Gabp Transcription Factor Is Required for the Development of Mouse Megakaryocytes.

Abstract 2604

GABP transcription factor has been implicated in the regulation of genes that are required for normal megakaryocytic differentiation. Megakaryocytes express several related ets factors, including Fli-1, ets2, and GABP, and it has been unclear if any single ets factor plays a non-redundant role in these cells. The tetrameric GABP transcription factor complex contains two molecules of GABPα, which binds DNA, and two molecules of GABPβ, which encodes the transcription activation domain. We created mice with loxP recombination sites which flank exons that encode Gabpa ets-related DNA-binding domains (floxed Gabpa, or Gabpa fl/fl), and bred them to mice that carry Mx1-Cre. In response to injection with the synthetic polynucleotide, pIC, these mice express Cre recombinase and efficiently delete Gabpa; these animals are referred to as knock-out (KO) mice. Control mice, which carry floxed Gabpa but lack Mx1-Cre, were treated identically with pIC. Platelet counts of KO mice declined to less than 50,000 within nine days, while platelet counts in control mice were unaffected. One half of KO mice died within two weeks of Gabpa deletion due to widespread visceral hemorrhage. Histologic examination of the bone marrow and spleen reveals a loss of megakaryocytes in KO mice, compared to control animals. Residual megakaryocytes in KO mice exhibit increased expression of platelet-specific antigens, CD41 and CD42, and a significant increase of DNA ploidy. Because Gabpa KO mice died with a striking loss of megakaryocytes and platelets, yet megakaryocytic differentiation appeared to be unimpaired, we sought to better define the nature of this defect. Bone marrow from Gabpa fl/fl mice was infected with a retrovirus that expresses Cre and green fluorescent protein (GFP), or control virus that expresses only GFP; grown for three days in liquid culture conditions that foster megakaryocytic differentiation; and analyzed for CD41 and CD42 expression, ploidy, and apoptosis. Gabpa was efficiently deleted by the Cre-bearing virus, and Gabpa deletion was associated with increased expression of CD41 and CD42, and increased DNA ploidy. However, Gabpa deletion was also associated with increased megakaryocytic-associated apoptosis, and in vitro megakaryocyte colony formation was dramatically reduced in Gabpα null cells. In summary, deletion of Gabpa in mice is associated with plummeting platelet counts, widespread visceral hemorrhage, and a loss of splenic and bone marrow megakaryocytes. In vitro analysis demonstrates intact megakaryocytic differentiation and a profound loss of megakaryocytic progenitor cells. The increased expression of megakaryocytic antigens and DNA ploidy may indicate that Gabpa deletion enhances megakaryocytic differentiation or, alternatively, it may represent selective loss of more immature megakaryocytic cells following Gabpa disruption. Data that directly test these alternative hypotheses will be presented. In summary, we demonstrate that GABP plays a non-redundant role in megakaryocyte development, that GABP is required for the proliferation of committed megakaryocytic progenitors, but that GABP is not required for the later stages of megakaryocytic maturation.