyses of Sox10 mutant mice reveal a significant shortening of the cochlear duct likely resulting from the progressive depletion of cochlear progenitors. While Sox10 appears dispensable for the differentiation and patterning of the inner ear prosensory progenitors, our data support a critical role for this transcription factor in the promotion of their survival. We provide genetic evidences that Sox10, in a concentration-dependant manner, could play a role in the regulation of Jagged1, a gene known to be important for inner ear prosensory development. Together, our results demonstrate that Sox10 regulates the biology of early cochlear progenitors during inner ear development, but, in contrast to neural crest-derived cells, this transcription factor is dispensable for their differentiation. Evidence also suggests that this effect occurs via the activation of the Jagged1 gene. [less ▲]

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See detailUltrastructural detection of nucleic acids within heat shock-induced perichromatin granules of HeLa cells by cytochemical and immunocytological methods.
Charlier, Christine; Lamaye, Françoise ULg; Thelen, Nicolas ULg et al

in Journal of Structural Biology (2009), 166(3), 329-36

The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock ... [more ▼]

The perichromatin granules (PGs) are enigmatic structures of the cell nucleus. The major drawbacks for a biological study are their rare occurrence and their small size in normal conditions. As heat shock has been shown to increase their number, we applied a hyperthermal shock on HeLa cells to investigate the nucleic acid content of PGs by means of cytochemical and immunocytological approaches. These heat shock-induced PGs (hsiPGs) appeared as clusters organized in the form of honeycomb structures and were always associated with some blocks of condensed chromatin, such as the perinucleolar chromatin shell. A stalk connecting the hsiPG to the chromatin could be observed. For the detection of RNA, we applied an immunocytological method involving two anti-RNA antibodies and quantified the gold labelling obtained. The results clearly revealed that hsiPGs contained RNA. Regarding to the detection of DNA, we used three different methods followed by quantitative analyses. The results seemed to indicate that a small amount of DNA was present in hsiPGs. Together, these findings suggest that hsiPGs might be RNP structures associated with particular regions of DNA. [less ▲]

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See detailLocalization of Nopp140 within mammalian cells during interphase and mitosis.
Thiry, Marc ULg; Cheutin, Thierry; Lamaye, Françoise ULg et al

in Histochemistry & Cell Biology (2009), 132(2), 129-40

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three ... [more ▼]

We investigated distribution of the nucleolar phosphoprotein Nopp140 within mammalian cells, using immunofluorescence confocal microscopy and immunoelectron microscopy. During interphase, three-dimensional image reconstructions of confocal sections revealed that nucleolar labelling appeared as several tiny spheres organized in necklaces. Moreover, after an immunogold labelling procedure, gold particles were detected not only over the dense fibrillar component but also over the fibrillar centres of nucleoli in untreated and actinomycin D-treated cells. Labelling was also consistently present in Cajal bodies. After pulse-chase experiments with BrUTP, colocalization was more prominent after a 10- to 15-min chase than after a 5-min chase. During mitosis, confocal analysis indicated that Nopp140 organization was lost. The protein dispersed between and around the chromosomes in prophase. From prometaphase to telophase, it was also detected in numerous cytoplasmic nucleolus-derived foci. During telophase, it reappeared in the reforming nucleoli of daughter nuclei. This strongly suggests that Nopp140 could be a component implicated in the early steps of pre-rRNA processing. [less ▲]

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See detailEarly identification of inner pillar cells during rat cochlear development.
Thelen, Nicolas ULg; Breuskin, Ingrid ULg; Malgrange, Brigitte ULg et al

in Cell & Tissue Research (2009), 337(1), 1-14

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 ... [more ▼]

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. [less ▲]

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See detailSpatiotemporal Distribution of Polysaccharides During the Mammalian Auditory Organ Development
Thelen, Nicolas ULg; Compère, Philippe; Malgrange, Brigitte et al

Poster (2008, October 31)

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See detailNew insights in mammalian auditory organ development
Thelen, Nicolas ULg; Malgrange, Brigitte ULg; Thiry, Marc

Conference (2008, October 30)

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Using cytochemical and immunohistochemical methods ... [more ▼]

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far from being elucidated. Using cytochemical and immunohistochemical methods at the light and electron microscope levels, we examined its spatiotemporal development in rats from embryonic day 16 (E16) to E19. At E16, whatever the region of the cochlear studied (base, middle, apex), the organ of Corti was not present. We demonstrate that the organ of Corti develops from a non-proliferative cell zone that is located in the junctional region between the greater epithelial ridge and the lesser epithelial ridge of the cochlear duct and that is characterized by the presence of numerous microvilli. Using the periodic acid-thiocarbohydrazide-silver proteinate method, we revealed that the first cells to develop in this zone are the inner pillar cells, a particular type of nonsensory supporting cells. They arise in the base of the cochlear duct at the boundary between the two ridges at E16. The cell differentiation in this prosensory region continues according to a base-to-apex gradient, the inner hair cells appearing in the greater epithelial ridge at E17 and the outer hair cells in the lesser epithelial ridge at E18. At E19, all the different cell types of the organ of Corti are well in place. We also showed that the development of the inner pillar cells within the prosensory region does not involve Notch1 signaling. These results highlight the central role that the inner pillar cells could play in the development of the organ of Corti. [less ▲]

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See detailStrategies to Regenerate Hair Cells: Identification of Progenitors and Critical Genes
Breuskin, Ingrid ULg; Bodson, Morgan ULg; Thelen, Nicolas ULg et al

in Hearing Research (2008), 236(1-2), 1-10

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 ... [more ▼]

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. [less ▲]

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See detailSox10 is not necessary for auditory neurons survival
Breuskin, I; Bodson, M; Thelen, Nicolas ULg et al

Poster (2008)

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See detailA protocol for studying the kinetics of RNA within cultured cells: application to ribosomal RNA.
Thiry, Marc ULg; Lamaye, Françoise ULg; Thelen, Nicolas ULg et al

in Nature Protocols (2008), 3(12), 1997-2004

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living ... [more ▼]

This protocol describes a nonisotopic method for high-resolution investigation of the kinetics of RNA within the cell. This involves the incorporation of bromouridine-5'-triphosphate into RNA of living cells by lipofection followed by immunocytological detection of BrRNAs. The use of the same antibody identified either with fluorescence or with gold particles revealed the three-dimensional organization of sites containing labeled RNAs or their precise localization by using confocal and ultrastructural microscopy, respectively. Comparison of three-dimensional reconstruction obtained from the series of optical sections and ultrathin sections was extremely fruitful to describe topological and spatial dynamics of RNAs from their synthesis site inside the nucleus to the cytoplasm. Combined with immunolocalization of proteins involved in different nuclear activities and with highly resolved three-dimensional visualizations of the labelings, this method should also provide a significant contribution to our understanding of the functional, volumic organization of the cell nucleus. The entire protocol can be completed in approximately 10 d. [less ▲]

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See detailAn atypical cell during development of the auditory organ : the inner pillar cell
Thelen, Nicolas ULg; Malgrange, Brigitte ULg; Thiry, Marc

Poster (2007, September)

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far to be elucidated. Using cytochemical and immunohistochemical methods at ... [more ▼]

Although the structure of the auditory organ in mature mammals, the organ of Corti, is clearly established, its development is far to be elucidated. Using cytochemical and immunohistochemical methods at the light and electron microscope levels, we examined its spatiotemporal development in rats from embryonic day 16 (E16) to E19. At E16, whatever the region of the cochlear studied (base, middle, apex), the organ of Corti is not present. We demonstrate that the organ of Corti develops from a non-proliferative cell zone that is located in the junctional region between the greater epithelial ridge and the lesser epithelial ridge of the cochlear duct and that is characterized by the presence of numerous microvilli. Using the periodic acid-thiocarbohydrazide-silver proteinate method, we reveal that the first cells to develop in this zone are the inner pillar cells, a particular type of nonsensory supporting cells; they arise in the base of the cochlear duct at the boundary between the two ridges at E16. The 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, all the different cell types of the organ of Corti are well in place. We also show that the development of the inner pillar cells within the prosensory region does not involve Notch1 signaling. These results highlight the central role that cells could play the inner pillar in the organ of Corti development. [less ▲]

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