| 1.Functional analyses of the hprg1 b gene and establishment of the hprg1 b stem cell lineThe hprg1 b gene of vertebrate is a homologue of the yeast GID2 gene.It encodes two homologous protein isoforms,Hprg1 a and Hprg1 b.In yeast,GID2 protein works as an component of E3 ubiquitin ligase and is involved in the regulation of the glucose metabolism.Furthermore,according to the large-scale screening of E3 ubiquitin ligase from human genome,it was found that human HPRG1 B protein interacts with E2 ubiquitin-binding enzyme through its R-type zinc finger domain to form a new enzyme protein complex.Recently,the research of Hprg1 protein in xenopus shows that frog's Hprg1 may serve as an E3 ubiquitin ligase to regulate the development of embryonic head.In addition,human embryos containing chromosomal fragment deletions of the hprg1 b gene are severely deformed.However,the role of hprg1 b gene in embryonic development remains unclear.The zebrafish genome encodes only one Hprg1 protein,which is the Hprg1 b protein.The results of multiple sequence alignment of Hprg1 a and Hprg1 b in human,mouse,turtle,chicken,xenopus and zerafish show that the similarity of protein sequences between zebrafish Hprg1 b protein and Hprg1 protein in other species analyzed is over 68%.Moreover,the gene structure of hprg1 a and hprg1 b in human,mouse,turtle,chicken,xenopus and zebrafish were analyzed,and we found that the splice sites and gene structures of these genes were highly conserved.In the evolutionary process,the hprg1 gene is highly conserved in protein sequence and gene structure,indicating that the function of this gene is highly conserved.In addition,we further studied the chromosomal syntenic relationship between hprg1 b gene and its nearby gene in human,mouse and zebrafish.And zebrafish hprg1 b gene displays syntenic relationship with human and mouse hprg1 b gene,indicating that the zebrafish hprg1 b gene is an orthologous gene of the human and mouse hprg1 b gene.Therefore,the study the role of hprg1 b gene in zebrafish in this paper helps to reveal the role of this gene in advanced vertebrates.Using the ddPCR,the expression of hprg1 b gene was accurately quantified during zebrafish embryonic development.The results showed that the hprg1 b gene had a strong maternal expression during zebrafish embryonic development.And hprg1 b was consistently highly expressed during embryogenesis.Moreover,in situ hybridization was used to analyze the temporal and spatial expression profiles of hprg1 b gene during zebrafish embryonic development.We found that,the expression region of hprg1 b gene completely coincide with the forming region of zebrafish brain and heart.The expression pattern of hprg1 b gene in zebrafish embryos suggests that hprg1 b gene may play a role in zebrafish embryogenesis,especially in the development of brain and heart.In order to study the role of hprg1 b gene in the development of zebrafish embryos,the translation blocking morpholino of the zebrafish hprg1 b gene was designed.And result of Western blotting demonstrated that the expression of Hprg1 b protein was significantly reduced in zebrafish injected with the hprg1 b morpholino.Blocking the expression of zebrafish hprg1 b gene during zygote stage led to a large number of embryonic deaths,and the mortality rate is up to 80%.The escapees of embryonic death showed abnormalities in brain and heart development,and the deformity rate is up to 93%.At 24 hpf,the forebrain of hpg1 b knockdown zebrafish was significantly smaller than control.A clear structure of the midbrain,hindbrain primordial and hindbrain regions were not visible.The head deformity was more obvious at 72 hpf.The whole head area became smaller and had no obvious morphological structure of the forebrain,midbrain and hindbrain.Meanwhile,the cardiac defects in hprg1 b knockdown zebrafish were analyzed.The main characteristics of cardiac defect were cardiac looping anomalies,and smaller atrial and ventricular chamber.Moreover,the number of cardiac cells of 72 hpf zebrafish was counted.The results showed that cardiac cells number of hprg1 b knockdown zebrafish was significantly reduced when compared with the control group.Furthermore,the expression of cmlc2 gene in the cardiac disc,heart tube,atrium,and ventricle during early embryonic stages was analyzed by in situ hybridization and ddPCR.Compared with the control group,the expression of cmlc2 in hprg1 b knockdown group were significantly lower than that of the control group,indicating that the hprg1 b gene could affect the zebrafish cardiogenesis by regulating the development of cardiomyocytes.Besides,the expression of bmp4 was also detected,an important regulator of atrioventricular channel development,at atrial and ventricular junctions at 72 hpf through in situ hybridization.The results showed that the expression of bmp4 in hprg1 b knockdown group was significantly lower than that in the control,indicating that hprg1 b gene might affect the development of the internal structure of zebrafish atrioventricular canal.In addition,the expression of the endocardial labeling factor has2 was also down-regulated in the 72 hpf hprg1 b gene knockout zebrafish,suggesting that hprg1 b knockdown led to abnormal endocardial development.Moreover,the expression of myosin heavy chain protein was down-regulated and part of the basic structure of cardiac sarcomere was absent in hprg1 b knockdown zebrafish,which further indicating that hprg1 b gene knockdown may affect the cardiac contraction.As knockdown of hprg1 b in zebrafish embryo led to a large-scale of embryonic death,ddPCR was used to investigate the expression of key regulators of zebrafish embryogenesis.The results showed that the expression of ectodermal markers(fgf8 and otx2),mesodermal labeling factors(gata4,gata5,and ntl),and endoderm marker(sox17)were significantly down-regulated in hprg1 b knockdown zebrafish when compared with the control group,suggesting that hprg1 b gene may play a role in zebrafish embryogenesis via regulating the expression of key embryonic genes.In addition,an hprg1b-egfp transgenic zebrafish embryonic stem cell line was constructed.This cell line shows high proliferation rate,high transcription efficiency,and a variety of core pluripotency genes were expressed in this cell line.Subsequently,the hprg1b-egfp transgenic zebrafish embryonic stem cells were transplanted into wild-type zebrafish and the developmental fate of Hprg1 b positive cells were tracked by confocal microscopy.At 24 hpf,exogenous hprg1b-egfp transgenic zebrafish cells were observed in the forebrain,midbrain,posterior brain primordium,hindbrain,and heart tube of chimeric zebrafish.At 48 hpf,transplanted cells were detected in the brain,ventricle,and atrium.Thus,this cell transplantation assay indicated that embryonic Hprg1 b positive cells were capable of differentiating and migrating into brain and heart cells during embryogenesis.Therefore,in this paper zebrafish model was used to study the function of hprg1 b gene in zebrafish early development.It was proved for the first time that the hprg1 b gene plays a role in brain and heart development.The hprg1b-egfp transgenic zebrafish embryonic stem cell line was established,which provided an ideal cell model for the further investigation of hprg1 b gene function and the molecular mechanism of early cardiogenesis.2.Identification of zebrafish magnetic protein and cryptochrome homologous geneMany animals use the magnetic field for orientation and navigation.Recent studies in Drosophila found that the complexes of magnetic receptor protein(MagR)and cryptochrome(Cry)were the molecular basis of magnetoreception.MagR and Cry proteins are widely expressed in a variety of animals.However,it is unknown whether they perform a conserved role in diverse animals.In order to explore the function of MagR and Cry in lower vertebrates,we identified and analyzed the expression of their homologous genes in zebrafish,respectively.The human genome contains one MAGR gene and two CRY genes.Zebrafish also has only one magr gene,but contains seven cry genes.By means of multiple protein sequence alignment,gene structure and chromosomal synteny analysis,we found that the magr gene was highly conserved during evolution and that the zebrafish magr gene was an orthologous to human MAGR.Zebrafish has four cry1 genes(cry1aa,cry1 ab,cry1ba and cry1bb)are homologous to human CRY1 gene and a single ortholog of human CRY2 as well as 2 cry-like genes(cry4 and cry5).RT-PCR analysis showed that the magr gene was expressed widely in the adult tissues and all cry genes were obviously expressed in the brain and eyes.The zebrafish magr gene and all cry genes except the cry1 aa have maternal supply in embryonic development.In addition,the zebrafish with magr deleption by translation blocking morphoino did not show significant changes in death and development,and the expression of embryonic developmental regulators fgf8,otx2,gata4,gata5,has2,ntl and sall4 did not change obviously in magr knockdown zebrafish.Taken together,magr and cry2 exist as a single copy gene,whereas cry1 exists as multiple gene duplicates in zebrafish.Our result suggests that magr may play a dispensable role in organogenesis and predicts a possibility to generate magr mutants for analyzing its role in zebrafish.3.Tmp3 gene knockout zebrafish RNAseq analysisIn recent years,with the rapid development of next-generation sequencing technology the requirement of time and money for sequencing are greatly reduced,thus genomic transcription sequencing(RNAseq)analysis has become the best choice for signal pathway investigation and target gene screening.In this paper,the blastula stage tmp3 gene knockout zebrafish was adopted for transcript sequencing.Using the tophat software,the sequencing results were mapped to the zebrafish reference genome.Subsequently,htseq-count software was to analyze the results of mapping to identify the splice site between the exons.In total,expression data of 24102 genes were obtained.Using the DESeq library package in the R language,we identified 2194 genes that differentially expressed in tmp3 gene knockout zebrafish compared with wild-type zebrafish.Among them,1231 genes are up-regulated and 963 genes are down-regulated.Next,this paper carried out the gene ontology(GO)enrichment analysis of the differentially expressed genes through the goseq software package.After analyzing the GO enrichment types of the down-regulated genes,we found that the GO enrichment type of the down-regulated genes was mainly associated with mesodermal development and cell proliferation in zebrafish.According to the severe cardiac development defects observed in tmp3 knockout zebrafish,the mesoderm development related genes dnarma,foxa2,lefty1,klf2 a,mespaa,noto,tll1,apela,tbx16 and tdgf1,and the cell proliferation related genes rrm2,gmnn,pold1,map2k2 b,ccnk,ccnl1 b,cdca8,aurka and cdk4 were selected from the down-regulated genes and served as candidate target genes of tmp3 during cardiogenesis.Thus,RNAseq analysis of tmp3 gene knockout zebrafish in this paper provides an indication for the further functional study of tmp3 gene. |
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