Inner ear cochlear spiral ganglion neurons (SGNs) transmit sound information to the brainstem. Recent single cell RNA-Seq studies have revealed heterogeneities within SGNs. Nonetheless, much remains unknown about the transcriptome of SGNs, especially which genes are specifically expressed in SGNs. To address these questions, we needed a deeper and broader gene coverage than that in previous studies. We performed bulk RNA-Seq on mouse SGNs at five ages, and on two reference cell types (hair cells and glia). Their transcriptome comparison identified genes previously unknown to be specifically expressed in SGNs. To validate our dataset and provide useful genetic tools for this research field, we generated two knockin mouse strains: Scrt2-P2A-tdTomato and Celf4-3xHA-P2A-iCreER-T2A-EGFP. Our comprehensive analysis confirmed the SGN-selective expression of the candidate genes, testifying to the quality of our transcriptome data. These two mouse strains can be used to temporally label SGNs or to sort them.

Comprehensive transcriptome analysis of cochlear spiral ganglion neurons at multiple ages.

In vivo genetic mutation has become a powerful tool for dissecting gene function; however, multi-gene interaction and the compensatory mechanisms involved can make findings from single mutations, at best difficult to interpret, and, at worst, misleading. Hence, it is necessary to establish an efficient way to disrupt multiple genes simultaneously. CRISPR/Cas9-mediated base editing disrupts gene function by converting a protein-coding sequence into a stop codon; this is referred to as CRISPR-stop. Its application in generating zygotic mutations has not been well explored yet. Here, we first performed a proof-of-principle test by disrupting Atoh1, a gene crucial for auditory hair cell generation. Next, we individually mutated vGlut3 (Slc17a8), otoferlin (Otof) and prestin (Slc26a5), three genes needed for normal hearing function. Finally, we successfully disrupted vGlut3, Otof and prestin simultaneously. Our results show that CRISPR-stop can efficiently generate single or triple homozygous F0 mouse mutants, bypassing laborious mouse breeding. We believe that CRISPR-stop is a powerful method that will pave the way for high-throughput screening of mouse developmental and functional genes, matching the efficiency of methods available for model organisms such as Drosophila.

Simultaneous zygotic inactivation of multiple genes in mouse through CRISPR/Cas9-mediated base editing.

The stria vascularis (SV) in the cochlea generates and maintains the endocochlear potential, thereby playing a pivotal role in normal hearing. Knowing transcriptional profiles and gene regulatory networks of SV cell types establishes a basis for studying the mechanism underlying SV-related hearing loss. While we have previously characterized the expression profiles of major SV cell types in the adult mouse, transcriptional profiles of rare SV cell types remained elusive due to the limitation of cell capture in single-cell RNA-Seq. The role of these rare cell types in the homeostatic function of the adult SV remain largely undefined. In this study, we performed single-nucleus RNA-Seq on the adult mouse SV in conjunction with sample preservation treatments during the isolation steps. We distinguish rare SV cell types, including spindle cells and root cells, from other cell types, and characterize their transcriptional profiles. Furthermore, we also identify and validate novel specific markers for these rare SV cell types. Finally, we identify homeostatic gene regulatory networks within spindle and root cells, establishing a basis for understanding the functional roles of these cells in hearing. These novel findings will provide new insights for future work in SV-related hearing loss and hearing fluctuation.

/pdf/characterization-of-rare-spindle-and-root-cell-2ku8u9el6g.pdf

Characterization of rare spindle and root cell transcriptional profiles in the stria vascularis of the adult mouse cochlea.

Mammalian cochlear outer hair cells (OHCs) are essential for hearing. OHC degeneration causes severe hearing impairment. Previous attempts of regenerating new OHCs from cochlear supporting cells (SCs) had yielded cells lacking Prestin, a key motor protein for OHC function. Thus, regeneration of Prestin+ OHCs remains a challenge for repairing OHC damage in vivo. Here, we reported that successful in vivo conversion of adult cochlear SCs into Prestin+ OHC-like cells could be achieved by simultaneous expression of Atoh1 and Ikzf2, two key transcriptional factors necessary for OHC development. New OHC-like cells exhibited upregulation of hundreds of OHC genes and downregulation of SC genes. Single cell transcriptomic analysis demonstrated that the differentiation status of these OHC-like cells was much more advanced than previously achieved. Thus, we have established an efficient approach to promote regeneration of Prestin+ OHCs and paved the way for repairing damaged cochlea in vivo via transdifferentiation of SCs.

/pdf/dual-expression-of-atoh1-and-ikzf2-promotes-transformation-3vdg1wel6x.pdf

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

Abstract Atoh1 is essential for the development of both outer hair cells (OHCs) and inner hair cells (IHCs) in the mammalian cochlea. Whereas Ikzf2 is necessary for OHC development, the key gene required for IHC development remains unknown. We found that deletion of Tbx2 in neonatal IHCs led to their transdifferentiation into OHCs by repressing 26.7% of IHC genes and inducing 56.3% of OHC genes, including Ikzf2. More importantly, persistent expression of Tbx2 coupled with transient Atoh1 expression effectively reprogrammed non-sensory supporting cells into new IHCs expressing the functional IHC marker vGlut3. The differentiation status of these new IHCs was considerably more advanced than that previously reported. Thus, Tbx2 is essential for IHC development and co-upregulation of Tbx2 with Atoh1 in supporting cells represents a new approach for treating deafness related to IHC degeneration.

/pdf/development-and-transdifferentiation-into-inner-hair-cells-2xprr2zx.pdf

Development and transdifferentiation into inner hair cells require Tbx2

Characterizing a novel vGlut3-P2A-iCreER knockin mouse strain in cochlea

Background Conditional loss-of-function studies are widely conducted using the Cre/Loxp system because this helps circumvent embryonic or neonatal lethality problems. However, Cre strains specific to the inner ear are lacking, and thus lethality frequently occurs even in conditional knockout studies. Results Here, we report a Rorb-IRES-Cre knockin mouse strain in which the Cre recapitulates the expression pattern of endogenous Rorb (RAR-related orphan receptor beta). Analysis of Rorb-IRES-Cre/+; Rosa26-CAG-LSL-tdTomato/+ cochlear samples revealed that tdTomato was expressed at the apical turn only by E12.5. TdTomato was observed in the apical and middle turns but was minimally expressed in the basal turn at E15.5, E18.5, and P5. However, most of the auditory hair cells (HCs) and supporting cells (SCs) in all three turns were tdTomato+ at P15 and P30. Intriguingly, no tdTomato+ vestibular cells were detected until P5 and a few cells were present at P15 and P30. Finally, we also confirmed Rorb mRNA and protein expression in cochlear HCs and SCs at P30. Conclusions We reveal that Rorb expression exhibits an apical-to-basal gradient in cochleae. The cochlear-specific and apical-to-basal-gradient Rorb Cre activity should enable discrimination of gene functions in cochlear vs vestibular regions as well as low-frequency vs high-frequency regions in the cochlea.

Fate-mapping analysis using Rorb-IRES-Cre reveals apical-to-basal gradient of Rorb expression in mouse cochlea.

http://www.cell.com/article/S2211124723005156/pdf

Epistatic genetic interactions between Insm1 and Ikzf2 during cochlear outer hair cell development.

Handle everyday research tasks with reliable, citation-backed results

Your personal Research Agent to handle research tasks with citation-backed results

Popular Tasks used by Researchers

How can I help with your research?

Meet SciSpace

Get more enhanced response by uploading the PDFs you want me to reference.

No relevant PDFs in your library

SciSpace is the AI research assistant for academics. Run systematic literature reviews on 280M+ papers, and write papers with cited sources. Trusted by 1M+ students, PhDs & researchers.

SciSpace | AI for Research

Analyze PDFs

Code & Manuscripts

Funding & Grants

Literature & Patents

Medical & Clinical Data

Systematic Review

Visualize & Present

Web & Data

Build a Google Scholar-like website for your research.

Build a website

Create charts and images for your research

Create a Chart

Write a paper for submission to a journal

Draft a manuscript

Patent Search

Design eye-catching scientific posters in minutes.

Scientific Poster Generation

Systematic Literature Review

One task is running at the moment. Your messages will be shown right after.

Drag and drop or click here to browse

Loved by <highlight>1 million+</highlight> researchers

Extract a list of specific topics and their sources from unstructured text

Topics

Compare and analyze relevant papers that matches with your search

Papers

Get insights from PDFs and bookmarked papers from your library

My library

Recent searches

Try searching for:

Catch AI-generated content in scholarly and non-scholarly content

{ai} Detector

Ai Writer

Get PDF Summaries, highlighted text explanations 

Chat with PDF

Effortlessly create in-text citations and bibliographies in APA and 2,500 other formats

Citation generator

Get explanations, summaries, and answers on academic papers

Ease up your research workflow with {scispace}'s cohort of exciting AI tools

Elevate your academic writing skills and convey your ideas the way you want

Paraphraser

Explore our range of reading and writing tools

Your file is being prepared and should be ready in a few minutes. If it's a large file, it might take a bit longer. You can close this window, and we'll email you the file when it's done.

You have reached a maximum limit of <strong>{limit}</strong> columns in the table. Remove at least <strong>1</strong> column to add or create another one.

Ying Lu

Author Tools

Chat about Author

Papers

Characterizing a novel vGlut3-P2A-iCreER knockin mouse strain in cochlea

Fate-mapping analysis using Rorb-IRES-Cre reveals apical-to-basal gradient of Rorb expression in mouse cochlea.

Epistatic genetic interactions between Insm1 and Ikzf2 during cochlear outer hair cell development.