The Role Of GMFB In Regeneration Of Neural Trauma In The Zebrafish Telencephalon

Central nervous systerm injury is a very serious and disabling disease in central nervous system(CNS)without effective treatment.After Central nervous systerm injury,patients often suffer from sensory and motion function disorders,which imposes a heavy burden on society and patients.Amphibians and bony fish has a magical regenerative ability completely.At present,a new popular animal model,zebrafish,which throughout the life of the central nervous system has full regeneration ability.So how highly regenerative potential of cellular and molecular mechanisms play a role.We could take advantage of this potential to improve the regeneration ability of mammals,especially to humans.Glia maturation factor beta(GMFB)is a sequence-conserved gene that is expressed predominately in the brain.GMFB is a novel ADF/cofilin superfamily protein involved in the reorganization of the F-actin cytoskeleton.GMFB has no actin binding or severing activity in in vitro assays.GMFB one of the isoforms of GMF,which plays an important role in branched actin regulation,lamellipodial dynamics,and directional migration.During neural development,the cytoskeleton of new neurons undergoes extensive and dynamic remodelling to facilitate the sequential steps of neurogenesis,cell migration and terminal differentiation.The neurogenesis regeneration of adult zebrafish also involves in actin remodeling.Therefore,it is not clear that how gmfb play a role in F-actin remodeling of neurogenesis regeneration of adult zebrafish telencephalon.ObjectiveTo built GMFB knockout model and to explore the mechanism of action in GMFB to how influence the morphologyof GFAP filaments and proliferation of radial glial cells after adult zebrafish telencephalon injury,finding the function and role of GMFB in the brain.2.Methods2.1 Fish maintenance and stab lesionsAdult zebrafish(Danio rerio)were maintained under standard conditions at 28.5? and 14 h light/10 h dark periods.To avoid pigment formation in larvae,0.003%w/v Phenylthiocarbamide(Sigma-Aldrich,USA)was added to the fish water at 24 hours post fertilization(hpf).Injuries to telencephalon were performed on 6-12 monthsold zebrafish,and left to regenerate for the desired time points.After completion of the surgical procedure,the fish was placed back into fresh fish water.2.2 CRISPR-Cas9 technology knock out gmfb geneFor microinjection,sgRNA and Cas9 mRNA were mixed in diethypyrocarbonate water to final concentrations of 100 ng/?l and 400 ng/?l,respectively.The solution was injected into 1-cell-stage zebrafish embryos derived from the AB line.The target sequence of sgRNA is located in the third exons of GMFB genome.We successfully knocked out 13 bases in GMFB genome.We confirmed that GMFB knockout was successful at a DNA level,mRNA level and protein level Additionally,the successful GMFB knockout was also confirmed in adult zebrafish telencephalon.2.3 Enzyme digestion experimentAdult zebrafish tail were placed in alkaline solution at 95? for 30min.Following this incubation,acid solution was added at the same volume and centrifuged for 5 minutes at 12000×g.The resultant clear liquid was used as a template for GMFB gene in PCR.Through the enzyme loci prediction of the gmfb gene,we choose the PVUII enzyme as the restriction enzyme.Enzyme-digested products were verified by nucleic acid electrophoresis and to select homozygous(gmfb(-/-)).2.4 Whole mount in situ hybridizationThe probe primers were designed to amplify the corresponding sequences from the total cDNA extracted from zebrafish embryos.All the probes contained the restriction enzyme sites of Bam HI and Eco RI.After enzyme digestion,the PCR products were cloned into the pBluescriptSK(+)vector.All the probe lengths were longer than 1000 bp,and the sequences subjected to BLAST analysis using the NCBI database to confirm their specificity.For whole mount in situ hybridization,digoxigenin-UTP rib probes were synthesized to detect GMFB mRNA according to the manufacturer's instructions.The colour reaction was performed using NBT/BCIP substrate.2.5 Tissue preparation and immunohistochemistryBriefly,specimens were deeply anesthetized in 0.2%tricaine(MS222,Sigma)in fresh water and fixed in 4%paraformaldehyde overnight.Thespecimenswas dehydrated,and embedded for sectioning,antigen repair.Their brains are dissected out and stained by fluorescent staining.Images were acquired with an Olympus microscope or a laser scanning confocal microscope.2.6 The observation of spontaneous movement in zebrafish larvaeEach larva was placed in a well.(n=12 larvae tails/group).The plate was placed onto the objective table of a Viewpoint behaviour machine.we used Viewpoint Life Sciences software control instrument motion video tracking system to capture the spontaneous movement video of zebrafish larvae each minute.The recorded larvae movement were transferred into a visual map,and total moving distance,average speed and other motion parameters exported.2.7 Statistical analysisThe statistical analyses were performed using SPSS 19 software(IBM,US A),data was presented as Mean ± SDand analysed using one-way ANOVA or independent-samples t test,as appropriate.A P value less than 0.05 was considered statistically significant.3.Results3.1 Expression of GMFB in zebrafish embryos and adult zebrafish telencephalonThe results revealed that GMFB was first expressed at 3 hours post fertilization and the hybridization signal was mainly localized to the brain by 3 days post fertilization.GMFB protein was expressed from 6 hpf and gradually increased until 3 dpf.We further examined the expression of GMFB in the adult zebrafish telencephalon and observed expression of GMFB protein throughout the ventricular domains of the telencephalon.In particular,high levels of GMFB were detected in the dorsomedial,dorsolateral ventricular zone and the dorsal region of the pallium.expression of GMFB was not detected in the ventricular zone of the subpallium.3.2 GMFB is upregulated in the dorsal ventricle after traumaticbrain injuryadult zebrafish telencephalon were stabbed with a needle and immunohistochemistry performed at the selected time points.The expression of GMFB was found to be slightly upregulated on the lesioned side at 1 day post lesion(dpl)and significantly upregulated at 3 dpl compared with the unlesioned side.3.3 GMFB mutant is defective in brain regeneration after injuryunder normal conditions,there was no obvious difference between the number of RGCs in telencephalons of wild type and GMFB mutant zebrafish as assessed by GFAP staining.However,the RGCs filaments were more sparse and disorganized in the telencephalon of GMFB mutant zebrafishcompared with the wild type zebrafish.the telencephalons of GMFB mutant and wild type zebrafish were injured with a needle and the RGCs were analysed for GFAP and SOX2staining.GFAP+radial filaments were increased on the lesioned side compared with the contralateral side in both wild type and GMFB mutant zebrafish at 3 dpl.However,the GFAP+radial filaments were thicker in wild type zebrafish than that in the GMFB mutant zebrafish.Staining with SOX2 antibody,an RGC soma marker,revealed that the number of SOX2 RGC increased in lesioned side compared to the control side in both wild type and GMFB mutant zebrafish at 3 dpl.However,the number of SOX2+RGC was higher in wild type zebrafish than that in GMFB mutant.Interestingly,the healing time of the wound in the telencephalon was delayed in GMFB mutants compared to wild types at 14 dpl.3.4 GMFB knockout abrogates the transformation of type I to type II RGCs during telencephalon regenerationNo significant differences were observed in the number of type ? and ? RGCs between GMFB mutant and wild type zebrafish in the medial zone of telencephalon under normal conditions.After injury,we found that the number of type ? RGCs were increased on the lesioned side compared with the uninjured side in both mutant and wild type at 5 dpl.However,the increase in the number of type ? RGCs was significantly less in the GMFB mutants compared to the controls.To view the relative contribution of GMFB in the increase of PCNA+cells,we counted the numbers of PCNA+GMFB+and PCNA+GMFB-cells under normal and injury conditions in wild type zebrafish telencephalon.In normal conditions,very few PCNA+cells were GMFB+,however both the numbers of PCNA+GMFB+and PCNA+GMFB-cells increased significantly after injury.The numbers of the two populations were comparable at 3 and 5 dpl.However,the increase in the number of PCNA+GMFB+cells was more significant compared with PCNA+GMFB-cells.Combined with the result that type ? RGCs were significantly decreased after GMFB knockout,this suggests that GMFB may be involved in regulating the transformation of type I to type ?I RGCs during regenerative neurogenesis.3.5 Inhibiting the ARP2/3 complex rescues malformed GFAP filaments in GMFB mutant larvaeWe treated larvae with ck666,an inhibitor of Arp2/3 complex,to observe the effects on GFAP filaments.GFAPfilament branches in GMFB mutant larvae were more complex than in wild type under normal condition.Accordingly,the branching of GFAP filaments was substantially inhibited by CK666 treatment in both wild type and mutant larvae.Taken together,these data suggest that the complexity of GFAP filaments in GMFB mutant larvae decreases after CK666 treatment.3.6 GMFB knockout in zebrafish causes behavioural alterationsResults demonstrated that the travel distances were significantly lower in the GMFB mutant group compared to the wild typegroup.Similarly,the velocity was also significantly lower in the mutant group,suggesting that the locomotion ability of zebrafish larva were impaired after GMFB knockout.4.conclusionIn this studywe propose a view:after brain injury in adult zebrafish,GMFB involved in nerve regeneration and GFAP fiber skeleton remodeling.