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. 2020 Feb 15;9(1):1-15.
eCollection 2020.

Characterization of the transcriptomes of Atoh1-induced hair cells in the mouse cochlea

Li-Man Liu  1 , Li-Ping Zhao  1   2 , Ling-Jie Wu  1   2 , Luo Guo  1   2 , Wen-Yan Li  1   2 , Yan Chen  1   2
Affiliations

Characterization of the transcriptomes of Atoh1-induced hair cells in the mouse cochlea

Li-Man Liu et al. Am J Stem Cells. .

Abstract

Postnatal mammalian cochlear hair cells (HCs) can be regenerated by direct transdifferentiation or by mitotic regeneration from supporting cells through many pathways, including Atoh1, Wnt, Hedgehog and Notch signaling. However, most new HCs are immature HCs. In this study we used RNA-Seq analysis to compare the differences between the transcriptomes of Atoh1 overexpression-induced new HCs and the native HCs, and to define the factors that might help to promote the maturation of new HCs. As expected, we found Atoh1-induced new HCs had obvious HC characteristics as demonstrated by the expression of HC markers such as Pou4f3 and Myosin VIIA (Myo7a). However, Atoh1-induced new HCs had significantly lower expression of genes that are related to HC function such as Slc26a5 (Prestin), Slc17a8 and Otof. We found that genes related to HC cell differentiation and maturation (Kcnma1, Myo6, Myo7a, Grxcr1, Gfi1, Wnt5a, Fgfr1, Gfi1, Fgf8 etc.) had significantly lower expression levels in new HCs compared to native HCs. In conclusion, we found a set of genes that might regulate the differentiation and maturation of new HCs, and these genes might serve as potential new therapeutic targets for functional HC regeneration and hearing recovery.

Keywords: Inner ears; RNA-Seq; cochlea; gene expression; hair cell maturation; hair cell regeneration; supporting cells.

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Conflict of interest statement

None.

Figures

Figure 1
Figure 1
Increased expression of Atoh1 in SCs induced new cochlear HCs in neonatal mouse. A. Diagram of the organ of Corti and scheme of Atoh1 overexpression. Cochlear hair cells are surrounded by SCs. Tamoxifen was injected intraperitoneally at the late stage of P3 and sensory epithelium was harvested at P7. B. In experimental mouse (Sox2-CreER/Atoh1-OE/Atoh1-nGFP/tdTomato mouse), the new HCs were labelled with green fluorescent protein(GFP) and tdTomato red fluorescent protein, native HCs were labeled with green fluorescent protein, SCs were labeled with tdTomato red fluorescent protein. OHC, outer hair cell; IHCs, inner hair cells; SCs, supporting cells; GER, greater epithelial ridge; LER, lesser epithelial ridge; P, postnatal; IF, immunofluorescence. Scale bar, 50 µm or 10 µm.
Figure 2
Figure 2
Screening new hair cells and native cochlear hair cells by flow cytometry. A. Scheme to perform flow cytometry. Tamoxifen was injected intraperitoneally at the late stage of P3 and sensory epithelium was harvested at P7. B. Wild type mice were used as negative control. C. Three groups of cells were obtained in experimental mice. D. Atoh1-eGFP mice were used as positive control. E. Immunofluorescent staining was used to detect the purity of sorted cells. F. qPCR was performed to validate the purity of sorted cells. FACS, fluorescence activated cell sorting. Scale bar, 50 µm.
Figure 3
Figure 3
Differentially expressed genes in native HCs and new HCs. A. All differentially expressed genes in native HCs and new HCs (fold change > 1.5, P < 0.05). The red dot represents the expression level of transcripts from native HCs, and the cyan dot represents the expression level of the same transcript from new HCs. B. The expression levels of HC and SC markers.
Figure 4
Figure 4
Top 200 genes expressed in native HCs and new HCs. A. The top 200 genes expressed in native HCs in decreasing order. The cyan numbers on the right side of each panel represent the ranking of the same genes in new HCs. B. Top 200 genes in new HCs in decreasing order. The red numbers on the right side of each panel represent the ranking of the same genes in native HCs.
Figure 5
Figure 5
Top differentially expressed genes highly enriched in native HCs and new HCs. A. Top 100 genes differentially expressed in native HCs and the black numbers on the right side of each panel represent the gene expression fold change in native HCs compared to new HCs. Total 52 differentially expressed genes highly enriched in new HCs and the black numbers on the right side of each panel represent the gene expression fold change in new HCs compared to native HCs. B. The top 20 genes exclusively expressed in native HCs or new HCs.
Figure 6
Figure 6
The results of RNA-Seq assay and qPCR assay. A. The differentially expressed genes related with HC differentiation and maturation from RNA-Seq data. B. The qPCR results of the differentially expressed genes.
Figure 7
Figure 7
GO analysis of differentially expressed genes in new HCs and native HCs. A. The functional gene groups were enriched in native HCs. B. The functional gene groups were enriched in new HCs. C. The STRING protein interaction analysis of genes that are differentially expressed in native HCs (red) and new HCs (blue).
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