Unknown,Transcriptomics,Genomics,Proteomics

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RNA-seq analysis of neonatal mouse cochlear supporting cells [NonTreated_JM]

ABSTRACT: This study examined transcripts that are enriched in neonatal mouse cochlear supporting cells at postnatal day 1 and postnatal day 6. Supporting cells were purified by FACS sorting for GFP fluorescence from the cochleas of transgenic mice in which a BAC including the LFng locus drives the expression of GFP. Two replicates of GFP+ supporting cells were compared with all other cochlear cell types that were GFP-. We performed this experiment at two different ages, postnatal day 1 and postnatal day 6. mRNA profiles of supporting cells (GFP+) and all other cochlear cell types (GFP-), two replicates each, at P1 and P6 mice were generated by deep sequencing using Illumna TruSeq.

ORGANISM(S): Mus musculus

SUBMITTER: Andrew Groves

PROVIDER: E-GEOD-83356 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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. Analysis was conducted on auditory nerve samples from stages encompassing maturation and onset of hearing. Cochleas were collected from euthanized mice (CBA/CaJ) at perinatal (P) stages P0, P3, P7, P10, P14 and P21. Cochleas underwent microdissection to remove the outer bony cochlear shell and cochlear lateral wall, thus preserving the modiolus portion of cochlea which contains mainly the auditory nerve. Paired cochleas (i.e., from left and right ears) from each mouse were pooled to make individual samples. All sample types were done in experimental duplicate (n=2).

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.

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RNA-seq analysis of neonatal mouse cochlear supporting cells [NonTreated_JM]

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