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

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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"

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