Functions Of C-Myb And C/ebp1 In Granulopoiesis And Diseases Formation

Background:Myeloid cells play a prominent and essential role in a number of important biological processes,including host defense,tissue regeneration and repairation.Myeloid cells can be devided into polymorphonuclear cells(Neutrophils,eosinophils,basophils and mast cells)and mono/macrophage(circulating macrophages,dendritic cells and tissue-specific macrophages).These two types of cells are in different shapes and functions.They all produce from two successive and partially overlapping hematopoietic stages:Primitive hematopoietic stage results in only limited myeloid cells and red blood cells;definitive hematopoiesis can continuously supply all the types of blood cells through a human’s life.Myelopoiesis is a process that including Mesodermal cells or HSCs specializing into myeloid progenitors directly,the cell fate choice into monocytes/macrophages or neutrophils and the hierarchy of differentiation and maturation processes from hematopoietic stem cells or myeloid progenitors.Myelopoiesis has been known to be a complex and orderly dynamic process,which requires a rigorous control by various different transcription factors.Any link error occurs will lead to disease.Thorough understanding of the regulation networks of the transcription factors will contribute a lot to demonstrate the basis of the occurrence of disease.Although a large number of related researches have been pay attention to the formation of myeloid cell development and maturation in the past decades,which were carried out on mice and cells.It’s very difficult to observe myeloid cells at embryonic stage in vivo because of opaque.And the transcriptional regulatory networks of early myeloid cell fate choice,specialization,differentiation and maturation are still currently unknown.Zebrafish has been used to be an animal model in different research areas(environmental safety,drug screening,developmental biology,immunology,human diseases)for more than 40 years due to its biological and genetic advantages such as small size,in vitro fertilization,the transparent body,strong reproductive capacity.Zebrafish is used to study myeloid cell development and myeloid-related diseases precisely because of its unique biological advantages and genetic advantages.There are also two hematopoietic stages in zebrafish which similar to the hematopoiesis process in mammal.Primitive erythropoiesis is produced from the inter media cell mass originated from the posterior lateral mesoderm while primitive myelopoiesis from anterior lateral mesoderm.At around 26h after fertilization,hematopoietic stem cells first came out at the dorsal aorta ventral region which marks the start phase of definitive hematopoiesis.These hematopoietic stem cells then gradually migrate to the caudal hematopoietic tissue or differentiate.Finally settle down at kidney which resembles human bone marrow.Furthermore,transcription factors in zebrafish that involved in many signaling pathways are very similar to mammalian.Thus,zebrafish can’t be a more excellent animal model for researching in myeloid formation,development and maturation,of which will help to delineate the networks of human myeloid formation.c-MYB is an important transcription factor,but also a proto-oncogene in hematopoiesis,that was first isolated from avian myeloid hyperplasia virus homologous with v-MYB.c-MYB is located at human chromosome 6,encoded c-MYB protein that contains highly conserved DNA domains:(1)N-terminal DNA binding domain,is capable of binding to specific DNA sequences;(2)transcription activation domain located in the center,which can facilitate transcription of downstream targets after binding to its promoter;(3)C-terminal negative regulatory region containing leucine zipper can inhibit transcriptional activation of genes.c-MYB is an important transcription factor involved in the development and differentiation of hematopoietic system that gradually decrease expression from hematopoietic stem cells and progenitor cells to differentiated more mature cells.It may be involved in the development of a series of human leukemia and other malignancies.c-MYB dysregulation will lead to some kinds of blood diseases such as myeloid dysplasia syndrome,leukemia,neutrophil-specific granule deficiency syndrome due to its functions in hematopoiesis.In poultry and mice,aberrant activation of c-MYB may cause leukemic transformation.Inhibition the activity of c-MYB in mice can inhibit acute myeloid leukemia.Clinically,high expressions of c-MYB were also found in leukemia cells in acute myeloid leukemia,acute lymphoblastic leukemia and chronic myeloid leukemia patients.All in all,c-MYB plays a key role in the development of blood formation and related diseases.However,in animal models,the underlying mechanism of c-MYB aberrant expression resulting in hematological disorders is still lack of evidence.Also the lack of a survival animal model with c-MYB activation leads to the difficulty of tracking diseases course of a c-MYB-related blood disease.c-MYB often acts with other transcription factors to show synergy and antagonism relationship on regulating proliferation and differentiation of cells.Primitive myeloid hematopoiesis is an important step in vertebrate embryonic development;it is still obscured that how the primitive myeloid development controlled despite several transcription factors have been reported.We found c-Myb also influence primitive myeloid development addition to the hematopoietic stem cells of definitive development which has not been mentioned in the relevant research on cell lines and mouse model.CCAAT enhancer binding protein family is one of a basic leucine zipper protein family member,played an important role in the formation,proliferation,differentiation of cells,oncology,immune,and metabolic.There are six members in mammals including C/EBPα,C/EBPβ,C/EBPγ,C/EBPδ,C/EBPε and C/EBPξ,and most of which can be found homolog in zebrafish.cebp1 encodes a protein homologous to mammalian C/EBPs containing a C-terminal basic leucine zipper region.However,the N-terminal region of C/ebp1 showed no significant homologous sequence with any of the human C/EBPs.Although it has been reported that cebp1 is expressed specifically in neutrophils related to lyz expression.But there are few reports about its functions.The myeloid-specific expression pattern of C/ebpl is similar to the expression of mammalian C/EBPε,which may perform some or all of the functions of the mammalian C/EBPε.Meanwhile,c-MYB as a proto-oncogene can be found abnormal expression in many blood diseases.The pathological mechanism is still obscured despite there are some research paper about c-MYB and MDS.This study was divided into three chapters:Chapter 1:Myeloid defect mutants obtained from forward and reverse genetic approaches.Chapter 2:The underlying mechanism of neutrophils maturation in zebrafish regulated by c-Myb and C/ebpl.Chapter 3:Aberrant c-myb and cebpl expression leads to myeloid disorder in zebrafish.In this chapter,we introduced a c-myb abnormal expression zebrafish transgenic line c-myb-gfp AE.Abnormal proliferation of myeloid cell in c-myb-gfp AE arises from embryos to adult fish which assemble human MDS.This model may provide a valuable platform for the further study of the role of c-Myb in leukemia pathogenesis and development of new anticancer drugs.This study may uncover an unreported machinery of primitive neutrophil development and should make its contribution on discovering the cause of human disease.Chapter 1:Myeloid defect mutants obtained from forward and reverse genetic approaches1.Objects1)This section we focus on the forward genetics scale ENU mutagenesisscreening of myeloid-specific gene lysozyme C defect mutants,the gene was named cmybhk=3 after the positional cloning.2)cebpl mutant was knockout by reverse genetics technology TALEN and named cebp1smul2.Methods1)ENU mutagenesis methods were used in this chapter.40 AB wild-type male zebrafish were treated and the survivals were mating with AB female to generate F1 generation.Then F2 generation was produced by F1 incrossing.Embryos were collected by F2 incrossing and screened by lysozyme C in situ hybridization for myeloid defects.Finally the affected gene was confirmed by mapping and sequencing.(I was only involved in large-scale mutation screening but not involved in the subsequent positional cloning)2)TALEN target was designed on the second exon of the cebpl gene with a FaqI recognized site used to identify mutations.Capped TALEN mRNA was synthesized in vitro by SP6 transcriptase after the plasmid was constructed.Then this mRNA was micro injected into single cell of zebrafish embryos which may lead to cebpl gene disruption and early termination of protein translation.Finally,cebpl mutant was screened by FaqI digestion.(Zhao Linfeng was involved in this section)3.Results1)After ENU treatment,the survivals of the AB wild-type male zebrafish were mated with female AB to generate F1 generation families which comprised of 20 F1 families of total 2000 zebrafish.1564 F2 families were then generated by F1 incrossing.Five myeloid-specific gene lysozyme C defect mutants were obtained by large-scale in situ hybridization screening of the 1383 F2 families identified with other blood lineage markers.One of the mutants firstly named 230 was then mapped and sequenced to determine the c-myb gene mutation and named cmybhk=32)TALEN target was designed on the second exon of the cebp1 gene containing a FaqI recognized site used to identify mutations.Capped TALEN mRNA was synthesized in vitro by SP6 transcriptase after the plasmid was constructed.Then this mRNA was micro injected into single cell of zebrafish embryos which may lead to cebpl gene disruption and early termination of protein translation.Finally,cebpl mutant was screened by FaqI digestion.4.Conclusions1)Five myeloid-specific gene lysozyme C defect mutants were obtained by large-scale in situ hybridization screening suggesting that ENU mutagenesis strategy is an efficient method.One of the mutants firstly named 230 was then mapped and sequenced to determine the c-myb gene mutation and named cmybhk=3.2)Using TALENs technology,we designed a TALEN to specifically recognize cebpl target sequence.mRN A encoding TALEN was microinjected into single cell stage zebrafish embryos after in vitro transcription.Finally we screened and obtained a cebp1 gene defective mutant by FaqI restriction enzyme digestion named cebp1smul.Chapter 2:The underlying mechanism of neutrophils maturation in zebrafish regulated by c-Myb and C/ebpl1.Objects1)We tried to characterize cmybhk=3 and cebp1smul mutants to initially identify the steps they act on primitive neutrophil development through a variety of phenotypic analysis and detection methods.2)We tried to illustrate the genetic networks in primitive neutrophil development through the molecular level,cellular level,and genetic pathway between c-Myb and C/ebpl.2.Methods1)We tried to characterize cmybhk=3 and cebp1smul mutants through whole mount in situ hybridization with different blood lineage markers including 21hpf pu.l marked myeloid progenitors,30hpf csflra marked macrophages,36hpf mfap4 marked macrophages,3dpf apoeb marked microglia,3dpf neutral red staining marked microglia,36hpf mpx,lyz marked neutrophils,22hpf cebp1maked neutrophil precursors.(Jin Hao was involved in this section)2)Differential interference contrast microscopy and Sudan Black B(SB)staining were used to observed neutrophil maturation status in mutants.3)Cebp1 was overexpressed in cebp1smul mutants by microinjecting pTol-hs70:cebp1-eGFP reconstructed plasmid to rescue cebp1smul mutant phenotypes.(Rescue experiment in cmybhk=3 mutant was completed by Professor Zhang Yiyue)4)lyz,mpx,Sudan Black B expression and DIC were observed in cmybhk=3 and cebp1smul double mutants compared with single mutants and wild type.5)Online software was used to predict c-Myb and C/ebpl binding sites at lyz promoter.Then GFP reporter vectors following by c-Myb and C/ebp1 binding sites with mutations were constructed by overlap PCR.The expression of GFP is evaluated between different groups.6)Furthermore,ChIP and co-IP were performed to confirm the interaction between c-Myb,C/ebp1 and lyz promoter,c-Myb and C/ebp1.(c-Myb related immunoprecipitation experiments were performed by Professor Zhang Yiyue)7)lyz was overexpressed in cebp1smul and cmybhk=3 mutants by microinjecting and calculated the number of SB positive cells.3.Results1)We tried to characterize cmybhk=3 and cebp1mul mutants through whole mount in situ hybridization with different blood lineage markers.It showed that mono/macrophages,myeloid progenitors,immature neutrophils were not affected but mature neutrophils were decreased in cmybhk=3 and cebp1smul mutants.2)Differential interference contrast microscopy and Sudan Black B staining were used to observed neutrophil maturation status in mutants.It showed that granules on neutrophils surface and SB positive cells became less obvious compared to its siblings.3)Rescue experiments indicate pTol-hs70:cebp1-eGFP can increase the expression of the lyz in cebp1smul mutant under heat shock activation using microinjection.4)lyz,mpo,Sudan Black B expression and differential interference contrast microscopy were observed in cmybhk=3 and cebplsmul double mutants compared to single mutants and wild type.And all of the markers show significant decrease in cmybhk=3 and cebp1smul double mutants than in single mutants and wilt type siblings.5)Online software was used to predict c-Myb and C/ebpl binding sites at lyz promoter.Then GFP reporter vectors following by c-Myb and C/ebpl binding sites with mutations were constructed by overlap PCR.The expression of GFP was evaluated between different groups.Results showed that GFP expression in c-Myb and C/ebpl binding sites mutation combined was the lowest one of the four groups,while the c-Myb binding sites mutation and C/ebpl binding sites mutation had lower GFP expression than non-mutated on lyz promoter.6)ChIP and co-IP experiments showed direct interactions between c-Myb,C/ebp1 and lyz promoter,c-Myb and C/ebp1.7)Results showed that overexpression of lyz can rescue SB positive cells in cebp1smul mutant but not in cmybhk=3 mutant.4.Conclusions1)Monocyte/macrophages,myeloid progenitors,immature neutrophils were not affected but mature neutrophils were decreased in cmybhk=3 and cebp1smul mutants.These data indicated that certain stage of granulopoiesis but not the upstream of myelopoiesis was blocked in cmybhk=3 and cebp1smul mutants.2)DIC and Sudan Black B staining showed that the remaining neutrophils in cmybhk=3 and cebplsmul mutants were really unmatured.3)All of the above phenotypes in cmybhk=3 and cebp1suml mutant were indeed due to the lack of c-myb and cebpl proved by rescue experiment.4)We found that in the process of primitive neutrophils maturation stage,c-Myb collaborates with C/ebpl in the same pathway proved by double mutant analysis.5)We may come to a conclusion that c-Myb and C/ebpl have a direct and synergistic effect on lyz mRNA expression.6)lysozyme C may be a key factor in the process of neutrophil maturation in cmybhk=3 and cebp1smul mutants.c-Myb is supposed to have other factors downstream directly or indirectly to affect maturation of granulocytes since the phenotypes can be rescued in cebp1smul mutants but not in cmybhk=3 mutanst.Chapter 3 Aberrant c-myb and cebpl expression leads to myeloid disorder in zebrafish1.Objects1)We tried to characterize aberrant c-myb expression zebrafish c-myb-gfp through whole mount in situ hybridization and SB staining.2)We hope to establish zebrafish animal disease models compared to human myeloid dysplasia using our aberrant c-myb and cebpl expression zebrafish.2.MethodsWe tried to characterize aberrant c-myb expression zebrafish c-myb-gfp through whole mount in situ hybridization and SB staining with different blood lineage markers.Including 36hpf c-myb,3dpf c-myb,5dpf c-myb,3dpf cebpa,3dpf mfap4,3dpf lyz,3dpf SB and 7dpf SB.3.Results1)We found that c-myb gene expression significantly increased in c-myb-gfp transgenic zebrafish compared with the wild-type by WISH.2)It showed that cebpa,lyz positive neutrophils increased significantly,while mfap4 positive macrophages reduced.The SB staining results not only show the increased number of granulocytes but also indicated the enlargement of neutrophil size,increase of granule,suggesting that the abnormal shape of neutrophils in c-myb-gfp embryos.4.Conclusions1)This chapter we introduce a c-myb aberrant activation transgenic zebrafish c-myb-gfp.c-myb abnormal expression can cause myeloid disorder in zebrafish embryonic stages.2)The phenotypes of cmybhk=3、cebp1smul mutants were similar to SGD in human while the phenotypes of c-myb-gfp transgenic zebrafish were similar with MDS in human.