The Establishment Of Real-time Monitoring Model For Neural Development And Nerve Injury Repair In Zebrafish

In recent years,many scholars have tried to use zebrafish as a model organism for the study of neurogenesis and nerve injury repair.The transcription factor GATA binding protein 3(Gata3)and Neurogenin 1(Neurogl)have been shown to play an important role in the generation,differentiationand migration of zebrafish newborn neurons,which are expressed only in the embryonic period and are rarely expressed in normal adult zebrafish.When the brain or spinal cord of adult zebrafish suffered serious damage,the expression of Gata3 genes would be significantly induced in cells,showing a high level of expression in the injury site as a result.And,the Neurogl gene is expressed in the proliferating and differentiating intermediate progenitor cells and promotes the differentiation of neurons.Therefore,it is of great significance to study the neural development of zebrafish embryos and the mechanism of nerve restoration after injury by labeling and tracking the expression of Gata3 and Neurogl genes in vivo.Previously,the establishment of zebrafish lines whose unique neural tissues or neurons were marked distinguishingly under gene trapping methods has played a critical role in the study of neurobiology zebrafish.However,there are defects in these strains more or less,such as inaccurate positioning.Fortunately,with the progress of gene editing technology,especially the advent of precise targeted knock-in technology for Clustered regularly interspaced short palindromic Repeats(CRISPR)/CRISPR-associated protein(Cas)system(referred to as CRISPR/Cas system),it is possible for us to mark specific genes accurately in zebrafish.To establish a real-time monitoring model for zebrafish neurodevelopment and nerve injury repair,we used the CRISPR/Cas9 system to fuse a p2A-mEOS3.2 reporter in-frame to the last codon of the Gata3 and Neurog1 genes,respectively.In this way,we could track the cells expressing Gata3 and Neurogl by capturing the mEOS3.2 signal.In this study,we successfully constructed the Gata3-p2A-mEOS3.2 and Neurogeninl-p2A-mEOS3.2 knock-in zebrafish lines and then screened the expression of Gata3 and Neurog1 genes in the early stage of zebrafish embryos in vivo,which has important significance for the study of early neural development in zebrafish.Meanwhile,we carried out the injury stimulation experiment targeting the adult zebrafish telencephalon.By capturing the fluorescence signal of mEOS3.2 protein,we successfully monitored the specifically expression of Gata3 and Neurogl at different time points.Furthermore,the establishment of real-time monitoring model for adult zebrafish nerve injury repair provided technical support for the study of neurogenesis under the damage repair process induced by injury.In preliminary exploration,it was found that Gata3 and Neurogl genes were abundantly expressed at the site of injury and the stem-cell enriched region.Surprisingly,it was also found that the cells expressing Neurogl gene displayed a obvious bridge acrossing the telencephalon injury area to the stem cells enriched area(telencephalon-midbrain junction),which may be the result of the migration of newbron neurons from the stem cell enriched area to the injury area.Overall,the research results of this project are of great significance for the study of early neurodevelopment,revealing the mechanism of adult neurogenesis and nerve injury repair in zebrafish in the future.