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

Neural Regeneration Research 2023年第3期18卷 577-581页

作者： Guo-Jian Shao Xin-Liang Wang Mei-Li Wei Da-Long Ren Bing Hu School of Life Sciences Division of Biomedical SciencesUniversity of Science and Technology of ChinaHefeiAnhui ProvinceChina Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-Breeding College of Animal Science and TechnologyAnhui Agricultural UniversityHefeiAnhui ProvinceChina

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.

关键词： axon regeneration central nervous system CRISPR/Cas9 DUSP2 JNK mauthner cell single-cell electroporation spinal cord injury two-photon axotomy zebrafish

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Dual Oxidase Mutant Retards mauthner-cell Axon Regeneration at an Early Stage via Modulating Mitochondrial Dynamics in Zebrafish

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Neuroscience Bulletin 2020年第12期36卷 1500-1512页

作者： Lei-Qing Yang Min Chen Da-Long Ren Bing Hu Eye Center The First Affiliated Hospital of USTCHefei National Laboratory for Physical Sciences at the MicroscaleChinese Academy of Sciences Key Laboratory of Brain Function and DiseaseSchool of Life SciencesDivision of Biomedical SciencesUniversity of Science and Technology of ChinaHefei 230026China Anhui Province Key Laboratory of Local Livestock and Poultry Genetic Resource Conservation and Bio-breeding College of Animal Science and TechnologyAnhui Agricultural UniversityHefei 230036China

Dual oxidase(duox)-deriyed reactive oxygen species(ROS)have been correlated with neuronal polarity,cerebellar development,and neuroplasticity.However,there have not been many comprehensive studies of the effect of individual duox isoforms on central-axon regenerationin vivo.Here,we explored this question in zebrafish,an excellent model organism for central-axon regeneration studies.In our research,mutation of the duox gene with CRISPR/Cas9 significantly retarded the singleaxon regeneration of the zebrafish mauthner cell in vivo.Using deep transcriptome sequencing,we found that the expression levels of related functional enzymes in mitochondria were down-regulated in duox mutant fish.In vivo imaging showed that duox mutants had significantly disrupted mitochondrial transport and redox state in single mauthner-cell axon.Our research data provide insights into how duox is involved in central-axon regeneration by changing mitochondrial transport.

关键词： duox Zebrafish mauthner cell Axon regeneration Mitochondrial dynamics

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In vivo imaging of calcium dynamics reveals the effect of activity in axonal regeneration of mauthner cell

In vivo imaging of calcium dynamics reveals the effect of ac...

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中国神经科学学会第十二届全国学术会议

作者： Min CHEN Rongchen HUANG Bing HU Laboratory of Neurodevelopment and Repair School of Life Sciences University of Science and Technology of China

Calcium is an important regulator of axon regeneration caused by laser axotomy, however its specific temporal dynamics during regeneration remain unclear. To clarify the role of calcium in axonal regeneration, we built an in vivo calcium imaging model of zebrafish mauthner cell and observed calcium dynamics at single-cell level in live zebrafish larvae during this process. Using electrical stimulation to induce calcium waves in zebrafish mauthner cell, we found that the cells labeled by plasmid of GCamp6 f showed more significant and various calcium spiking patterns than those labeled by calcium indicator（calcium green）. Consequently, we monitored that the mauthner cell axons with different regeneration ability displayed different calcium spiking patterns after axotomy. In summary, the cells with poor regeneration ability showed a gradually weaking calcium spiking pattern, while the cells with strong ability showed a similar spiking pattern with undamaged ones. What’s more, we inadvertently found an interesting phenomenon that the capacity of regeneration of axon correlated with the peak amplitude of calcium spiking before axotomy. The positive correlation became more significant when we exchanged the short stimulus with a serious combined stimuli, suggesting that a higher peak amplitude of calcium spiking predicting a stronger regeneration ability. To test the conjecture, we overexpressed Kir2.1 a to inhibit the calcium spiking then observed its effect on regeneration. As expected, Kir2.1 a overexpression decreased the regenerated length, a response that was paralleled by decreased mitochondrial motility which is proved to be related with axonal regeneration.

关键词： calcium imaging regeneration mauthner cell zebrafish activity

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In vivo imaging of mauthneraxon regeneration, remyelination and synapse reestablishment after laser axotomy in zebrafish larvae

In vivo imaging of Mauthneraxon regeneration, remyelination ...

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中国神经科学学会第十二届全国学术会议

作者： Bing-bing Hu Min Chen Yu-bin Huang Rong-chen Huang Wu Yin Yang Xu Lei Li Bing Hu Chinese Academy of Sciences Key Laboratory of Brain Function and Disease Hefei National Laboratory for Physical Sciences at the Microscale School of Life SciencesUniversity of Science and Technology of China Institute of Biomedical and Health Engineering Shenzhen Institutes of Advanced Technology Chinese Academy of Sciences

Objective Zebrafish is an excellent model to study central nervous system（CNS） axonal degeneration and regeneration since we can observe these processes in vivo and in real time in transparent larvae. Previous studies have shown that mauthner cell（M-cell） axon regenerates poorly after mechanical spinal cord injury. Inconsistent with this result, however, we have found that M-cell possesses a great capacity for axon regeneration after two-photon laser ablation. Methods By using ZEISS LSM 710 twophoton microscope, we performed specific unilateral axotomy of GFP labeled M-cells in the Tol-056 enhancer trap line larvae. Results Our results showed that distal axons almost degeneratedcompletelyat 24 hours after laser axotomy. After that, the proximal axons initiated a robust regeneration and many of the M-cell axons almost regenerated fully at 4 days post axotomy. Furthermore, we also visualized that regenerated axons wereremyelinated when we severed fluorescent dye labeled M-cells in the Tg（mbp:EGFP-CAAX） line larvae. Moreover, by single M-cell co-electroporation with Syp:EGFP and Ds Red2 plasmids we observed synapses reestablishment in vivo during laser injury-induced axon re-extension which suggested re-innervation of denervated pathways. In addition, we further demonstrated that microtubule destabilization with pharmacological treatment of nocodazole could completely abolish this regeneration capacity. Conclusion These results together suggested that in vivotime-lapse imaging of M-cell axon laser injury may provide a powerful analytical model for understanding the underlying cellular and molecular mechanisms of the CNS axon regeneration.

关键词： mauthner cell laser axotomy degeneration regeneration remyelination synapse reestablishment microtubule

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