| BackgroundGenome editing is attractive to basic research and clinical treatment.With the development of artificial designer nuclease targeting specific DNA sequences,genome editing is coming true.TALEN is one of genome-editing technique.There are two main domain of TALEN:TALE arm to identify and bind DNA and FokI to cleavage DNA.At first,TALE identify and bind the targeted DNA,and then the FokI nuclease create double-strand breaks(DSBs)in vivo.DSBs are repaired in nearly all cells by two highly conserved processes,non-homologous end joining(NHEJ)and Homologous recombination(HR).NHEJ often results in small insertions or deletions and targeted gene can be disrupted.There are many advantages in TALEN compared with ZFN:simple design,convenient assembling,high specificity and low toxicity.Rising CRISPR/Cas9 is more convenient to use than TALEN,However,there is lower off-target rate of TALEN than CRISPR/Cas9.As a rising model organism,zebrafish shares almost 87%homology with human genome.Molecular pathways participated in embryonic development are quite conserved.The zebrafish model shows many unique biological advantages such as its small size,large number of progeny,short life cycle,external fertilization and development,transparent embryos.So zebrafish is applicable to knock out and generate mutants.The zebrafish models become increasingly popular in the recently years.Various study models of zebrafish has been gradually established,such as leukemia model,immune system model and cardiovascular development and disease model.These models greatly improve our understanding of the mechanisms of related disease.Zebrafish,as a vertebrate,is similar to mammals in the process of hematopoiesis,which occurs in two waves,primitive and definitive hematopoiesis.Primitive hematopoiesis cells consist of primitive red cells and primitive microphages.The primitive erythropoiesis is developing at the same stages as primitive myelopoiesis,and mainly originate from the intermediate cell mass(ICM)of ventral mesoderm.Primitive myeloid progenitors originate from rostral blood island(RBI)of the anterior lateral medsoderm(ALIM).Before the onset of blood circulation,some early macrophages differentiate in the yolk sac,then,spread in the head mesenchyme,from there invade epithelial tissues:retina and brain(especially the optic tectum).At 60 hpf,all macrophages in the brain and retina undergo a specific transformation into "early microglia".From 3 days postfertilization(dpf)on,the majority of the microglial population in the CNS are localized within the grey matter of the optic tectum in the dorsal midbrain in WT zebrafish.Definitive hematopoiesis starts around 30hpf,when hematopoietic stem cells(HSCs)are believed to initiate from the ventral wall of DA(VDA).By 2 dpf,these HSCs in the VDA migrate to the caudal hematopietic tissue(CHT),and finally home to kidney,the adult hematopoietic organ in zebrafish,by 5dpf.Definitive hematopoiesis cells include:neutrophils,basophils,eosinophils,mast cells,microphages,erythrocytes,magakaryocytes,thrombocytes,T and B lymphocytes and nature killer(NK)cells.All these different type of hematopoietic cells are derived from a common ancestor known as HSCs.All of them play important roles to maintain the normal physiological function.Hematopoiesis is regulated by multiple factors with temporal and spatial variation complicatedly and well-organized.There are various markers expressed specially in every hematopoietic cell during hematopoietic development.During primitive hematopoiesis,transcription factors Gatal and Gfilaa regulate erythropoiesis and transcription factors Pu.l and C/ebpl regulate myeloid hematopoiesis.Gatal and Pu.1 exhibit a cross-inhibitory relationship to determine further specification towards erythroid or myeloid fates.During definitive hematopoiesis,transcription factors Runx1 and Cmyb,which represent the marker genes of HSCs,determine the production of HSCs.Besides,mpx and lyz mark neutrophils,mfap4 and mpegl mark macrophages,?e1 and ?e1 mark the definitive erythroid linceage,ikaros?rag1 and rag2 mark the lymphoid lineage,c-mpl marks thrombocytes.Retinoblastoma1(Rb1)was identified and cloned as the first tumor suppressor gene 29 years ago,which was first found based on its mutation in retinoblastoma.The retinoblastoma(RB)family of proteins play a key role in regulating advancement of the cell division cycle from the G1 to S-phases and are critical regulators of cell proliferation.Inactivation of RBI will lead to dysregulated cell proliferation and/or chromosomal instability,which are strongly linked to cancer development.RBI is considered mainly regulating the development of the retina,but in recent years,more and more evidence show that RB1 functions widely.RB1 can participate in multiple biochemical processes and play a variety of roles in biology.Retinoblastoma protein(pRB)is functionally inactivated in a large number of tumors including retinoblastoma,osteosarcoma,small-cell lung carcinoma,as well as bladder,breast and prostate cancers.RB1 is related to hair cells regeneration after injuration.In addition,RB1 is contributed to acute lymphoid leukemia and chronic myeloid leukemia and is closely related to erythrocytes maturation.In zebrafish,rb1 gene is usually utilized as a tumor suppressor in mutants or models.Howerver,the biological functions of rbl in zebrafish remains poorly understood.Apoptosis is a basic feature of life in a cell,which ha important significance in the field of biology.Apoptosis is highly regulated by multiple genes which are conserved in vertebrate,such as bcl2a family,apoptosis Inducing Factor(AIF)Bax,caspase family and tp53.There are various methods to detect apoptosis,including morphologic detection,whole-mount terminal deoxynucleotidyl nick end labeling(terminal deoxynucleotidyl dUTP nick end-1),and Acridine orange(AO staining)in vivo.In this study,gene editing technique,transcription activator-like(TAL)effectors nuclease(TALEN),was used to knock out four genes related to hematopoiesis including gfilaa,c-mpl,c/ebpl and rbl in wild type(AB stain)embryos(F0).And somatic mutations of target genes were generated in F0.F0 were mated with wild type fish to generate F1 which were performed screening in order to identify stable and heritable heterozygous F1.And then these F0 were mated with wild type fish to generate F2.Then,we further characterized this mutant,with multiple functional assay and gene expression detection such as Neutral Red(NR)staining,Sudan Black B(SBB)staining,and whole-mount in situ hybridization(WISH).Finally,we carried out molecular biology experiment to analyze the mechanism of zebrafish rblsmu.This study is divided into three parts:part one,generation of zebrafish mutants by TALEN;part two,characterization of rb1smu;part three,functional analysis of rb1smu Part one:Generation of zebrafish mutants by TALENObjective:We used reverse genetics technology TALEN to target rbl in wild type zebrafish embryos,in order to mutate rbl isolate novel zebrafish mutants with hematopoietic defects by large-scale screening.Then stable germ line rbl chimera mutants(F0)were screened by PCR-restriction enzyme digestion and sequencing.Gene editing technique,transcription activator-like(TAL)effectors nuclease(TALEN),was used to knock out four genes related to hematopoiesis including gfi1aa,c-mpl,c/ebp1 and rb1 in wild type(AB stain)embryos(F0).Then stable mutated germ lines were supposed to be found by genotyping.These mutants will be used for subsequent further research work.Method:We constructed TALEN using the Unit Assembly method.TALEN mRNA was microinjected into wild type AB zebrafish embryos to obtain F0 generation.Surviving F0 were mated with wild-type AB to generate F1.Collecting F1 embryos,mutated germ line F0 were screened by PCR-restriction enzyme digestion.These FO will be used to generate F1,and then stable mutated germ lines F1 were screened by genotypingResults:F0 were generated by TALEN mRNA treatment.Then the enzyme digestion and sequencing were carried out to screen.At last,four mutant lines of c/ebp1,six mutant lines of c-mpl,five mutant lines of gfi1aa and five mutant lines of rbl were screened successfully.These mutants will be used to study the phenotype and gene functions in subsequent work.Part two:Characterization of rblsmuObjective:The developmental status of were observed the mutant rblsmu.Multiple functional assay and gene expression detection were employed to further characterize rb1smu to find the hematopoietic defects.Rescue experiment was carried out,so as to confirm the correspondence between the phenotype and rbl.Method:Stereo microscope were utilized to observe the development of rblsmu and survival curves plotted were plotted.Two independent assay,including sudan black B staining and Neutral Red staining,were performed to detect the number and function of granulocytes and macrophages in rblsmu.Whole-mount in situ hybridization and quantitative fluorescence polymerase chain reaction(QF-PCR)were utilized to identify the defect of primitive and definitive hematopoiesis.Rescue experiment was carried out by using mRNA.Results:1)Rb1smu mutants develop as normally as wildtype at the same developmental stages before 4 days post-fertilization.However,from 4 days post-fertilization on,there are obvious differences between the mutants and wildtype embryos:wildtype embryos can swim independently and forage on the dish,but the mutants lie underwater and move rarely;most wildtype embryos can develop normally to sexual maturity,but the mutants begin to die from the lOdpf and up to 13dpf,the mutants die up.2)The number of neutrophils is not changed and peroxidase activity is normal in rb1smu.In rb1smu,macrophages appears normal at 4.5 dpf in PBI,but microglial cells increase in the midbrain and hindbrain significantly.3)Normal expression of multiple hematopoietic genes,such as pu.1,gatal,c-myb,lyz,mfap4 and ?e1,showed primitive hematopoiesis,definitive stem cell development,definitive myloid development and definitive erythropoiesis in rblsmu were unaffected.However,decreased expression of the lymphoid specific gene,rag1,shown lymphoid development was defect in rb1smu.The expression of apoeb was significantly increased in the midbrain and hindbrain,suggesting microglia significantly increased in rb1smu.4)Direct injection with rbl mRNA could partly restore the phenotype of rb1smu.Part three:apoptosis detection and analysis of rblsmu.Objective:The reasons for increased microglia and reduced lymphocytes were explored.Then apoptosis mechanisms associated with phenotypes were assayed.Method:Whole-mount BrdU assay was carried out to detect the reason for increased microglia.Whole-mount TUNEL and Whole-mount acridine orange staining were utilized to analyse apoptotic cells in midbrain and hindbrain.Transgenic line Tg(NBT:bcl2a)was introduced into rb1smu mutants,in which neuronal cells overexpress bcl2a,and then microglia were assayed by Neutral Red staining.P53 deficient was introduced into rb1smu mutants,and then microglia were detected by Neutral.Red staining in double mutants.Results:1)Whole-mount BrdU assay showed that increased microglia is derived from proliferating.2)Whole-mount TUNEL and Whole-mount acridine orange staining revealed apoptotic cells increased significantly in the midbrain and hindbrain.3)Overexpression bcl2a in neurons of rblsmu lead to decreased microglia and phenotype was partially restored.But there was no significant differences of microglia between tp 53M214Krb1smu mutants and rb1smu mutants,suggesting that increased microglia is independent of p53.Conclusions:1)We obtained 20 mosaic F0 by TALEN targeted rb1,which were germ lines.These mutants were then classified into five types based on genotypes.Further work is ongoing to explore the mechanisms of apoptosis and to establish diseases models.2)Rb1smu can not grow to sexual maturity and from 4 days post-fertilization on,its motion was abnormal.Increased microglia were present in midbrain and hindbrain,but microglial progenitors-primitive myeloid cells were normal.Reduced lymphocytes were present in thymus,but other lineages could develop normally.This suggest that rbl could play a role of lymphocyte development and neuron developmental,and rb1smu is available for studying the function of its mechanism.3)Whole-mount BrdU assay and Whole-mount TUNEL showed that increased microglia is derived from proliferating.Apoptosis increased significantly,which isIndependent of p53;Overexpressing bcl2a in neurons can restore phenotype partially,suggesting that increased apoptosis appear in neurons,and bcl2a participates in the pathway of apoptosis. |
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