Although erythroid cells and megakaryocytes derive from a common progenitor and share many essential transcription factors, their terminal maturation follows very different paths: erythroid cells undergo cell cycle exit and enucleation while megakaryocytes continue to progress through the cell cycle, but skip late stages of mitosis to become polyploid cells. In our efforts to identify genes that participate in this process, we discovered that survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express survivin throughout their maturation, up through the terminal orthochromatic stage of differentiation. In contrast, purified murine megakaryocytes express nearly 4-fold lower levels of survivin mRNA compared to erythroid cells and no detectable protein. To investigate whether survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring the human survivin cDNA or control retrovirus, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of survivin antagonized megakaryocyte development, but did not affect the terminal differentiation of red blood cells. In contrast, a 50% reduction in survivin mRNA, caused by a heterozygous loss of survivin, blocked erythroid, but not megakaryocyte, development in vitro. Thus, our preliminary data support a physiologic role for persistent survivin expression in erythroid cells and a significantly reduced level of expression in megakaryocytes. Based on these findings, we hypothesize that persistent survivin expression is required for differentiation and/or survival of erythroid cells, while its reduction is essential for terminal maturation of megakaryocytes. In this application, we propose to investigate how survivin contributes to erythroid cell and megakaryocyte development. Specifically, we plan to 1) Determine the requirement for survivin in red cell and megakaryocyte development by conditional gene targeting in mice, 2) Characterize the mechanism by which survivin participates in erythroid cell differentiation by analyzing the phenotype of cultured erythroid cells with reduced survivin expression and by identifying survivin protein complexes in erythroid cells, and 3) Investigate why persistent survivin expression is inhibitory to megakaryocyte polyploidization and maturation by analysis of transgenic mice that ectopically express survivin in megakaryocytes and erythroid cells. A more detailed assessment of how survivin contributes to hematopoiesis will aid in our understanding of the role of cell cycle regulation and apoptosis in normal blood cell development, and may shed light into the mechanism by which erythroid cells and megakaryocytes arise from a common precursor. ? ? ?
