山东大学耳鼻喉眼学报 ›› 2019, Vol. 33 ›› Issue (3): 49-55.doi: 10.6040/j.issn.1673-3770.1.2018.039

• 论著 • 上一篇    下一篇

致聋基因 EYA4 在斑马鱼胚胎发育中的时空表达特征分析

钱方舟1,2,黄啸博1,魏钦俊3,陈智斌1()   

  1. 1. 南京医科大学第一附属医院 江苏省人民医院耳鼻咽喉科，江苏 南京210029
    2. 江阴市人民医院耳鼻咽喉科，江苏 无锡214400
    3. 南京医科大学基础医学院医学遗传学系，江苏 南京211166
  • 收稿日期:2018-11-12 修回日期:2018-12-21 出版日期:2019-05-20 发布日期:2019-08-07
  • 通讯作者: 陈智斌 E-mail:czbnj@163.com
  • 基金资助:
    江苏省重点研发计划（社会发展）项目(BE2016762)

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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摘要： 目的

研究致聋基因EYA4在斑马鱼胚胎发育不同时期的表达及其定位，为进一步利用斑马鱼模型探索其可能的致病机制奠定基础。

方法

利用生物信息学分析斑马鱼与人EYA4基因的同源性，同时通过整胚原位杂交和半定量PCR分析斑马鱼eya4基因在早期64细胞胚胎、oblong?sphere、50%?epiboly、15?somite、24 hpf、36 hpf、48 hpf、60 hpf、72 hpf和1周的时空表达分析。

 结果

生物信息学分析显示斑马鱼eya4与人EYA4基因高度同源。半定量PCR结果显示斑马鱼eya4在胚胎发育早期（64细胞~50%?epiboly）不表达，15?somite期逐渐开始表达并于24~48 hpf达到高峰，此后表达水平稍降低并维持在一定水平。全胚原位杂交检测结果显示eya4在斑马鱼64-细胞，oblong?sphere和50%?epiboly时期均未表达，提示该基因为非母源性表达，在15?somite期开始出现可检测的表达，24 ~ 48 hpf期表达水平达到顶峰，并且在头部神经系统、听囊内耳毛细胞和侧线系统中均有表达，至60 hpf~1?week期表达水平减弱并维持稳定在一定水平。

 结论

斑马鱼eya4与人致聋基因EYA4具有高度同源性，通过研究eya4在斑马鱼胚胎早期发育中的时空表达模式，可为后续利用斑马鱼作为动物模型深入研究人类EYA4在胚胎发育中的作用及其突变致聋机制奠定重要的实验基础。

关键词: EYA4基因, 斑马鱼, 胚胎发育, 整胚原位杂交, 时空表达

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

中图分类号: 

  • R764.43

表1

体外转录反应体系"

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

图1

斑马鱼和人EYA4蛋白结构域比较"

图2

人、黑猩猩、小鼠、大鼠和斑马鱼EYA4蛋白同源比对"

图3

EYA4蛋白系统进化树"

图4

RT-PCR检测斑马鱼不同发育时期eya4基因表达"

图5

斑马鱼胚胎不同发育时期eya4基因整胚原位杂交"

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