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See detailEvidence for a partial epithelial-mesenchymal transition in postnatal stages of rat auditory organ morphogenesis
Johnen, Nicolas ULg; Cloes, Marie ULg; Thelen, Nicolas ULg et al

Poster (2012, May 04)

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During ... [more ▼]

An epithelial-mesenchymal transition is a biological process that allows a polarized epithelial cell to undergo multiple biochemical changes that enable it to assume a mesenchymal cell phenotype. During this process, epithelial cells loosen cell-cell adhesion, module their polarity and rearrange their cytoskeleton: intermediate filaments typically switch from cytokeratin to vimentin. They also enhance their motility capacity. The epithelial-mesenchymal transition plays key roles in the formation of the body plan and in the differentiation of multiple tissues and organs but it is also involved in tissue repair, tissue homeostasis, fibrosis, and carcinoma progression. Until now, epithelial-mesenchymal transition has been rarely mentioned in the inner ear organogenesis. In chick, epithelial-mesenchymal transition has been reported as a possible mechanism of semicircular canal morphogenesis. More recently, an in vitro study has also indicated that sensory epithelial cells from mouse utricle can undergo an epithelial-mesenchymal transition to become cells expressing features of prosensory cells. By contrast, epithelial-mesenchymal transition has never been observed during auditory organ morphogenesis. The auditory organ, the organ of Corti, is a highly specialized structure composed by specific cellular types. The sensory cells 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 organ of Corti: inner and outer pillar cells, phalangeal cell and Deiter’s cells. The inner pillar cells and outer pillar cells combine to form the tunnel of Corti, a fluid filled triangular space that separates the single row of inner hair cells from the first row of outer hair cells. The Nuel spaces are another interval in the organ of Corti that is situated between the outer pillar cells and the different rows of outer hair cells and Deiters cells. To determine whether an epithelial-mesenchymal transition may play a role in the morphogenesis of the auditory organ, we studied the spatial localization of several epithelial-mesenchymal transition markers, the cell-cell adhesion molecules and intermediate filament cytoskeletal proteins, in epithelium of the dorsal cochlea during development of the rat organ of Corti from 18th embryonic day until 25th postnatal day. We examined by confocal microscopy immunolabelings on cryosections of whole cochleae with antibodies anti-cytokeratins as well as with antibodies anti-vimentin, anti-E-cadherin and anti-beta-catenin.Our results showed a partial loss of E-cadherin and beta-catenin between supporting cells at P8 and P12, respectively, and a temporary appearance of vimentin in pillar cells and Deiters between P8 and P10. Our results show a local loss of adhesion between supporting cells of the OC from P8, an increase expression of cytokeratins in supporting cells around P10 and a temporary appearance of vimentin in supporting cells at P8-10. These observations suggest that a partial epithelial-mesenchymal transition might be involved in the remodeling of the Corti organ during the postnatal stages of development in rat. [less ▲]

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See detailSpatio-temporal localization of the cytoskeleton during auditory organ development in mammalia
Johnen, Nicolas ULg; Thelen, Nicolas ULg; Cloes, Marie ULg et al

Poster (2011, March 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 (SC) can be identified in the OC: inner and outer pillar cells (PC), phalangeal cell and Deiter’s cells. Sensory and supporting cells possess characteristic cytoskeleton proteins in direct relation with their morphological features and their development. Indeed, this organ had morphological changes such as the setting up of the sensory epithelium after the birth or the openings of the Corti’s tunnel at P8 and of the Nuel’s spaces at P10. In the present study, by using confocal microscopy, we investigated the spatio-temporal localization of the three cellular cytoskeletal filaments : microtubules (β-1, 2, 3, 4-tubulin), microfilaments (cytoplasmic β- and γ-actin) and intermediate filaments (CK4, 5, 7, 8, CKpan and vimentin) during the development of the OC in rat from the embryonic day 18 (E18) to the post-natal day 25 (P25). The immunolabellings indicated clearly that β-1, 2, 3-tubulins were only present the SC and nervous fibers during development whereas β-4-tubulin was found firstly in the HC and then in the SC. The two actin-isotypes were detected in the HC apex but were also seen in the PC from P8 to P25 for β-actin isoform and in the basal membrane from E18 to P8 for the γ-actin isoform. All intermediate filament proteins were only found in the SC, especially between P8 and P12. Our results show that the localization of the cytoskeleton proteins during the auditory organ development depends on the cellular type and the developmental stage. A profound modification of cytoskeleton occurs between P8 and P12. [less ▲]

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See detailNucleolar structure across evolution : the transition between bi- and tricompartmentalized nucleoli lies within the class Reptilia
Franck, Claire; Lamaye, Françoise; Thelen, Nicolas ULg et al

Poster (2011, January 31)

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See detailSpatio-temporal localization of the glial fibrillary acidic protein (GFAP) in the spiral ganglion from the 16th embryonic day until the 25th postnatal day in rats
Cloes, Marie ULg; Thelen, Nicolas ULg; Johnen, Nicolas ULg et al

Poster (2011, January 31)

The spiral ganglion (SG) is responsible for the conduction of the information between the sensory epithelium of the auditory organ (the organ of Corti) and the central nervous system. The origin and ... [more ▼]

The spiral ganglion (SG) is responsible for the conduction of the information between the sensory epithelium of the auditory organ (the organ of Corti) and the central nervous system. The origin and nature of the SG glial cells in mammals are barely known, although glial cells are essential to the development and the working of the nervous system. Using confocal microscopy, we studied the spatio-temporal distribution of the GFAP in the rat SG from the 16th embryonic day (E16) until the 25th postnatal day (P25). We performed a double-labelling experiment targeting GFAP- and betaIIItubulin-positive cells. BetaIII-tubulin is used for the labelling of (pro)neural cells, according to a previous preliminary study from our team. Our first results show clearly that the GFAP is expressed in the SG from P0 until P25. A homogenous labelling is found in the cytoplasm of a few dispersed unidentified cells among the (pro)neurons, whereas a granular labelling appears among a group of cells neighbouring the bundle of fibers innervating the organ of Corti. The identity of the GFAP-positive cells will be further investigated by electron microscopy. The reason why the labelling of the GFAP adopts those two different aspects is still unknown. Moreover, it seems that, surprisingly, some cells of the ganglion are not labelled by either marker. The possibility that such cells correspond to fibroblasts will be tested thanks to the labelling of vimentin. [less ▲]

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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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