Journal of Otolaryngology and Ophthalmology of Shandong University ›› 2019, Vol. 33 ›› Issue (3): 49-55.doi: 10.6040/j.issn.1673-3770.1.2018.039

• Original Article • Previous Articles     Next Articles

Temporal and spatial expression of the deafness-causing gene EYA4 during the embryonic development of zebrafish

Fangzhou QIAN1,2,Xiaobo HUANG1,Qinjun WEI3,Zhibin CHEN1()   

  1. 1. Department of Otorhinolaryngology, The First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, Jiangsu, China
    2. Department of Otorhinolaryngology, Jiangyin People′s Hospital, Wuxi 214400, Jiangsu, China
    3. Department of Medical Genetics, School of Basic Medicine, Nanjing Medical University, Nanjing 211166, Jiangsu, China
  • Received:2018-11-12 Revised:2018-12-21 Online:2019-05-20 Published:2019-08-07
  • Contact: Zhibin CHEN E-mail:czbnj@163.com

RichHTML

14

PDF (PC)

460

Abstract: Objective

To study expression and localization of the deafness-causing gene EYA4 in different developmental stages of the zebrafish embryo and to lay a foundation for further investigation of the possible underlying mechanism of deafness using the zebrafish model.

Methods

Bioinformatics tools were used to analyze the homology of the zebrafish and human EYA4 gene. Temporal and spatial expression of the eya4 gene in the early stages of development of the zebrafish embryo (64-cell, oblong-sphere, 50%-epiboly, 15-somite, 24 hpf, 36 hpf, 48 hpf, 60 hpf, 72 hpf, and 1-week) was analyzed using whole-embryo in situ hybridization and semi-quantitative PCR.

 Results

Results of bioinformatics analysis showed that the eya4 gene of zebrafish and the human EYA4 gene are highly homologous. Semi-quantitative PCR results showed that the eya4 gene of zebrafish was not expressed at the early stage of embryonic development (64-cell~50%-epiboly), and gradually began to be expressed at the 15-somite stage; its expression was highest at 24-48 hpf, after which expression levels slightly reduced and then stabilized. Results of whole-embryo in situ hybridization showed that the eya4 gene was not expressed at the following three stages of zebrafish development: the 64-cell stage, oblong-sphere stage, and 50 %-epiboly stage, which indicated that the eya4 gene shows non-maternal expression. At the 15-somite stage, low expression of the eya4 gene was detected. Expression levels of eya4 were highest during 24 to 48 hpf, and eya4 was expressed in parts of the nervous system in the head, the auditory hair cells of the inner ear, and the lateral line system. Expression levels of eya4 reduced and remained stable at 60 hpf to 1-week post fertilization.

 Conclusion

The zebrafish eya4 gene and the human EYA4 gene are highly homologous. Studying the temporal and spatial expression patterns of the EYA4 gene during early development of zebrafish embryos will help determine its function in embryonic development and the mechanism of the deafness caused by the mutation of the EYA4 gene using the zebrafish model.

Key words: EYA4, Zebrafish, Embryonic development, Whole-embryo in situ hybridization, Spatial and temporal expression

CLC Number: 

  • R764.43

Table 1

In vitro transcriptional reaction system"

成分 体积 终浓度
Template DNA 5 μL 300~500 ng
5 Transcription buffer 2 μL 1×
DTT (0.1M) 1 μL 10 mM
DIG-RNA labeling mix (10×) 1 μL 1×
RNAsein 0.5 μL 20 U
T7 RNA polymerase (20 U/μL) 0.5 μL 10 U

Fig.1

Comparative analysis of the domains of zebrafish and human EYA4 proteins"

Fig.2

Homology of the human, chimpanzee, mouse, and zebrafish EYA4 protein sequences"

Fig.3

Phylogenetic tree generated using sequences of EYA4 proteins"

Fig.4

Expression levels of the eya4 gene in zebrafish at different developmental stages detected using RT-PCR"

Fig.5

In situ hybridization of the eya4 gene in different developmental stages of zebrafish embryos"

1 Meena R , Ayub M . Genetics of human hereditary hearing impairment[J]. J Ayub Med Coll Abbottabad, 2017, 29(4): 671-676.
2 Egilmez OK , Kalcioglu MT . Genetics of nonsyndromic congenital hearing loss[J]. Scientifica (Cairo), 2016, 2016: 7576064. doi:10.1155/2016/7576064 .
doi: 10.1155/2016/7576064
3 Stelma F , Bhutta MF . Non-syndromic hereditary sensorineural hearing loss: review of the genes involved[J]. J Laryngol Otol, 2014, 128(1): 13-21. doi:10.1017/S00222-15113003265 .
doi: 10.1017/S00222-15113003265
4 O′Neill ME , Marietta J , Nishimura D , et al . A gene for autosomal dominant late-onset progressive non-syndromic hearing loss, DFNA10, maps to chromosome 6[J]. Hum Mol Genet, 1996, 5(6): 853-856.
5 Wayne S , Robertson NG , DeClau F , et al . Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus[J]. Hum Mol Genet, 2001, 10(3): 195-200. doi:10.1093/hmg/10.3.195 .
doi: 10.1093/hmg/10.3.195
6 Liu F , Hu JJ , Xia WJ , et al . Exome sequencing identifies a mutation in EYA4 as a novel cause of autosomal dominant non-syndromic hearing loss[J]. PLoS One, 2015, 10(5): e0126602. doi:10.1371/journal.pone.0126602 .
doi: 10.1371/journal.pone.0126602
7 Tan MX , Shen XF , Yao J , et al . Identification of I411K, a novel missense EYA4 mutation causing autosomal dominant non-syndromic hearing loss[J]. Int J Mol Med, 2014, 34(6): 1467-1472. doi:10.3892/ijmm.2014.1939 .
doi: 10.3892/ijmm.2014.1939
8 Nicolson T . The genetics of hair-cell function in zebrafish[J]. J Neurogenet, 2017, 31(3): 102-112. doi:10.1080/01677063.2017.1342246 .
doi: 10.1080/01677063.2017.1342246
9 Chitramuthu BP , Bennett HP . High resolution whole mount in situ hybridization within zebrafish embryos to study gene expression and function[J]. J Vis Exp, 2013(80): e50644. doi:10.3791/50644 .
doi: 10.3791/50644
10 Thisse C , Thisse B . High-resolution in situ hybridization to whole-mount zebrafish embryos[J]. Nat Protoc, 2008, 3(1): 59-69. doi:10.1038/nprot.2007.514 .
doi: 10.1038/nprot.2007.514
11 孙菲菲, 胡松群, 唐艳, 等 . 高通量基因捕获测序技术在12个耳聋家庭中的应用[J]. 山东大学耳鼻喉眼学报, 2017,31(5):45-49.doi:10.6040/j.issn.1673-3770.0.2017.351 .
doi: 10.6040/j.issn.1673-3770.0.2017.351
SUN Feifei , HU Songqun , TANG Yan , et al . A next-generation sequencing gene panel for molecular diagnosis in twelve Chinese families with non-syndromic sensorineural hearing loss[J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2017, 31(5): 45-49. doi:10.6040/j.issn.1673-3770.0.2017.351 .
doi: 10.6040/j.issn.1673-3770.0.2017.351
12 Venkatesh MD , Moorchung N , Puri BP . Genetics of non syndromic hearing loss[J]. Med J Armed Forces India, 2015, 71(4): 363-368. doi:10.1016/j.mjafi.2015.07.003 .
doi: 10.1016/j.mjafi.2015.07.003
13 He YZ , Bao BE , Li HW . Using zebrafish as a model to study the role of epigenetics in hearing loss[J]. Expert Opin Drug Discov, 2017, 12(9): 967-975. doi:10.1080/17460441.2017.1340270 .
doi: 10.1080/17460441.2017.1340270
14 Ernest S , Rauch GJ , Haffter P , et al . Mariner is defective in myosin VIIA: a zebrafish model for human hereditary deafness[J]. Hum Mol Genet, 2000, 9(14): 2189-2196.doi:10.1093/hmg/9.14.2189 .
doi: 10.1093/hmg/9.14.2189
15 Abdelhak S , Kalatzis V , Heilig R , et al . A human homologue of the Drosophila eyes absent gene underlies branchio-oto-renal (BOR) syndrome and identifies a novel gene family[J]. Nat Genet, 1997, 15(2): 157-164. doi:10.1038/ng0297-157 .
doi: 10.1038/ng0297-157
16 Schönberger J , Wang LB , Shin JT , et al . Mutation in the transcriptional coactivator EYA4 causes dilated cardiomyopathy and sensorineural hearing loss[J]. Nat Genet, 2005, 37(4): 418-422. doi:10.1038/ng1527 .
doi: 10.1038/ng1527
[1] ZHONG Xiaosheng, YANG Haidi, ZHENG Yiqing. Treatment and outcome of tinnitus in sudden deafness [J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2019, 33(4): 43-46.
[2] ZHANG Junjun, LIU Haili, LIU Jiafu, QIN Xiaofeng, DU Hongming, WANG Houhui. Clinical efficacy and safety of different methods of administration of glucocorticoids in the treatment of sudden deafness [J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2019, 33(4): 52-55.
[3] TONG Qiaozhen, ZHAO Yan, CHENG Sihua. The constructive and mediating effects of knowledge/attitude/belief/practice interference on negative emotion in patients with sudden deafness [J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2019, 33(4): 162-164.
[4] Xingkuan BU. WHO program on the prevention of deafness and China's response and role [J]. Journal of Otolaryngology and Ophthalmology of Shandong University, 2019, 33(3): 3-8.
[5] XU Ming, LUO Xinggu, TANG Hongbo, JIANG Qingshan. Clinical characteristics and prognostic factors for sudden idiopathic sensorineural hearing loss in children [J]. J Otolaryngol Ophthalmol Shandong Univ, 2018, 32(5): 41-44.
[6] CHEN Wei, HU Zhongnan, TONG Zhaojun, LI Xiangyu, LIU Wenjun. Characteristics of auditory brainstem response to high-frequency stimulus in young and middle-aged patients with sudden deafness and no history of vertigo or cardiocerebrovascular disease [J]. J Otolaryngol Ophthalmol Shandong Univ, 2018, 32(5): 45-49.
[7] ZHU Wenyan, JIN Xin, SHE Wandong, MA Yongchi. Assessment of psychopathological characteristics in 30 patients with sudden sensorineural hearing loss [J]. J Otolaryngol Ophthalmol Shandong Univ, 2018, 32(5): 50-52.
[8] HUANG Fang, ZHENG Zhijuan, XIE Lei. Relationship of anxiety and depression with illness stage in patients with sudden hearing loss and its influencing factors [J]. J Otolaryngol Ophthalmol Shandong Univ, 2018, 32(4): 1-6.
[9] SONG Feng, GAN Bin, XU Anting, WANG Jian. Long-term low intensity noise exposure reduce the number of ribbon synapses in guinea pigs. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2017, 31(5): 41-44.
[10] ZHANG Yubo, YUE Liyan, YIN Xiaoyan, LÜ Zhe, SHAN Chunguang. Effect of tinnitus treatment instrument combined with Ginkgo biloba extract on hearing and hemorheology in patients with sudden deafness and tinnitus. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2017, 31(5): 50-53.
[11] ZHANG Zhuan, LIU Tao, BAI Zhili, ZHOU Changming. Evolution of oxidative stress in the pathogenesis and treatment of noise-induced hearing loss. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2017, 31(5): 101-103.
[12] . Evaluation of hearing levels, speech functions and cognitive abilities of cochlear implantation in deaf children with abnormal white matter. [J]. J Otolaryngol Ophthalmol Shandong Univ, 2017, 31(2): 43-48.
[13] MA Rongfeng, ZHU Fugao, SUN Meihong, FU Tao, ZHANG Xiaoheng, CAO Lin. Analysis on the relevant prognostic factors of sudden deafness and selection of course of treatment. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2016, 30(4): 69-74.
[14] SUN Changzhi, LIU Wenlong, LI Yan, LUO Renzhong. Diagnosis and treatment of otitis media with effusion in children with bilateral sensorineural deafness. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2015, 29(4): 11-14.
[15] ZHONG Xiaosheng, YANG Haidi, ZHENG Yiqing. Characteristics of tinnitus in patients with idiopathic sudden sensorineural hearing loss. [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2015, 29(4): 15-18.
Viewed
Full text


Abstract

Cited
  Shared   
  Discussed   
[1] LIN Bin,WANGHui-ge . Functional endoscopic sinus surgery, FESS[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2006, 20(6): 481 -487 .
[2] . [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2006, 20(1): 84 -87 .
[3] NIU Shanli,CHAI Maowen,LI Zhenxiu . Endoscopic rhinoplasty of inferior turbinate in 60 patients with chronic hypertrophic rhinitis[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2006, 20(1): 16 -18 .
[4] . [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2006, 20(1): 90 -91 .
[5] . [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(3): 200 -203 .
[6] JI Xiao-bin,DENG Jia-de,ZANG Lin-quan,WANG Lei,XIE Jun . Blood histamine in guinea pigs with allergic rhinitis[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(3): 228 -230 .
[7] BAI Guang-ping,LI Jun,DONG Pin . Endscopic surgery for chronic nasosinusitis and nasal polyps in 180 cases[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(3): 242 -243 .
[8] . [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(3): 248 -249 .
[9] . [J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(3): 285 -285 .
[10] ZHANG Ji-zhong,LI Da-jian . Chondroseptum or ethmoid in repairing of nasal septum perforation under endoscopy[J]. JOURNAL OF SHANDONG UNIVERSITY (OTOLARYNGOLOGY AND OPHTHALMOLOGY), 2008, 22(4): 336 -337 .
Website Copyright: Editorial Office of Journal of Otolaryngology and Ophthalmology of Shandong University
Address: No.27 Shanda South Road, Jinan, Shandong, China. Post code: 250100 Tel: +86-0531-88366912 Email: ebhxb@sdu.edu.cn Supported by Beijing Magtech Co.Ltd