中国神经再生研究(英文版) ›› 2023, Vol. 18 ›› Issue (3): 577-581.doi: 10.4103/1673-5374.350208

• 原著:脊髓损伤修复保护与再生 • 上一篇    下一篇

以CRISPR/Cas9敲除双特异性磷酸酶2可促进斑马鱼早期Mauthner细胞轴突的再生

  

  • 出版日期:2023-03-15 发布日期:2022-08-26
  • 基金资助:

    中国国家自然科学基金项目(82071357),中国科学技术部项目(2019YFA0405600)

DUSP2 deletion with CRISPR/Cas9 promotes Mauthner cell axonal regeneration at the early stage of zebrafish

Guo-Jian Shao1, #, Xin-Liang Wang1, #, Mei-Li Wei2, Da-Long Ren2, *, Bing Hu1, *   

  1. 1School of Life Sciences, Division of Biomedical Sciences, University of Science and Technology of China, Hefei, Anhui Province, China; 2Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding, College of Animal Science and Technology, Anhui Agricultural University, Hefei, Anhui Province, China
  • Online:2023-03-15 Published:2022-08-26
  • Contact: Da-Long Ren, PhD, rendl@ustc.edu.cn; Bing Hu, PhD, bhu@ustc.edu.cn.
  • Supported by:
    This work was granted by the National Natural Science Foundation of China, No. 82071357 and Ministry of Science and Technology of China, No. 2019YFA0405600 (both to BH).

摘要:

既往研究显示,JNK可在轴突再生中起重要作用,而双特异性磷酸酶2位于细胞核中,已被证明可在体内介导JNK的去磷酸化;然而,其介导的JNK去磷酸化是否对轴突再生有影响,目前还无共识。为此以CRISPR/Cas9技术敲除斑马鱼的双特异性磷酸酶2基因发现,(1)出生后早期(8d龄内)dusp2-/-斑马鱼的Mauthner细胞轴突可以再生,而dusp2+/-则不能。通过单细胞电穿孔过表达双特异性磷酸酶2会抑制Mauthner细胞轴突的再生。(2)进一步蛋白印迹结果显示,双特异性磷酸酶2敲除可略微增加JNK的磷酸化水平,从而促进Mauthner细胞轴突的延伸。(3)这一实验结果表明,双特异性磷酸酶2可能通过增强JNK磷酸化来促进斑马鱼轴突的再生。

https://orcid.org/0000-0001-8627-5272 (Bing Hu); 

https://orcid.org/0000-0002-9210-0863 (Da-Long Ren)

关键词: CRISPR/Cas9技术, 双光子轴突横切, 中枢神经系统, 脊髓损伤, 轴突再生, 双特异性磷酸酶2, JNK, 单细胞电穿孔, Mauthner细胞, 斑马鱼

Abstract: Axon regeneration of central neurons is a complex process that is tightly regulated by multiple extrinsic and intrinsic factors. The expression levels of distinct genes are changed after central neural system (CNS) injury and affect axon regeneration. A previous study identified dusp2 as an upregulated gene in zebrafish with spinal cord injury. Here, we found that dual specificity phosphatase 2 (DUSP2) is a negative regulator of axon regeneration of the Mauthner cell (M-cell). DUSP2 is a phosphatase that mediates the dephosphorylation of JNK. In this study, we knocked out dusp2 by CRISPR/Cas9 and found that M-cell axons of dusp2–/– zebrafish had a better regeneration at the early stage after birth (within 8 days after birth), while those of dusp2+/– zebrafish did not. Overexpression of DUSP2 in Tg (Tol 056)  zebrafish by single-cell electroporation retarded the regeneration of M-cell axons. Western blotting results showed that DUSP2 knockout slightly increased the levels of phosphorylated JNK. These findings suggest that knocking out DUSP2 promoted the regeneration of zebrafish M-cell axons, possibly through enhancing JNK phosphorylation. 

Key words: axon regeneration, central nervous system, CRISPR/Cas9, DUSP2, JNK, Mauthner cell, single-cell electroporation, spinal cord injury, two-photon axotomy, zebrafish