The Functional Study Of Transcription Factors Irf8 And Pu.1 In Zebrafish Myeloid Leukemogenesis

Defects in the developmental hierarchy of mononuclear (macrophage/monocytes) and polymorphonuclear (granulocytes) phagocytes may lead to serious myeloid disorders, including, in particular, myeloproliferative disease or myelodysplastic syndrome and, more severely, myeloid leukemia, and it is characterized by the extensive expansion of myeloid cells at different developmental stages. Although multiple cues, such as tumor suppressor gene mutations and active oncogenic genes, orchestrate the entire process, the transcription factors that are crucial for myeloid development play indispensable roles.IRF8, also called ICSBP, is a critical transcription regulator for myeloid lineage commitment. Loss of IRF8 leads to the expansion of neutrophils with a significant reduction in macrophages and dendritic cells. Consistent with its function in myelopoiesis, IRF8 plays critical roles in the pathogenesis of myeloid leukemia. In mice with an Irf8 defect, a chronic myeloid leukemia (CML)-like disease develops. In CML and AML patients, the compromised expression of IRF8 has been suggested to be a pathogenic factor. Meanwhile, the down regulation of IRF8 is necessary for the development of BCR-ABL-inducible CML disease. Therefore, Irf8 is recognized as a tumor suppressor in myeloid leukemia. However, the exact role and mechanism underlying IRF8 in myelopoiesis, particularly in leukemogenesis in vivo, remain poorly understood.PU.1, an Ets family member, is another factor that is important for myelopoiesis, it is also involved in leukemogenesis. Interestingly, IRF8 typically works with PU.1 via the IRF association domain (IAD) to regulate myeloid development. Meanwhile, this complex exerts a regulatory role on the promoter of p15mk4b, an important cyclin-dependent kinase inhibitor that is frequently inactivated in AML. Therefore, both factors most likely have synergistic roles in leukemogenesis. Unfortunately, the in vivo evidence about their roles and relationships in myelopoiesis, particularly leukemogenesis, is still minor, which is partially due to the lethality of Pu.l mutant mice, preventing the ability to generate Pu.l and Irf8 double mutant animals.Recently, zebrafish (Danio rario) have been utilized as a popular model for studying myelopoiesis due to several merits. Zebrafish are an ideal platform for building a leukemia model that is suitable for a large-scale chemical screen that can be used to identify candidate drugs. In zebrafish, the entire output of myeloid cells is generated by successive waves of haematopoiesis. A previous study reported that zebrafish Irf8 was a crucial factor that functioned downstream of Pu.l to control primitive myeloid progenitor fate determination.In this study, several irf8 mutant alleles were created via TALENs. In mutants, we found the significant reduction of macrophages occurred throughout the lifespan, which was consistent with the essential roles of IRF8 in macrophage formation and maintenance. The cell cycle analysis and apoptotic assay results indicated that Irf8 tightly regulated the myeloid proliferation at the larval and adult stages; therefore, the myeloproliferative syndrome quickly developed in irf8-/-. With the progression of time, this myeloproliferative syndrome worsened to transplantable myeloid leukemia and high lethality ensued. This study was the first to develop a myeloid leukemia zebrafish model that was caused by endogenous gene mutation, providing an important supplement to transgenic approach-based assays that could serve as a useful tool for studying leukemogenesis. The expansion of the neutrophils was well attenuated by topotecan treatment. Therefore, irf8â–³57/â–³57 might serve as a valuable platform for large scale chemical screens that are used to identify functional compounds in the regulation of myelopoiesis and leukemogenesis.At the same time, we found a more serious granulocytes infiltration happened in irf8â–³57/â–³57 /pu.1G242D/G242D,and with an abundant dead leukocytes emerged in gut and liver. To our surprise, the frequency of the blasts was significantly alleviated in double mutants, and finally result in an earlier death compared with irf8 mutants. Therefore, Pu.l and Irf8 synergistically regulate the progression of myeloid leukemia via different mechanisms.In addition, we performed ENU screening to obtain some mutations associated with myeloid cells development, and I have screened six mutants, fortunately, one of them is a ir/Smutant, the start codon "ATG" mutate into "ACG", and lead to a function loss of Irf8. The phenotype analysis showed that a similar abnormality appeared in macrophages and granulocytes compared with TALEN mutants.