| BackgroundBlood system diseases(such as the Mediterranean anemia,immunodeficiency and leukemia,etc)are severe diseases that affect human health and their high incidence brings heavy burdens to the medical security system and the whole society.To its deep reasons,high rate of blood diseases reflected that human are not aware of the molecular and pathological mechanisms of hematopoieisis.To make blood system function normally and to maintain a steady state,it depends on the normal development of various blood cells,but hematopoietic process defects can lead to a variety of developmental disabilities in blood cells or blood vessels.As a result,to better understand the development of hematopoiesis from the molecular and cellular level will help us clarify the mechanisms of blood diseases pathogenesis and reveal potential application in treatment of clinical diseases.In vertebrates,hematopoiesis is a complicated and dynamic process controlled by multiple factors.All kinds of blood cells are growing and maturing in this process.Although different components of blood have different physiological functions,they have the same ancestry:hematopoietic stem cell(HSC).Early development of hematopoietic stem cell is the core process of hematopoiesis.It is indispensable for embryonic organogenesis,maintenance of homeostasis and recovery of damage under the circumstances of production,maintenance,proliferation,migration and positioning of HSC.[1,2]During the process of differentiation,HSCs first become hematopoietic progenitors and hematopoietic precursors,eventually become all lineage cells.During the differentiation of hematopoietic stem cells into all kinds of lineage,the participation of many kinds of molecular switches is required.Transcription factors play an important regulatory role in this process and interact with each other to form a complex regulatory network.Transcription factors play an important role in the process of development and differentiation in hematopoiesis.Different levels of expression of transcription factors in different spatio-temporal stages during development make hematopoietic stem cells or progenitor cells differentiate toward a particular lineage.GFI1,a transcriptional repressor,participates in hematopoietic regulation.In the patients with GFI1 gene deletion or mutation,granulocytes were being blocked in promyelocytic stage.Although GFI1 gene knockout mice did not develop into leukemia,due to the loss of function,knockout of GFI1 gene may contribute to the occurrence of leukemia,especially myeloid leukemia cells,such as the AML.Classical models of organisms such as worms or fruit flies are poorly related to human homologically.Traditional model of mammals,due to their large size and low reproductive rate,are difficult for hematopoietic high-throughput screening.Also,mammal development occurs in womb,so it is hard to catch live images for changing phenotype during embryonic hematopoiesis.In terms of genome,proteome,embryonic development,as well as mechanisms of diseases and so on,zebrafish as a kind of vertebrates shows to be highly consistent with human.In particular,early development of blood and cardiovascular system in zebrafish,including components of blood,is very similar with human.What's more,the defective mutants in development can still live for a few days,so using zebrafish as model is better for people to study early embryonic hematopoiesis.[3]The first part of our paper is to apply TALEN and CRISPR-Cas9 gene knockout technologies to knockout gfilaa and gfilb genes in the wild type AB zebrafish,respectively.We obtained gfi1aako mutants and gfi1bko mutants with a variety of different genotypes successfully.And study of spatio-temporal expression of each lineage marker in hematopoiesis(primitive hematopoiesis and definitive hematopoiesis)in these mutants and the development of various lineage cells in detail were carried out in order to investigate regulatory mechanism of gfilaa and gfi1b during erythropoiesis development.The latter part of the paper is about the effect of a-catenin,?-catenin on embryonic stem cell migration and their related cellular and molecular regulation mechanism.Content of the thesis is divided into two parts:the first part:Participation in the national project "Knockout all genes in chromosome 1 of zebrafish" and mechanism of gfilaa gene in regulation of hematopoietic development in zebrafish;The second part:The role of a-catenin and ?-catenin in embryonic hematopoietic stem cell migration.Part I:Participation in the national project "Knockout all genes in chromosome 1 of zebrafish" and mechanism of gfilaa gene in regulation of hematopoietic development in zebrafishObjective:This section introduces CRISPR-Cas9 and TALEN gene editing technologies,then use zebrafish as a model organism to complete the following two jobs with these two technologies:1)Using the CRISPR-Cas9 technology,participating in the national project"Knockout all genes in chromosome 1 of zebrafish" project,knockout 11 genes(vps37c,tmem132a,dnajal,smulb,rps6,btr01,clh20orf27,mucl3a,si:ch211-239f4.6,mucmsl,si:ch211-239f4.4)on chromosome 1 with ZKO numbers of 863?873 and screening stable genetic mutants from F2 generation.2)We will introduce TALEN targeted gene knockout technology in zebrafish in order to obtain zebrafish gfi1aako mutants with defects in hematopoietic development.Then taking advantage of CRISPR-Cas9 knockout technology,gfi1b,another family gene of gfi1,its mutants were got.Finally,we used these two mutants to study their phenotypes and functions,and to explore regulation and development of hematopoietic cells and the molecular mechanism of gfi1 gene.Method:1)We mainly use the CRISPR-Cas9 genome editing system with its main methods of gRNA target recognition and Cas9 nucleic acid cutting action.At first,we searched the basic information of genes numbered 863?873 in ZKO by NCBI,Ensembl and Smart web site,then we designed gene knockout targets of these related genes and synthesized of corresponding primers.Secondly,we obtained corresponding gRNA and Cas9 mRNA in vitro transcription.Then mixed these RNAs together and microinjected into one-cell stage of wild type zebrafish embryos.FO chimeric generations of zebrafish were got.Finally,we screened out FO adult zebrafish with targeted point mutations in reproductive cells by the method of PCR,enzyme digestion and sequencing.Such FO fish outcrossed with AB to get offsprings(F1),then screened zebrafish individuals which can stable heritable through cutting tail and sequencing.2)With TALEN and CRISPR-Cas9 gene knockout technologies,we wanted to knockout genes of gfi1aa and gfi1b in the wild type AB zebrafish,respectively.And study of spatio-temporal expression of each lineage marker in hematopoiesis(primitive hematopoiesis and definitive hematopoiesis)in these mutants and the development of various lineage cells in detail was carried out in order to investigate regulatory mechanism of gfi1aa and gfi1b during erythropoiesis development.Results:1)Using Cas9 gene knockout technology,we obtain single mutation genotype F1 generation zebrafish.Then outcross with AB to get reared offspring(F2)and use PCR,enzyme digestion and sequencing to obtain hybrid F2 generation mutant zebrafish,and submit to the national resource center for zebrafish.Intercross of F2 mutant zebrafish,phenotypic screening by methods such as in situ hybridization,Sudan black B dyeing and neutral red staining.2)Using TALEN targeted gene knockout technology to knockout gfi1aa gene in zebrafish,we obtained gfi1aako mutants with 23 missing bases(5'-TCTTCTCAACAGTCCCCGCTGAG-3').Phenotypic analysis indicates that,in the early stage of hematopoiesis,gfi1aa affect the development of erythrocytes and neutrophils,not macrophages.The gene gfilaa in gfi1aako mutants influence early development of erythrocytes mainly because it inhibits proliferation of erythrocytes.But it does not affect the erythroid precursors and cell morphology of erythrocytes.It was interesting that expression level of hemoglobin in gfilaako homozygotes was recovered equal to siblings at the period of 50 hpf.gfi1b is homologous gene of gfi1aa in zebrafish,it is also high expressed in erythrocytes and megakaryocytes.In order to prove that definitive hematopoiesis of erythrocytes is regulated by gfi1b,we used the CRISPR-Cas9 knockout technology to obtain gfi1bko mutants and continue to explore the development and function of erythrocytes.Double mutants experimental results found that in the definitive hematopoiesis,gfi1b,a family gene of gfil,would compensate the function of gfi1aa gene.Part II:The role of a-catenin and ?-catenin in embryonic hematopoietic stem cell migrationObjective:Hematopoietic stem cell(HSC)developmental defects will cause all kinds of developmental disorders in blood cells or blood vessels,and this defect is closely related to many diseases of the blood system.Embryonic HSCs migration is necessary for development of HSC.However,knowledge about the mechanism of embryonic migration of HSC is very limited.To date,the molecules that associate with embryonic hematopoietic stem cell migration are only a few,such as adhesion molecules(such as VE-cadherin),cytokines and chemokines.[4]?-catenin as a cellular skeleton protein,can bind to VE-cadherin with a-catenin and y-catenin and play an important role as a protein complex in intercellular adhesion.[5]Takahashi found that silencing ?-catenin in malignant melanoma B16 cells could promote migration of tumor cells.[6]cMyb in embryonic stage can regulate HSCs migration we reported before,is the research of cmyb unprecedented discovery.In this section,we used cmybthk=3 mutants of zebrafish as defect models to further study mechanisms of a-catenin and ?-catenin in embryonic HSCs migration and positioning.Moreover,improvement of molecular and signal networks regulated by embryonic HSC development will be carried out gradually.Method:First of all,in order to detect whether a-catenin and ?-catenin in HSC were influenced by mutation of cmyb,we use Tg(cd41:eGFP)transgenic line which HSC was marked by Cd41-eGFP to compare ?-catenin(ctnnal),?-catenin(ctnnb1)expression level in HSC between cmybhk=3 mutants and their siblings by QRT-PCR.We found that expression levels of ctnna1 and ctnnb1 in cmybhk=3 mutant were significantly higher than in their siblings.Secondly,rescue experiments were taken by microinjection of ctnnal-MO and ctnnbl-MO to knockdown its relevant gene so as to compare the numbers of total HSC in VDA region and the numbers of HSC emigrating from VDA region per unit time(4h)between cmybhk=3 mutants and their siblings.Finally,chloroquine(CQ),inhibitor of ?-catenin was used to treat zebrafish in order to observe the number of HSCs in VDA region in cmybhk=3mutants.Results:By QRT-PCR experiments,we found that expression of ctnnal and ctnnbl in cmybhk=3 mutants are higher than their siblings.It implied that a-catenin and?-catenin were probably downstream genes of cmyb and they probably regulate migration of HSC together with cMyb protein.It was found that the number of HSCs in VDA region in cmybhk=3 homozygous mutants was decreased to a considerable level with their siblings,and the number of HSCs emigrating per unit time was also increased after MO microinjection.After treatment of CQ,it could promote HSCs stranded in VDA in cmybhk=3mutamts migration and reduce the number of HSCs in VDA region.ConclusionPart one1)Using the CRISPR technology,mediated by Cas9 genome fixed point mutation experiment,knockout 11 genes on chromosome 1 with ZKO number of 863?873 and screening stable genetic mutants from F2 generation.2)During early hematopoietic development in zebrafish,it is mainly gfi1aa gene rather than gfi1b gene to regulate erythropoiesis;3)The gene gfilaa mainly affects early erythropoiesis by regulating proliferation of red blood cells,while their precursors and cell morphology of red blood cells were normal in gfi1aa-null zebrafish.4)During definitive hematopoiesis,gfi1b,a gene of gfi1 family,will compensate functions of gfi1aa.Part two5)?-catenin and ?-catenin were probably downstream genes of cmyb and they probably regulate migration of HSC together with cMyb protein.6)?-catenin and ?-catenin protein mainly affect embryonic HSC migration through changing the combination with cytoskeleton. |
PDF Full Text Request