Functional Analysis of Transcription Factor c-Maf in Embryonic Hematopoiesis.

Abstract 3646

Poster Board III-582

c-Maf is one of the large Maf (musculoaponeurotic fibrosarcoma) transcription factors that belong to the AP1 super family of basic leucine zipper proteins. c-Maf has been shown to be important for the regulation of IL-4 and IL-21 gene expression in T-helper-2 cells and the formation of the lens structure. A recent report has demonstrated that the augmentation of c-Maf gene expression was observed in myeloma cell lines and bone marrow samples of multiple myeloma patients. It suggests that c-Maf plays important role in multiple myeloma pathogenesis. However, the physiological roles of c-Maf in normal hematopoiesis are largely unknown and have never been analyzed in vivo setting. In this study, we investigated that c-Maf is indispensable for hematopoesis via analyzing c-Maf deficient (c-Maf^−/−) mice. In the C57BL/6J background, c-Maf^−/− mice were embryonic lethal and it died between embryonic day (E) 15 and E18. c-Maf^−/− embryo looked pale and had evidence of anemia. In c-Maf^−/− peripheral blood at E14.5, the number of enucleated erythrocytes was decreased compared that in wild-type (WT) littermates. Flow cytometric analysis of fetal liver cells indicated that mature red blood cells were reduced in c-Maf^−/− embryos. In addition, Annexin-V positive apoptotic cells were increased in c-Maf^−/− fetal liver. Interestingly, there was no significant difference between WT and c-Maf^−/− fetal liver cells in erythroid colony counts on the colony-forming unit assay. These results indicated that c-Maf^−/− erythroid cells can develop to mature cells, and the impaired definitive erythropoiesis in the c-Maf^−/− mice is due to a non-cell-autonomous effect. Immunohistochemical staining of WT fetal liver using anti-c-Maf antibody showed that c-Maf was highly expressed in macrophage but not in erythroid cells. For this reason, we hypothesized that c-Maf^−/− macrophage was responsible for anemia of c-Maf^−/− embryo. The erythropoietic microenvironment in fetal liver mainly consists of clusters of erythroblastic islands that made up of central macrophage and surrounding erythroblasts. The compromise of erythroblasitc islands through defect of macrophage causes anemia and embryonic lethality. To verify whether c-Maf^−/− macrophage cause fail to form erythroblasitic island, we isolated the erythroblasitc island from WT and c-Maf^−/− fetal liver. As a result, the number of erythroblast surrounding the macrophage in erythroblastic island was significantly reduced in c-Maf^−/− mice. Moreover, in vitro reconstitution experiments showed that c-Maf^−/− erythroblasts were able to form erythroblastic island with WT macrophages, while c-Maf^−/− macrophages and WT erythroblasts could not form erythroblastic island. These results strongly suggest that the cause of anemia in c-Maf^−/− mice is due to abnormal function of macrophages in forming erythroblastic island. Finally, to clarify the molecular mechanism in formation of erythroblastic island, we found the target gene of c-Maf in fetal liver macrophages. The DNA microarray and real time RT-PCR analyses showed that the decreased expression of vascular adhesion molecule 1 (VCAM-1), known as an adhesion molecule expressed on macrophage of erythroblastic island, was observed in c-Maf^−/− fetal liver macrophage. We conclude that c-Maf is indispensable for definitive hematopoiesis and erythroblastic island formation through crucial roll in fetal liver macrophages.