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Single cell transcriptional profiles of control and Ikzf2-transfected mouse cochlear cells

ABSTRACT: In order to determine the transcriptional effect of Ikzf2 overexpression in mammalian auditory hair cells, mouse cochleae were transfected with either a control GFP or a Ikzf2 virus between postnatal day 1 and 3 (P1-3) and then harvested for single cell gene expression profiling at postnatal day 8 (P8) on the 10X Genomics Single Cell 3' v2 platform.

ORGANISM(S): Mus musculus

PROVIDER: GSE120462 | GEO | 2018/11/28

REPOSITORIES: GEO

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Ikzf2/helios mutant cochlear RNA-seq analysis

Project description:Given the potential role of the helios transcription factor in postnatal outer hair cell gene regulation indicated by our RiboTag translatome analyses, our next goal was to identify and validate genes that are regulated by helios in outer hair cells by performing RNA-seq on P8 cello mutant animals compared to wild-type littermates.

2018-11-28 | GSE116702 | GEO

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)

Project description: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.

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Ikzf2/helios mutant cochlear RNA-seq analysis

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

Project description:This study examined transcripts that are enriched in neonatal mouse cochlear supporting cells at postnatal day 1 and postnatal day 6 after inhibition of the Notch signaling pathway. Cochleas from postnatal day 0 and postnatal day 5 were cultured for 24 hours in the gamma secretase inhibitor DAPT or DMSO as a vehicle control. 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 0+24 hours culture and postnatal day 5 + 24 hours culture. (corresponding to P1 and P6).

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Dual expression of Atoh1 and Ikzf2 promotes transformation of adult cochlear supporting cells into outer hair cells

Project description:Mammalian cochlear outer hair cells (OHCs) are essential for hearing. Severe hearing impairment follows OHC degeneration. Previous attempts at regenerating new OHCs from cochlear supporting cells (SCs) have been unsuccessful, notably lacking expression of the key OHC motor protein, Prestin. Thus, regeneration of Prestin+ OHCs represented a barrier to restoring auditory function in vivo. Here, we reported the successful in vivo conversion of adult mouse cochlear SCs into Prestin+ OHC-like cells through the concurrent induction of two key transcriptional factors known to be necessary for OHC development: Atoh1 and Ikzf2. Single cell RNA sequencing reveals the upregulation of 729 OHC genes and downregulation of 331 SC genes in OHC-like cells. The resulting differentiation status of these OHC-like cells was much more advanced than previously achieved. This study thus established an efficient approach to induce the regeneration of Prestin+ OHCs, paving the way for in vivo cochlear repair via SC transdifferentiation.

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