Mechanism Of Gfi1 In Zebrafish Hematopoiesis And Establishment Of C-myb Zebrafish Leukemia Model

Hematopoiesis is a complex and well-organized dynamic process.Accurate regulation of different transcription factors is essential for normal hematopoiesis.The occurrence of disease is always closely associated with the abnormality of transcription factors.Gfi1 and Gfi1b,as transcription repressors,play crucial roles in hematopoiesis.Gfil could restrict HSCs proliferation,regulate myelopoiesis and lymphopoiesis.Gfi1b is essential for erythroid progenitor cells terminal differentiation and megakaryocytes differentiation.Mutation of GFI1 gene associates with severe congenital neutropenia,while GFI1B gene mutations associate with platelet deficiencies and bleeding disorders.The c-MYB transcription factor is a crucial regulator of hematopoietic cell proliferation and differentiation.Dysregulation of cMYB activity is often associated with various hematological disorders,such as leukemia.In this study,we investigate how gfi1 regulates zebrafish hematopoiesis and establish a c-mybhyper zebrafish leukemia model.In the first part of this study,we investigate how gfi1 regulates zebrafish hematopoiesis.Gfil and Gfib are indispensable for endothelial to hematopoietic transition in the emergence of EMP and HSC.However,until now,the role of Gfi1 and Gfi1b in primitive hematopoietic commitment is still not well understood.We established a heritable zebrafish gfi1aa mutant using TALEN technology.We found that gfi1aa mutant showed decreased primitive erythropoiesis and increased primitive myrlopoiesis,indicating gfi1aa is indispensable for primitive erythropoiesis and primitive myelopoiesis.Then we detected the expression of hemangioblast makers in gfi1aa mutant,gfi1aa mutant showed increased expression of hemangioblast makers etv2.Moreover,etv2 MO knockdown could rescue the hematopoietic defect in gfi1aa mutant.We found that Gfi1 could bind to etv2 and alter the H3K4me1 level of etv2 intron2 region.Furthermore,we established a heritable zebrafish gfi1b mutant by CRISPR/cas9.We found that gfi1b was dispensable for primitive erythropoiesis.However,gfi1b and gfi1aa double mutant showed severely primitive erythropoiesis defect.Our studies reveal that Gfilaa-dependent downregulation of etv2 is indispensable for hemangioblasts differentiation,erythropoiesis,myelopoiesis and gfi1b function synergistically with gfi1aa in primitive erythropoiesis.The second part,we report a detail characterization of c-myb-gfp transgenic zebrafish harboring c-Myb hyperactivity(named c-mybhyper).c-MYB is expressed at high levels in most AML and ALL patients.However,the animal model to reveal the mechanism of c-MYB aberrant expression associated with hematological disorders is still lacking.For the lack of a survival animal model with c-MYB aberrant leads to the difficulty of tracking disease course of a c-MYB-related blood disease.The c-mybhyper zebrafish exhibited abnormal granulocyte expansion in embryonic stage that resembled human myelodysplastic syndrome(MDS)from embryonic stage to adulthood.Strikingly,a small portion of c-mybhyper adult fish developed acute myeloid leukemia-like or acute lymphoid leukemia-like disorders from MDS with age.Moreover,c-mybhyper adult fish leukemias were transplantable.The myeloid and lymphoid malignancies in c-mybhyper adult fish were likely caused by the hyperactivity of c-myb,resulting in the dysregulation of a number of cell-cycle-related genes.Finally,treatment with c-myb target drug flavopiridol could relieve the MDS-like symptoms in c-mybhyper adult fish.Our study establishes a zebrafish model for studying the cellular and molecular mechanisms underlying c-Myb-associated leukemogenesis as well as for anti-leukemic drug screening.