EDNRB2 regulates fate determination, migration and maturation of hair-cell precursors in regenerating avian auditory epithelia
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PROVIDER: PRJNA1213701 | ENA |
REPOSITORIES: ENA
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Project description:The mammalian cochlea loses the ability for hair cell (HC) regeneration after birth, while in the avian auditory epithelium, namely the basilar papilla (BP), supporting cells (SCs) retain the capability for HC regeneration throughout life. Our previous study using single-cell RNA sequencing indicated stepwise fate conversion of SCs to HCs via the precursor state, in which EDNRB2 exhibited specifically high expression, in the process of HC regeneration in chick BP. The present study aimed to reveal a distinct role of EDNRB2 in HC regeneration in chick BP. During HC regeneration in chick BP explant cultures, EDNRB2 expression emerged in a part of ATOH1-expressing SCs immediately after HC loss and decreased according to the progress of HC regeneration process. In embryonic chick BP, EDNRB2 expression was specifically identified in the precursor cell state, which confirmed that mature SCs were reprogrammed to the precursor state in response to HC loss. Pharmacological inhibition of endothelin receptor type B (EDNRB) signaling decreased regenerated HCs in chick BP explant cultures. RNA sequencing revealed that inhibition of EDNRB signaling downregulated genes for HC differentiation and maturation together with genes for cell migration. Histological assessments clarified the deterioration of migration of HC precursors and the delay of HC regeneration processes by inhibition of EDNRB signaling. These results indicate that the expression of EDNRB2 contributes to the fate determination of HCs and that EDNRB signaling regulates the migration and maturation of HC precursors during chick HC regeneration.
2025-06-21 | GSE287538 | GEO
Project description:Regenerating hair cells in human vestibular sensory epithelia
| PRJNA430413 | ENA
Project description:In order to gain insight into the molecular events which underlie auditory hair cell regeneration in chicken, we compared gene expression in chicken basilar papillae after 24, 48, and 72 hours in culture with or without forskolin (100uM).<br><br>Under sterile conditions, cochlear ducts containing the basilar papillae were carefully dissected out of ~4-day-old chicks and then individually cultured in DMEM with 10% fetal bovine serum with or without forskolin (final concentration 100 ?M, delivered in 1% DMSO) for either 24, 48, or 72 hours at 37ï¾°C. Control samples received DMSO at 1% as a vehicle control. At the end of 3 days, the tegmentum vasculosum was dissected off to expose the auditory epithelium, which was delicately freed from the underlying cartilaginous plates. All explants were kept in culture for 3 days because new hair cells are first seen in basilar papillae treated with forskolin after ~3 days of exposure to the drug [19]. Therefore, at even earlier time points in culture the molecular events that subserve hair cell proliferation are well underway. Each sample was comprised of 3 auditory epithelia from 3 different chicks which were put in ~100 ?L of DMEM and then immediately frozen at -80ï¾°C until RNA isolation could be performed. There were a total of 24 samples in this experiment: six 72-hour forskolin, six 72-hour control, three 48-hour forskolin, three 48-hour control, three 24-hour forskolin, and three 24-hour control.
2010-06-09 | E-MEXP-2642 | biostudies-arrayexpress
Project description:The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these sensory hair cells lack the capacity for regeneration and if damaged lead to hearing or balance disorders. However, non-mammalian vertebrates such as birds maintain their regenerative abilities throughout their life. In a previous study we conducted a gene expression profiling time course of regenerating sensory epithelia (SE) in avian cochlea and utricle on a custom transcription factor microarray following damage by both laser and chemical ablation. We identified several known signaling cascades such as The Pax-Eya-Six-Dach pathway, Ap-1 pathway, the Tgf-β pathway and sonic hedgehog signaling that are differentially expressed during SE regeneration. In this study we selected 27 of these genes for knockdown by siRNA or small molecule inhibition to determine their requirement for SE regeneration and identify downstream targets. We assessed phenotypes using a 96 well proliferation assay and expression profiled each knockdown on a custom transcription factor microarray. Using these techniques we have determined several genes that are required for SE proliferation and identified novel epistatic relationships between many of these genes. Pure sensory epithelia was isolated from avian utricles. Sensory epithelia was physically dissociated and grown in 96 well cultures for 3 days. Prior to confluency, dissociated sensory epithelia were transfected with siRNAs (12 pmol/well) or small molecule inhibitor in 0.1% DMSO. RNA was isolated 24 hrs post transfection and assayed on a custom oligonucleotide transcription factor microarray compared to controls (GFP siRNA or 0.1% DMSO only). siRNA: CEBPG, JunD, BTAF1, LRP5, PAX2, PAX5, PAX7, Wnt4, BCL11A, CBX3, CBX4, CTNNB1, CUTL1, MYT1L, RARA, TIMELESS, TRIP15, PAX3, EZH2, HES1, ID1, CDKN1B, and PPARGC1 small molecule inhibition: IGF, MAPK, SHH, and JNK
2010-07-02 | E-GEOD-16842 | biostudies-arrayexpress
Project description:Sensorineural hearing loss is included in the most common disabilities, and often caused by loss of sensory hair cells in the cochlea. Hair cell regeneration has long been a main target for developing novel therapeutics for sensorineural hearing loss. In the mammalian cochlea, hair cell regeneration occurs in very limited situations, while auditory epithelia of non-mammalians retain the capacity for hair cell regeneration. In the avian basilar papilla, an auditory sensory epithelium, supporting cells, which are sources for regenerated hair cells, are usually quiescent, while hair cell loss induces both direct transdifferentiation of supporting cells and mitotic division of supporting cells. In the present study, we aimed to establish an explant culture model for hair cell regeneration in chick basilar papillae, and validated usefulness of our model to investigate the initial phase of hair cell regeneration. Histological assessments demonstrated that hair cell regeneration via direct transdifferentiation of supporting cells occurred in our model. Labeling assay using 5-ethynyl-2'-deoxyuridine (EdU) revealed the occurrence of mitotic division of supporting cells in the specific location in basilar papillae, while no EdU labeling was identified in newly generated HCs. RNA sequencing indicated alterations in known signaling pathways associated with hair cell regeneration, which is consistent with previous findings. In addition, unbiased analyses of RNA sequencing data indicated novel genes and signaling pathways that could be related to the ignition of supporting cell activation in chick basilar papillae. These results indicate the advantages of our model using explant cultures of chick basilar papillae for exploring molecular mechanisms for hair cell regeneration. Further studies such as single-cell RNA sequencing will allow us to capture the spatiotemporal information by using our explant culture model.
2020-10-23 | GSE154375 | GEO
Project description:We characterized the proteome of the auditory brainstem of a chick embryo on embryonic day 13, when apoptosis occurs in auditory nuclei. We identified caspase substrates by searching the peptidome for peptides C-terminal to caspase-typical cleavage sites.
2022-04-01 | PXD030697 | Pride
Project description:Sound localization requires extremely precise development of auditory brainstem circuits, the molecular mechanisms of which are largely unknown. We previously demonstrated a novel requirement for non-apoptotic activity of the protease caspase-3 in chick auditory brainstem development. Here, we used mass spectrometry to identify proteolytic substrates of caspase-3 during chick auditory brainstem development. Functional annotation analysis revealed that our caspase-3 substrates were enriched more than two-fold for proteins associated with extracellular vesicles (EVs), membrane-bound nanoparticles that function in intercellular communication. The proteome of EVs isolated from the auditory brainstem contained caspase-3 and was highly enriched for the caspase-3 substrates identified here. Additionally, we identified two caspase-3 substrates with known functions in axon guidance, namely Neural Cell Adhesion Molecule (NCAM) and Neuronal-glial Cell Adhesion Molecule (Ng-CAM), that were found in auditory brainstem EVs and expressed in the auditory pathway alongside cleaved caspase-3. Taken together, these data suggest a novel developmental mechanism whereby caspase-3 influences auditory brainstem circuit formation through the proteolytic cleavage of EV proteins.
2020-10-16 | PXD021728 | Pride
Project description:The inner ear utilizes sensory hair cells as mechano-electric transducers for sensing sound and balance. In mammals, these sensory hair cells lack the capacity for regeneration and if damaged lead to hearing or balance disorders. However, non-mammalian vertebrates such as birds maintain their regenerative abilities throughout their life. In a previous study we conducted a gene expression profiling time course of regenerating sensory epithelia (SE) in avian cochlea and utricle on a custom transcription factor microarray following damage by both laser and chemical ablation. We identified several known signaling cascades such as The Pax-Eya-Six-Dach pathway, Ap-1 pathway, the Tgf-β pathway and sonic hedgehog signaling that are differentially expressed during SE regeneration. In this study we selected 27 of these genes for knockdown by siRNA or small molecule inhibition to determine their requirement for SE regeneration and identify downstream targets. We assessed phenotypes using a 96 well proliferation assay and expression profiled each knockdown on a custom transcription factor microarray. Using these techniques we have determined several genes that are required for SE proliferation and identified novel epistatic relationships between many of these genes.
2010-07-02 | GSE16842 | GEO
Project description:Spiral ganglion neurons (SGNs) and the associated components of the auditory nerve are primary carriers of auditory information from hair cells to the brain. Loss of SGNs occurs with many pathological conditions, resulting in permanent sensorineural hearing loss. Neural stem/progenitors (NSPs) have been well-characterized in several locations of adult brain and retina. However, it is unclear whether NSPs are present in the adult auditory nerve. Here we examined the self-renewal potential of the adult auditory nerve using ouabain application as a well-established mouse model of acute SGN injury. The observed increase in cell proliferation, alteration in enchromatin/heterochromatin ratio and down-regulation of histone deacetylase expression in glial cells suggest that the quiescent glial cells convert to an activated state after SGN degeneration. This was further confirmed by global gene expression analysis of injured auditory nerves, which showed up-regulation of numerous neurogenesis- and/or development-associated genes shortly after ouabain exposure. These genes include molecular markers commonly used for the identification of NSPs. Under a strict culture regimen, auditory nerve-derived cells of adult mouse ears gave rise to neurospheres, suggesting that multipotent NSPs are present in adult cochlear nerve. Neurosphere assays on Sox2 transgenic mice revealed that Sox2+ glial cells are the source for NSPs. Our data also showed that acute injury or hypoxia enhances neurosphere formation. Taken together, our study revealed that glial cells of adult cochlea exhibit several NSP characteristics, and hence these mature non-neuronal cells may be important targets for promoting self-repair of degenerative auditory nerves. Auditory nerves were removed from the temporal bones of adult CBA/CaJ mice, aged 8 to 12 weeks. Tissues were either collected and used directly as the tissue samples or dissociated and used for the cell culture samples. Dissociated auditory nerve cells were propagated and grown to full confluency (5-7 days), constituting the cultured cell samples. For neurosphere samples, growth medium was changed to neurosphere formation medium and the cells were cultured for an additional 12 days. All samples were prepared in triplicate (n=3).
2015-04-29 | E-GEOD-65249 | biostudies-arrayexpress
Project description:Spiral ganglion neurons (SGNs) and the associated components of the auditory nerve are primary carriers of auditory information from hair cells to the brain. Loss of SGNs occurs with many pathological conditions, resulting in permanent sensorineural hearing loss. Neural stem/progenitors (NSPs) have been well-characterized in several locations of adult brain and retina. However, it is unclear whether NSPs are present in the adult auditory nerve. Here we examined the self-renewal potential of the adult auditory nerve using ouabain application as a well-established mouse model of acute SGN injury. The observed increase in cell proliferation, alteration in enchromatin/heterochromatin ratio and down-regulation of histone deacetylase expression in glial cells suggest that the quiescent glial cells convert to an activated state after SGN degeneration. This was further confirmed by global gene expression analysis of injured auditory nerves, which showed up-regulation of numerous neurogenesis- and/or development-associated genes shortly after ouabain exposure. These genes include molecular markers commonly used for the identification of NSPs. Under a strict culture regimen, auditory nerve-derived cells of adult mouse ears gave rise to neurospheres, suggesting that multipotent NSPs are present in adult cochlear nerve. Neurosphere assays on Sox2 transgenic mice revealed that Sox2+ glial cells are the source for NSPs. Our data also showed that acute injury or hypoxia enhances neurosphere formation. Taken together, our study revealed that glial cells of adult cochlea exhibit several NSP characteristics, and hence these mature non-neuronal cells may be important targets for promoting self-repair of degenerative auditory nerves.
2015-04-29 | GSE65249 | GEO