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Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss

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Abstract

Hair cells play key roles in hearing and balance, and hair cell loss would result in hearing loss or vestibular dysfunction. Cellular and molecular research in hair cell biology provides us a better understanding of hearing and deafness. Zebrafish, owing to their hair cell-enriched organs, have been widely applied in hair cell-related research worldwide. Similar to mammals, zebrafish have inner ear hair cells. In addition, they also have lateral line neuromast hair cells. These different types of hair cells vary in morphology and function. However, systematic analysis of their molecular characteristics remains lacking. In this study, we analyzed the GFP+ cells isolated from Tg(Brn3c:mGFP) larvae with GFP expression in all hair cells using single-cell RNA-sequencing (scRNA-seq). Three subtypes of hair cells, namely macula hair cell (MHC), crista hair cell (CHC), and neuromast hair cell (NHC), were characterized and validated by whole-mount in situ hybridization analysis of marker genes. The hair cell scRNA-seq data revealed hair cell-specific genes, including hearing loss genes that have been identified in humans and novel genes potentially involved in hair cell formation and function. Two novel genes were discovered to specifically function in NHCs and MHCs, corresponding to their specific expression in NHCs and MHCs. This study allows us to understand the specific genes in hair cell subpopulations of zebrafish, which will shed light on the genetics of both human vestibular and cochlear hair cell function.

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Availability of data and materials

All the high-throughput sequencing data generated in this study have been deposited in the Gene Expression Omnibus database under accession number GSE221471 and are available at the following URL: https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE85337. All the experimental materials generated in this study are available from the corresponding authors upon reasonable request.

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Funding

This work was supported in part by grants from the National Natural Science Foundation of China, http://www.nsfc.gov.cn (2018YFA0801004 and 81870359 received by Dong Liu; 31900484 to Gangcai Xie); Natural Science Foundation of Jiangsu Province, http://kjjh.jspc.org.cn (BK20180048 and BRA2019278 received by Dong Liu; BK20190924 to Gangcai Xie; BK20190920 to Guanyun Wei). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. Fuping Qian, Guanyun Wei, Yajing Gao and Xin Wang have contributed equally to this work.

Authors and Affiliations

  1. School of Life Sciences, Nantong Laboratory of Development and Diseases, Nantong University, Nantong, 226019, China

    Fuping Qian, Guanyun Wei, Jie Gong, Chao Guo, Cheng Wang & Dong Liu

  2. Co-Innovation Center of Neuroregeneration, School of Life SciencesKey Laboratory of Neuroregeneration of Jiangsu and Ministry of Education, Nantong University, Nantong, 226019, China

    Yajing Gao, Xin Wang, Xiaoning Wang, Xu Zhang, Jinxiang Zhao, Mengting Xu, Yuebo Hu, Guoli Yin, Renjie Chai & Dong Liu

  3. State Key Laboratory of Bioelectronics, Co-Innovation Center of Neuroregeneration, Department of Otolaryngology Head and Neck Surgery, Zhongda Hospital, Jiangsu Province High-Tech Key Laboratory for Bio-Medical Research, School of Life Science and Technology, Southeast University, Nanjing, 210096, China

    Renjie Chai

  4. Institute for Stem Cell and Regeneration, Chinese Academy of Science, Beijing, 100864, China

    Renjie Chai

  5. Beijing Key Laboratory of Neural Regeneration and Repair, Capital Medical University, Beijing, 100069, China

    Renjie Chai

  6. Department of Otolaryngology Head and Neck Surgery, Sichuan Provincial People’s Hospital, University of Electronic Science and Technology of China, Chengdu, 610072, China

    Renjie Chai

  7. Institute of Reproductive Medicine, Medical School, Nantong University, Nantong, 226001, China

    Jiahui Kang & Gangcai Xie

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  1. Fuping Qian
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  4. Xin Wang
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Contributions

FQ, DL, GX, and RC conceived and designed the experiments, and wrote the manuscript. FQ, YG, XW, JG, CG, XW, XZ, JZ, CW, MX, YH, and GY performed the experiments. FQ, GW, JK, GX, DL, and RC analyzed the data. All authors read and approved the final manuscript.

Corresponding authors

Correspondence to Renjie Chai, Gangcai Xie or Dong Liu.

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The authors declare that they have no conflicts of interest.

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All zebrafish experimentation was carried out in accordance with the NIH Guidelines for the care and use of laboratory animals (http://oacu.od.nih.gov/regs/index.htm) and ethically approved by the Administration Committee of Experimental Animals, Jiangsu Province, China (Approval ID: 20180405-039).

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18_2022_4410_MOESM1_ESM.pdf

Supplementary file1 Fig. S1. The procedure of single cell preparation and cell sorting using the FACS method. Fig. S2. The FACS sorting data. Cells of Tg(Brn3c:mGFP) zebrafish at 6 dpf were sorted. Fig. S3. The expression pattern of GFP, tectb and zpld1a gene in sequenced cells. The feature plot and violin plot are shown. Fig. S4. The expression patterns of the cluster 0- and cluster 7-enriched genes. The feature plot and violin plot are shown for each gene. Fig. S5. The expression pattern of the cluster 1-enriched gene. The feature plot and violin plot are shown for each gene. Fig. S6. The expression patterns of the cluster 9-enriched genes. The feature plot and violin plot are shown for each gene. Fig. S7. The expression patterns of some hair cell marker genes. The feature plot and violin plot are shown for each gene. Fig. S8. The expression pattern of capgb and mb gene. The feature plot and violin plot are shown for each gene. Fig. S9. The verification of gene knockdown using RT-PCR. The gene expression was reduced in capgb-morphants and mb-morphants compared to the control (Ctrl). The white dotted boxes indicate the gene expression detected. (PDF 1521 kb)

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Qian, F., Wei, G., Gao, Y. et al. Single-cell RNA-sequencing of zebrafish hair cells reveals novel genes potentially involved in hearing loss. Cell. Mol. Life Sci. 79, 385 (2022). https://doi.org/10.1007/s00018-022-04410-2

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