DNA methylation in the mouse cochlea promotes maturation of supporting cells and contributes to the failure of hair cell regeneration
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ABSTRACT:
PROVIDER: PRJNA931748 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| Other | ||||
| SRR23344100_1.fastq.gz | Fastqsanger.gz | |||
| SRR23344100_2.fastq.gz | Fastqsanger.gz | |||
| SRR23344101_1.fastq.gz | Fastqsanger.gz | |||
| SRR23344101_2.fastq.gz | Fastqsanger.gz |
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Project description:Purpose - To profile epigenetic changes in cochlear progenitor cells, hair cells, and supporting cells across postnatal development. Methods- Cochlear cell types were obtained from fluorescently labeled transgenic mice. E13.5 cochlear prosensory progenitor cells (E13.5_PG, p27Kip1-GFP+), P1 hair cells (P1_HC, Atoh1-GFP+), P1 supporting cells (P1_SC, Lfng-GFP+), and P6, P8, P21 supporting cells (P6_SC, P8_SC, P21_SC, Lfng-creER, NuTRAP lineage-traced) were FACS purified for epigenetic profiling using WGBS (DNA methylation), CUT&Tag (H3K4me1, H3K4me3, H3K27me3, CUTAC H3K4me2). DNA demethylation of hair cell-specific promoters was interrogated by comparing % mCpG profiles of transdifferentiating supporting cells (P1_SC_tdt_gfp, Lfng-creER/TdTomato+, Atoh1-GFP+) against non-transdifferentiating supporting cells (P1_SC_tdt, P6_SC_tdt, Lfng-creER/TdTomato+) purified via FACS. P1 and P8 wildtype cochleas were enzymatically dissociated and used directly for scMultiome simultaneous profiling of RNA and ATAC at the single cell level (P1_scMultiome, P8_scMultiome, 10x Genomics). For scMultiome of P70 wildtype (P70_wt_scMultiome, Lfng-CreERT2, NuTRAP) and P70 deafened (P70_deaf_scMultiome, Lfng-CreERT2, NuTRAP, Pou4f3DTR) supporting cells, mice received 100 mg/kg tamoxifen (Sigma-Aldrich T5648) at P21, 0.01 mg/kg Diptheria toxin (Sigma-Aldrich D0564) at P28, and allowed to mature to P70, at which point cochlear tissues were harvested, FACS purified for NuTRAP+ supporting cells, and inputted into a scMultiome reaction. Results- WGBS highlighted differentially methylated regions (DMR) across E13.5_PG, P1_HC, P1_SC, P6_SC, and P21_SC. Pre-established DMRs become hypermethylated in supporting cells between P1 and P21. This corresponds with increasing heterochromatin characteristics, such as loss of accessibility (CUTAC), loss of H3K4me1, and switch between H3K27me3-mediated repression to DNA methylation-mediated silencing. WGBS of P1_SC_tdt_gfp compared to P1_SC_tdt and P6_SC_tdt showed DNA demethylation of hair cell-specific DMRs, suggesting that DNA demethylation is required for transdifferentiation of supporting cells into hair cells. P1_scMultiome, P8_scMultiome, P70_wt_scMultiome, and P70_deaf_scMultiome revealed changes in chromatin accessibility associated with age, as well as from long-term deafening at the single cell level. scMultiome datasets also highlight cell type-specific enhancers active during different stages of postnatal development.
2023-02-07 | GSE224563 | GEO
Project description:Cochlear supporting cells are critical for proper hearing function and regeneration of hair cells, which are responsible for perceiving sound in the inner ear. In the neonatal mouse cochlea, when supporting cells are ablated, they can regenerate through progenitor cells in the cochlea. Here, we show through single cell RNA sequencing and in vivo fate mapping, that upon supporting cell ablation in the neonatal cochlea, inner phalangeal cells regenerate through two independent mechanisms; one which requires proliferation and the other by which transdifferentiation (i.e. regeneration without mitotic division) of cochlear progenitor cells occurs. These dual mechanisms are spatially distinct where transdifferentiation occurs in all three turns of the cochlea, while proliferation-induced regeneration occurs exclusively in the apical and middle turns. Inhibiting proliferation using aphidicolin, slightly decreased the number of regenerated IPhCs in the apex middle turns, but not the base, after damage. We also observed a medial to lateral transition in the compartmentalization of the GER after supporting cell damage, suggesting de-differentiation of these domains. Overall, our study reveals GER cells as supporting cell progenitors that undergo dual mechanisms of regeneration (mitotic and non-mitotic). Additionally, we uncover transcriptomic changes after supporting cell damage in the neonatal mouse cochlea that could be used as gene targets in therapies for hearing loss.
2026-01-12 | GSE291228 | GEO
Project description:Mechanosensory hair cells of the mature mammalian organ of Corti do not regenerate, and loss of hair cells leads to permanent hearing loss. Although non-mammalian vertebrates are able to regenerate hair cells by the division and transdifferentiation of adjacent supporting cells, many humans with severe hearing loss completely lack hair cells and supporting cells, with the organ of Corti being replaced by a flat epithelium of non-sensory cells To determine if mature non-sensory cells can produce hair cells in vivo, we reprogrammed non-sensory cells of the mature cochlea with three hair cell transcription factors, Gfi1, Atoh1, and Pou4f3. We were able to generate significant numbers of hair cell-like cells in the non-sensory inner and outer sulci of the cochlea after 2 weeks of reprogramming. New hair cells continued to be added to the non-sensory regions of the cochlea over the next seven weeks, and expressed hair cell proteins such as Myosin VIIa, parvalbumin, and prestin and formed stereociliary hair bundles. We confirmed the identity of these cells by high depth single cell RNA-seq. Significantly, cells adjacent to converted hair cells expressed markers of supporting cells, suggesting that transcription factor reprogramming of non-sensory tissue in the adult animal can generate mosaics of sensory cells similar to those seen in the organ of Corti. Generating such sensory mosaics by reprogramming may represent a potential strategy for hearing restoration in humans.
2024-01-03 | GSE224627 | GEO
Project description:In contrast to mammals, the avian cochlea, specifically the basilar papilla, can regenerate sensory hair cells, which involves fate conversion of supporting cells to hair cells. To determine the mechanisms for converting supporting cells to hair cells, we used single-cell RNA sequencing during hair cell regeneration in explant cultures of chick basilar papillae. We identified dynamic changes in the gene expression of supporting cells, and the pseudotime trajectory analysis demonstrated the stepwise fate conversion from supporting cells to hair cells. Initially, supporting cell identity was erased and transition to the precursor state occurred. A subsequent gain in hair cell identity progressed together with downregulation of precursor-state genes. Transforming growth factor beta receptor 1-mediated signaling was involved in induction of the initial step, and its inhibition resulted in suppression of hair cell regeneration. Our data provide new insights for understanding fate conversion from supporting cells to hair cells in avian basilar papillae.
2023-01-10 | GSE209791 | GEO
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:Follistatin promotes LIN28B mediated supporting cell reprogramming and hair cell regeneration in the murine cochlea
| PRJNA729762 | ENA
Project description:Signal transducer and activator of transcription 3 (Stat3) plays a role in various cellular processes such as differentiation, inflammation, cell survival and microtubule dynamics, depending on the cell type and the activated signaling pathway. Stat3 is highly expressed in the hair cells and supporting cells of the cochlea and is essential for the differentiation of mouse hair cells in the early embryonic stage. However, it is unclear how Stat3 contributes to the correct function of cells in the organ of Corti postnatally. To investigate this, an inducible Cre/loxp system was used to knock out Stat3 in either the outer hair cells or the supporting cells. The results showed that the absence of Stat3 in either the outer hair cells or the supporting cells resulted in hearing loss without altering the morphology of the organ of Corti. Molecular analysis of the outer hair cells revealed an inflammatory process with increased cytokine production and upregulation of the NF-kB pathway. However, the absence of Stat3 in the supporting cells resulted in reduced microtubule stability. In conclusion, Stat3 is a critical protein for the sensory epithelium of the cochlea and hearing and functions in a cell and function-specific manner
2024-10-30 | GSE278228 | GEO
Project description:Latent transdifferentiation potential exists in supporting cells at neonatal stage in the organ of Corti in mouse, but this plasticity is lost rapidly during the first week of postnatal maturation, leading to the failure of sensory hair cell regeneration. To investigate the molecular mechanism underlying the loss of transdifferentiation potential, we compared epigenetic changes in E17.5, P1 and P6 supporting cells. In E17.5 and P1 supporting cells, hair cell gene enhancers are kept in a primed state (H3K4me1+ H3K27ac-) which can be readily activated during transdifferentiation induced by Notch blocking. As supporting cells mature, a substantial amount of hair cell gene enhancers are decommissioned by H3K4me1, leading to the loss of transdifferentiation potential. In contrast, hair cell gene enhancers retain their commissioned epigenetic status in mature utricular supporting cells, sustaining the transidifferentiation potential in utricular supporting cells in mature animals. Our findings reveal the epigenetic mechanisms underlying the failure of sensory hair cell regeneration in mammalian cochlea.
2021-07-09 | GSE150010 | GEO
Project description:In adult mammals, hair cell loss is irreversible and may result in hearing and balance deficits. In contrast, birds can regenerate hair cells through differentiation of supporting cells and restore inner ear function, suggesting that hair cell progenitors are present in the population of supporting cells. We used microarrays to identify novel genes related to the regeneration in the chicken utricle. Supporting cell and hair cell populations of chicken utricle obtained by laser capture microdissection, following to do RNA extraction and hybridization on Affymetrix microarrays.
2016-05-17 | E-GEOD-81461 | biostudies-arrayexpress
Project description:Background: Understanding the developmental mechanisms that regulate hair cell differentiation in the cochlea is essential to designing genetic therapies for acquired hearing loss due to hair cell loss or damage. We have previously identified Fibroblast Growth Factor 20 (FGF20) as having a key role in hair cell and supporting cell differentiation and patterning in the mouse cochlear sensory epithelium. To investigate the genetic landscape regulated by FGF20 signaling in hair cell and supporting cell progenitors, we employ Translating Ribosome Affinity Purification (TRAP) combined with Next Generation mRNA Sequencing (TRAPseq). Methods: In mice, we used Fgf20-Cre to activate ROSA-fsTRAP in hair cell and supporting cell progenitors, and then collected translating mRNA using TRAP from Fgf20+/- (control) and Fgf20-/- cochleae. Library preparation and sequencing were done in two separate experiments, each with 12 samples that included 2 pre-TRAP (pre-immunoprecipitation) Fgf20+/- samples, 2 TRAP Fgf20+/- samples, 4 pre-TRAP Fgf20-/- samples, and 4 TRAP Fgf20-/- samples. Samples were sequenced via Illumina HiSeq 3000. We compared pre-TRAP (pre-immunoprecipitation) samples with TRAP mRNA samples to validate the TRAP technique as well as identify genes enriched in our target cell population. We also compared Fgf20+/- and Fgf20-/- TRAP mRNA samples to identify differentially expressed genes downstream of FGF20 during hair cell and supporting cell differentiation. Results: TRAPseq targeting the prosensory cell population effectively enriches for translating mRNA within this rare cell population. TRAPseq comparing Fgf20+/- and Fgf20-/- samples identified differentially expressed genes downstream of FGF20. These included FGF-response genes Etv4, Etv5, Etv1, and Dusp6, as well as genes associated with cochlea development and hearing, such as Hey1, Hey2, Heyl, Tectb, Fat3, Cpxm2, Sall1, and cell cycle regulators such as Cdc20.
2020-04-10 | GSE148380 | GEO