The Biological Function Of Irf2bp2 In Zebrafish Embryonic Hematopoietic Development

Chapter I The Biologic function of Irf2bp2a on neutrophil apoptosis in zebrafish embryonic development.Interferon regulatory factor 2 binding protein 2(IRF2BP2)was first named for its specific binding to the transcription factor IRF2 and its corepressor.IRF2BP2 belongs to the IRF2BP family.The family consists of three members:IRF2BP1,IRF2BP2 and IRF2BPL,all of which are evolutionarily conserved.They contain an N-terminal C4 zinc finger domain and a C-terminal C3HC4 type ring finger domain.Unlike humans and mice,which have only one IRF2BP2 gene,there are two paralogs of irf2bp2a and irf2bp2b in the zebrafish genome.By utilized whole mount in situ hybridization to illustrate the temporal and spatial expression of irf2bp2 paralogs in zebrafish.Both of them were found in the pectoral fin,eyes,pharynx and neural system,in addition to their unique expression patterns.Irf2bp2a was found to be obviously expressed in PBI during early embryonic development,suggesting that irf2bp2a may play an important role in embryonic hematopoietic development.Therefore,we constructed the irf2bp2a knock-out lineage by CRISPR Cas9 technique,and defective granulopoiesis was displayed in irf2bp2a-/--mutants.Further molecular studies elucidate that the activated apoptosis pathway mediated by p53 is implicated in defective granulopoiesis of irf2bp2a-/-mutantsChapter II The biologic function of Irf2bp2b in the cell fate choice of NMPs in zebrafish.Proper cell fate choice of neutrophil-macrophage progenitors(NMPs)is essential for adequate myeloid sub-populations during embryonic development and in adulthood.The network governing NMPs cell fate is composed of several key determinants such as myeloid master regulators C/EBPa and PU.1.Nevertheless,more regulators remain to be identified and characterized.To ensure balanced commitment of NMPs toward each lineage,the interplay among these determinants is not only synergistic,but also antagonistic.Depletion of Interferon regulatory factor 2 binding protein 2b(Irf2bp2b),a well known negative transcription regulator,results in a bias in NMP cell fate choice which favors macrophages at the expense of neutrophils during the zebrafish definitive myelopoiesis stage in deficient embryos.Mechanistic studies indicate that Irf2bp2b acts as a downstream target of C/ebpa,to repress pu.l expression,and that SUMOylation confers the repressive function of Irf2bp2b.Thus,Irf2bp2b is a novel determinant in the cell fate choice of NMPsChapter III Primitive macrophages are dispensable for HSPC mobilization and definitive hematopoiesisHematopoietic development is a process in which hematopoietic stem and progenitor cells(HSPCs)proliferate and differentiate into the full complement of hematopoietic cells In vertebrates,haematopoietic stem/progenitor cells(HSPCs)first emerge in the aorta-gonad-mesonephros(AGM)before colonizing transitory and subsequently definitive haematopoietic organs allowing haematopoiesis throughout adult life.The molecular mechanisms affecting the migration of HSPCs and colonization of hematopoietic organs remain unknown.It has been reported that the migration and difinitive hematopoietic development of HSPCs are affected in the absence of primitive macrophage deletion,but in our C/EBPa knock out fish line(cebparj31),with the absence of primitive macrophages,the migration and colonization of HSPCs and the difinitive hematopoietic development of hematopoiesis were not affected.We therefore concluded that the absence of primary macrophages has no significant effect on the migration and colonization of HSPCs and the development of definitive hematopoiesis.