| objective :(1)Construct a reliable and stable Single-cell RNA sequencing(Sc RNA-seq)workflow for the adult mouse cochlear and vestibular cells by optimizing the previous Sc RNA-seq method.(2)Identification of various cochlear and vestibular cell types and their marker genes,reveal the localization and expression of the known deafness genes in the inner ear.(3)In-depth analysis of the marker genes and biological processes of different types of hair cells(HCs),reveal the similarity and specificity of HCs,reveal the expression patterns of known HC-related genes in HCs.(4)In-depth analysis of the differentially expressed gene(DEG)Cdh23 in cochlear and vestibular Sc RNA-seq data,reveal the effect of Cdh23 mutation on the survival of the HCs in C57BL/6 aging mice.Method :(1)We optimized the previously published dissection,digestion,and cell dissociation process of cochlear and vestibular cells.We dissected the cochlea and vestibular end organs of 10-week-old adult CBA mice and obtained the tissues mainly containing the cochlear basilar membrane and vestibular macula of the utricle and saccule.Collagenase IV was used for the digestion to obtain cochlear and vestibular single-cell suspensions.We made the c DNA library immediately after completing the single-cell suspension according to the relevant manual of 10 x Genomics,and sent it to Shanghai Illumina Company for sequencing using Nextseq 6000.Seurat was used to perform data quality control and clustering analysis using R.(2)We screened the known marker genes of different types of cochlear and vestibular cells based on various published literatues,and checked the marker genes of each cell cluster to identify the cell types in our Sc RNA-seq data.We compared the transcriptome of each cell type of the cochlea or vestibule with the transcriptome of all other cell types in the same data,identified the marker genes of different cell types;We summarized the known deafness genes and showed their expression pattern in different inner ear cell types.(3)We compared the transcriptome between each type of HCs and all other types of cells in the cochlea/vestibule,performed the in-depth literature review for the top 20 HC marker genes,and analyzed their enriched biological process(BP).We conducted two independent analyses to uncover the transcriptomic similarity among HC types,1.Cochlear and vestibular Sc RNA-seq data were merged and re-analyzed for cell type identification and 3 dimension Principal Component Analysis(3d PCA)revealment.2.All HCs were subsetted and merged for DEGs identification and 3d PCA revealment.Through the comparison of every two types of HCs,the DEGs among HCs were identified,and the representative DEGs were further validated by RNAscope in situ hybridization.(4)As the most representative DEGs,the expression of Cdh23 was further characterized in our data;the Cdh23 mutation P30 and 1-year-old mice were used for testing the survival of the inner ear HCs based on the Myo6 immunofluorescence staining.Result:(1)We successfully detected the normal morphology of various inner ear sensory epithelial cells: inner hair cells(IHC),outer hair cells(OHC),type I HC,type II HC,Neurons,and various supporting cell types.The quality control of our Sc RNA-seq data was good,which enabled the identification of 29 and 22 cell clusters from cochlear and vestibular cells,respectively.(2)25 and 17 cell types were identified from cochlear and vestibular data,respectively;maker genes were characterized for all cell types,and 138 deafness genes were mapped to different cell types of cochlea and vestibule.(3)The top 20 marker genes of IHC,OHC,type I HC,and type II HC were revealed,containing the classic known marker genes,genes that have been popularly studied in recent years,and novel marker genes;we also showed the top 10 activated and suppressed BP in all HCs.For the first time,we revealed the similarities among cochlear and vestibular HCs from the perspective of the overall transcriptome;OHC is closer to type I HC,and IHC is closer to type II HC.Pairwise comparisons among HCs revealed their differences,and we validated m RNA transcription of 12 typical DEGs in the Organ of Corti(OC)and utricle macula.The expression pattern of232 HC related genes in 4 HCs were shown in the heatmap.(4)Cdh23 is a marker gene of cochlear and vestibular hair cells,and its expression level in the cochlea is significantly higher than in vestibular HCs.The cochlea of Cdh23 gene mutant mice has severe hair cell loss at one year after birth,and the loss of OHC is mainly in the high-frequency region;the loss of vestibular HC in 1-year-old gene mutant mice was not observed.Conclusion: We successfully constructed a reliable workflow for Sc RNA-seq on cochlear and vestibular cells from 10-week-old adult mice,identified different inner ear cell types in our Sc RNA-seq data,and mapped the expression of deafness genes in the inner ear.The transcriptome characteristics of different types of HCs were revealed in detail;Their similarities and differences at the transcriptome level were comprehensively analyzed.Compared with vestibular hair cells,the Cdh23 gene is more important for the survival of cochlear hair cells. |
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