| The damage or loss of human organs/limbs has serious impact on their daily life and even physical and mental health.Therefore,studies on the mechanisms and methods of organ and limb regeneration has became the priority in medical and scientific research field.The so-called regeneration refers to the process of structural and functional restoration of the damaged organs and tissues,in which a variety of biological events,such as immune response,cell proliferation,apoptosis and differentiation are involved.It has been reported that the non-coding RNAs(ncRNAs)micro RNAs(mi RNAs)play an important regulatory role in the caudal fin regeneration of zebrafish,but the function of long non-coding RNA(lncRNAs)is largely unknown.Therefore,the present study aimed to screen out the differentially expressed ncRNAs and m RNAs during the fish caudal fin regeneration by whole transcriptome sequencing analysis using the world-recognized model organism zebrafish as an experimental model.The newly discovered differentially expressed ncRNAs and m RNAs were selected as the desired genes to explore their regulatory effects and molecular mechanism in the process of caudal fin regeneration.This study might provide a good experimental basis for clarifying the mechanism of caudal fin regeneration,and also provides a theoretical basis for broadening the research of limb regeneration and seeking the effective drug target for limb regeneration.The main research contents and experimental results are as the following:(1)The morphological and histological characteristics of the caudal fin regeneration of zebrafishFirst of all,the morphological and histological of changes during the zebrafish caudal fin regeneration were record by using the optical microscopy,histological sections,and HE staining techniques.The process roughly includes four stages as follows:(a)wound healing;(b)AEC formation;(c)the formation of blastma;and(d)the regenerative growth.Particularly,1-3 dpa was the key period for zebrafish caudal fin regeneration.In order to further explore the apoptosis and proliferation during the caudal fin regeneration of zebrafish,TUNEL(Terninal-deoxynucleotidy1 transferase mediated nick end labeling,TUNEL)and Ki67 labeling technology were used in this study.The result showed that apoptosis mainly occured in the wound healing period(1 dpa)which was the early stage of regeneration however cell proliferation began at 1 dpa,but reached the highest at 3 dpa.This result indicted that zebrafish caudal fin regeneration was a typical way of regeneration that depends on bud-based tissues,and apoptosis and cell proliferation were involved in the critical period(1-3 dpa)of zebrafish caudal fin regeneration.(2)Screening of the key genes during zebrafish caudal fin regeneration based on RNA-seqIn order to further systematically explore the molecular mechanism of zebrafish caudal fin regeneration and to screen out key genes related to caudal fin regeneration,the whole-transcriptome sequencing(RNA-seq)was used to explore the transcriptome level expression at three key periods(0 dpa,3dpa,7 dpa)during the process of caudal fin regeneration,and in which 0 dpa was used as a control group.The results showed that the number of differentially expressed genes(DEG)was significantly different between different groups.For example,compared with the control group(0 dpa),5 621 differentially expressed m RNAs,136 differentially expressed lncRNAs,and 153 differentially expressed mi RNAs were screened at 3 dpa.However,at 7 dpa,only 1 513 differentially expressed m RNAs,41 differentially expressed lncRNA and 81 differentially expressed mi RNAs were found.This phenomenon indicated that complex genetic changes was experienced during caudal fin regeneration,especially at 3 dpa.Real-time fluorescent quantitative PCR(RT-q PCR)was used to verify the expression levels of differentially expressed m RNAs and ncRNAs at different stages of zebrafish caudal fin regeneration,and the verification results were basically consistent with the RNA-seq data.In order to further understand the specific role of these DEGs in the process of caudal fin regeneration,we conducted GO(Gene Ontology,GO)and KEGG analysis on DEGs between different groups.The result of GO analysis indicated that among the 20 biological processes with significant enrichment of DEGs among different groups,all of them included GO terms related to caudal fin regeneration such as cellular processes,developmental processes,growth,and immune system processes.The results of KEGG pathway analysis showed that DEGs between different groups were significantly enriched in classic signaling pathways related to the regeneration process,such as wnt signaling pathway,JAK-STAT signaling pathway,Hedgehog signaling pathway,p53 signaling pathway,cytokine-cytokine receptor interactions,etc.In addition,the ce RNA regulatory network of LncRNA-mi RNA-m RNA was constructed to further explore its mutual regulation mechanism in the process of caudal fin regeneration,which might provide a certain theoretical and experimental basis for future functional analysis.(3)The expression and function analysis of related lncRNAs during caudal fin regenerationBased on the RNA-seq analysis of the process in zebrafish caudal fin regeneration,we used NCBI,UCSC,Ensemble,and ZFIN to conduct bioinformatics analysis on the selected lncRNAs and m RNAs.And in situ hybridization technology was also used to analyze its expression and location during the caudal fin regeneration.The results of bioinformatics analysis showed that lncRNA-156279 was a ～931bp transcript located on chromosome 6 of zebrafish(chromosome 6: 27620687-27623283),belonging to intronic lncRNA(lincRNA)according to the position in protein-coding gene.The transcript of its adjacent protein coding gene slco2a1-201 was about 2 350 bp in length located on chromosome 6 of zebrafish(chromosome 6: 27624023-27664382)with 14 exons.LncRNA-154324 had a total length of about 916 bp and was located on zebrafish chromosome 15(chromosome 15: 17376367-17378063),belonging to lincRNA.The transcript of its adjacent protein coding gene vmp1-201 was about 2 135 bp in length located on chromosome 15 of zebrafish(chromosome 15:17343319-17373352)with 12 exons.It was inferred that lncRNA-154324 and vmp1-201 and lncRNA-156279 and slco2a1-201 might be involved in zebrafish caudal fin regeneration according to the results of bioinformatics analysis and spatial expression detection by in situ hybridization.Based on the results of bioinformatics analysis,this experiment used CRISPR/Cas9 gene knockout technology to study the function of two lncRNA-cis relationship pairs(lncRNA-154324 and vmp1-201 and lncRNA-156279 and slco2a1-201).The results showed that 4 genes play an important role in the growth of zebrafish caudal fin,especially the slco2a1-201 gene plays an indispensable role in the growth of zebrafish caudal fin,which may play a role by influencing marker genes in caudal fin regeneration.(4)The expression and function analysis of genes related to caudal fin regenerationIn this study,the expression characteristics and possible functions of cytokines in zebrafish caudal fin regeneration were explored by using bioinformatics and RT-q PCR.The results of the study showed that the expression heat map of the differentially expressed cytokines in the samples showed two expression trends: high-low-slightly high and low-high-low.The results of enrichment analysis of GO and KEGG pathway of differentially expressed cytokines showed that most genes were significantly enriched in GO terms related to caudal fin regeneration and classical signaling pathways related to immune,development and regeneration processes.The expression of immune-related cytokines during the critical period of caudal fin regeneration was determined by RT-q PCR.The results show that these inflammatory cytokines may be involved in the regeneration of the caudal fin.The pro-inflammatory cytokines IL-1β,IL-6,IL-8 and TNF-α may mainly play a role in inducing inflammation,while the functions of the anti-inflammatory cytokines IL-10 and TGF-β may focus on damage repair.The balance expressed between the two maintains the regeneration efficiency of the caudal fin,otherwise the regeneration of the caudal fin may be inhibited.In addition,during fin regeneration,the expression of JAK2α and STAT1 b in the JAK-STAT signaling pathway also increased significantly,indicating that the inflammatory response in caudal fin regeneration may be related to the JAK-STAT signaling pathway,which is consistent with previous studies.The expression and bioinformatics of Hsp90 during the regeneration of the caudal fin of zebrafish were also studied in this work.Firstly,the bioinformatics analysis of Hsp90 with NCBI,UCSC,Ensemble and ZFIN showed that Hsp90 was a ～2.5 Kb protein-coding gene located on zebrafish chromosome 20.The results of blast and phylogenetic analysis showed that the protein has a high level of conservation among species.RT-q PCR and WB were used to study the expression patterns of Hsp90 during caudal fin regeneration,it was found that Hsp90 began to express at the early stage of caudal fin regeneration(1-3dpa),reached the highest at 3 dpa,and then began to decline.The results of in situ hybridization confirmed that Hsp90 was expressed at 1 dpa during the process of caudal fin regeneration,then gradually increased,and reached a peak at 3 dpa.This result was consistent with the results of RT-q PCR and western blot.It was worth noting that Hsp90 was mainly expressed on the wound epidermis at 1 dpa.From 2 dpa to 3 dpa,the expression of Hsp90 was mainly located near the bud base tissues and blood vessels.The above results indicated that Hsp90 was expressed in the key regenerative cells of caudal fin regeneration and may play a role in the initiation and proliferation of fin regeneration.In order to further study the function of Hsp90 in caudal fin regeneration,this experiment used CRISPR/Cas9 gene editing knockout technology to select the target site on the second exon of the zebrafish Hsp90 gene.Through in vitro transcription,the synthesized sg RNA and Cas9 protein were mixed according to the concentration and then microinjected into the 1-cell zebrafish embryo.After micro-injection,the 24 hpf embryos were subjected to PCR,sequencing detection and in situ hybridization to obtain the positive F0 generation individuals with single target mutation of Hsp90 gene.After Hsp90-Cas9 knockout injection of Zebrafish embryo,observe the knockout efficiency after knockout injection and its effect on zebrafish embryo death,deformity rate and caudal fin regeneration-related marker genes(Bmp1b,lef1,msx1b).The results showed that Hsp90 knockout caused changes in the body’s phenotypic growth process,indicating that this gene has a certain function in zebrafish.Among them,making the tail curl up again proves that Hsp90 has an important regulatory role in the growth of the caudal fin. |
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