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Single cell transcriptomic analysis of P8 and P15 mouse cochlea (control and three overexpression conditions) and simultaneous single cell transcriptomic and accessible chromatin analysis of P1 and P8 mouse cochlea (wildtype)

ABSTRACT: Purpose - To determine transcriptomic variations in the mouse cochlea in response to three different transcription factor overexpression conditions and no induction control and to determine transcriptomic and accessible chromatin variations in the mouse cochlea between postnatal day 1 (P1) and postnatal day 8 (P8) stages of development. Methods - SC RNA seq performed on FACS enriched cells obtained from experimental animals (Sox9CreER; Rosa26A/GA/GAP/+; Rosa26 tdtomato). Same samples at two timepoints 1 week (P8) and 2 week (P15) ages. scMultiome ATAC + Gene Expression (10x Genomics) experiments on enzymatically dissociated and unenriched cochlear nuclei from wildtype mice in a mixed background of CD1 and FVB/NJ, and C57BL/6. Samples were collected at P1 and P8. Gene expression data was used to annotate clusters. Chromatin accessibility determined by ATAC was compared between P1 and P8 stages. Results - The SC RNA seq highlighted differential transcriptomic changes between the two ages and the three overexpression conditions. Hair cell loci become less epigenetically accessible in supporting cells and GER cells between P1 and P8. Conclusions - Our study highlights the variability in transcriptomic response to three transcription factor combination mediated reprogramming cues at two different ages. The SC RNA seq confirmed a variety of our biological observations with regard to the hair cell reprogramming process in the mouse cochlea. We also demonstrate variability in chromatin accessibility between P1 and P8, which may explain why GER and supporting cells of the cochlea become more resistant to transcription factor reprogramming into hair cells during the first postnatal week.

ORGANISM(S): Mus musculus

PROVIDER: GSE182202 | GEO | 2022/08/01

REPOSITORIES: GEO

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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.

Project description:Purpose: Wnt activation can induce regeneration in the early postnatal cochlea, but the regenerative capacity of the postnatal cochlea diminishes between P0 to P5. The goal of this study was to utilize RNA-Seq for the overall evaluation of expression patterns, comparing P1 and P4 newborn cochlea with and without Wnt activation. Methods: Sox2-CreER; β-cateninflox(Exon3)/+ mice were used to stabilize β-catenin in Sox2+ cochlear supporting cells. Tamoxifen was injected to the mother at either P1 or P4 to induce Cre activation in the pups. mRNA libraries were generated from treated and untreated pups 3 days after injection in triplicates (overall 12 samples) using ThruPLEX DNA-seq kit (Rubicon Genomics) and then sequenced using Illumina NextSeq500 Single-End 75bp. Reads were aligned to mm10 and quality control was assessed using FastQC and Qualimap followed by FeatureCounts, Sailfish v0.10.1 and tximport. Differential expression was evaluated using DESeq2 and subsequent pathway analysis was performed using Ingenuity Pathway Analysis Software (Qiagen). Results: 40-50 million reads were detected per sample. PCA and Pearson correlation demonstrated a clear segragation of the samples by age that was supported by the identification of 2914 differentially expressed genes between P1 and P4 samples. Additional affect of treatment resulted is 530 differentially expressed genes between treated and untreated samples at P1 and 304 genes were differentially expressed between treated and untreated samples at P4. Clear activation of the Wnt pathway was apparent at both time-points after treatment. However, activation of downstream pathways that may be involved in regeneration was detected at P1 but not at P4, mainly the involvement of cell-cycle and proliferation as well as Erbb2 associated genes. Conclusions: Our results suggest that the Wnt pathway is still active and can be further induced at early and late postnatal stages in a similar manner. However, at early stages, exogenous Wnt activation potentially promotes an arrest of maturation progression or even partial dedifferentiation of supporting cells into a less mature state that enables regeneration, while the downstream affect is not as prominenet later on. Proliferative capacity in the early postnatal cochlea is correlated with a unique transcriptional response that diminishes in the older cochlea, including a robust increase in the expression of cell cycle genes and cellular growth and development genes. Our results suggest that the downstream transcriptional response to Wnt activation is the key factor mediating a regenerative response, or a lack-thereof, in the mammalian cochlea and that efforts to stimulate regeneration in the adult auditory organ may require recapitulating the transcriptional response of the early neonatal cochlea to exogenous Wnt activation and subsequent alterations of silenced state of Wnt target genes. .

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Single cell transcriptomic analysis of P8 and P15 mouse cochlea (control and three overexpression conditions) and simultaneous single cell transcriptomic and accessible chromatin analysis of P1 and P8 mouse cochlea (wildtype)

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