The transcription factor Gata1 is expressed in several hematopoietic lineages and plays essential roles in normal hematopoietic development during embryonic stages. The lethality of Gata1-null embryos has precluded determination of its role in adult erythropoiesis. Here we have examined the effects of Gata1 loss in adult erythropoiesis using conditional Gata1 knockout mice expressing either interferon-or tamoxifen-inducible Cre recombinase (Mx-Cre and Tx-Cre, respectively). Mx-Cre-mediated Gata1 recombination, although incomplete, resulted in maturation arrest of Gata1-null erythroid cells at the proerythroblast stage, thrombocytopenia, and excessive proliferation of megakaryocytes in the spleen. Tx-Cremediated Gata1 recombination resulted in depletion of the erythroid compartment in bone marrow and spleen. Formation of the early and late erythroid progenitors in bone marrow was significantly reduced in the absence of Gata1. Furthermore, on treatment with a hemolytic agent, these mice failed to activate a stress erythropoietic response, despite the rising erythropoietin levels. These results indicate that, in addition to the requirement of Gata1 in adult megakaryopoiesis, Gata1 is necessary for steady-state erythropoiesis and for erythroid expansion in response to anemia. Thus, ablation of Gata1 in adult mice results in a condition resembling aplastic crisis in human. IntroductionProduction of mature blood cells from the pluripotent hematopoietic stem cells is continuous throughout life. This process is controlled by combinatorial functions of lineage-specific and ubiquitous transcription factors. The zinc finger transcription factor Gata1 is a prototype of lineage-specific transcription factors, with restricted expression in several myeloid lineages. 1,2 Gene ablation studies of Gata1 in mice revealed its essential role in erythroid cell development. Embryos lacking Gata1 die at approximately embryonic day 11.5 because of arrested maturation of primitive erythroid cells. 3 A similar phenotype was observed in embryos bearing a Gata1 knockdown mutation (Gata1.05) that causes 95% reduction of Gata1 mRNA level in erythroid cells. 4 A critical role for Gata1 in megakaryocyte maturation and platelet formation has been shown in mice with megakaryocyte-specific Gata1 deficiency. 5,6 A remarkable phenotype of these mutant mice is that defective maturation of megakaryocytes is associated with excessive proliferation, suggesting that Gata1 inhibits cell proliferation during megakaryocytic maturation.Although roles for Gata1 in postnatal megakaryopoiesis have been thoroughly investigated, the embryonic lethality caused by Gata1 deficiency in erythroid precursors has precluded studies of its role in postnatal erythropoiesis. In adults, the rate of red cell production in the steady-state condition is much lower than that in the fetal and neonatal stages. However, in response to erythropoietic stress, the rate of erythropoiesis is rapidly increased. 7 The plasma levels of erythropoietin (Epo), the central regulator of erythr...

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Erythroid progenitors have the potential to proliferate rapidly in response to environmental stimuli. This process is referred to as stress erythropoiesis, with erythropoietin (EPO) playing central roles in its promotion. In this study, we wanted to elucidate the molecular mechanisms governing the regulation of stress erythropoiesis and the maintenance of red-cell homeostasis. This was achieved by our development of a noninvasive real-time monitoring system for erythropoiesis using transgenic mouse lines expressing luciferase under the control of the mouse Gata1 hematopoietic regulatory domain (G1-HRD-luc) or human ␤-globin locus control region (Hbb-LCR-luc). Optical bioluminescence images revealed that the luciferase was specifically expressed in spleen and bone marrow and was induced rapidly in response to anemia and hypoxia stimuli. The G1-HRD-luc activity tracked the emergence and disappearance of proerythroblast-stage progenitors, whereas the Hbb-LCR-luc activity tracked erythroblasts and later stage erythroid cells. Increased plasma EPO concentration preceded an increase in G1-HRD-luc, supporting our contention that EPO acts as the key upstream signal in stress erythropoiesis. Hence, we conclude that G1-HRD-luc and Hbb-LCR-luc reporters are differentially activated during stress erythropoiesis and that the transgenic mouse lines used serve as an important means for understanding the homeostatic regulation of erythropoiesis. IntroductionThe lifespan of circulating erythroid cells is limited, rendering continuous erythropoiesis a necessity throughout the life of the animal. Erythroid progenitors have the potential to proliferate rapidly in response to anemia and hypoxia stimuli, a process referred to as stress erythropoiesis and mediated by erythropoietin (EPO). 1,2 EPO is a glycoprotein hormone, 3 and its expression is induced by various erythropoietic stimuli. During erythropoiesis, hematopoietic stem cells (HSCs) first give rise to common myeloid progenitors (CMPs) with the potential to differentiate toward erythroid, megakaryocytic, and granulocytic/ monocytic cell lineages. 4 CMP progenitors differentiate into bipotential megakaryocytic and erythroid progenitors (MEPs). 4 The earliest erythroid-committed progenitors are those corresponding to erythroid burst-forming units (BFU-Es), which further differentiate through erythroid colony-forming units (CFU-Es) and proerythroblasts into erythroblasts. These erythroid progenitors, which account for a rare population of hematopoietic cells under steady state conditions, have the potential to proliferate rapidly in response to acute erythropoietic stimuli. [5][6][7] In particular, the number of CFU-Es increases markedly during stress erythropoiesis. The receptor for erythropoietin (EpoR), which is expressed abundantly in CFU-E stage progenitors, 8 plays a crucial role in promoting the erythropoietic response. 9,10 Erythropoietic stimuli result in tissue hypoxia and induce EPO gene expression through the activation of hypoxia-inducible transcription factors. EP...

Transcription factor GATA-1 is essential for erythroid cell differentiation. GATA-binding motifs have been found in the regulatory regions of various erythroid-specific genes, suggesting that GATA-1 contributes to gene regulation during the entire process of erythropoiesis. A GATA-1 germ-line mutation results in embryonic lethality due to defective primitive erythropoiesis and GATA-1-null embryonic stem cells fails to differentiate beyond the proerythroblast stage. Therefore, the precise roles of GATA-1 in the later stages of erythropoiesis could not be clarified. Under the control of a GATA-1 gene hematopoietic regulatory domain, a GATA-1 mutant lacking the N-finger domain (∆ ∆ ∆ ∆ NF mutant) was over-expressed in mice. These mice exhibited abnormal morphology in peripheral red blood cells (RBCs), reticulocytosis, splenomegaly, and erythroid hyperplasia, indicating compensated hemolysis. These mice were extremely sensitive to phenylhydrazine (PHZ), an agent that induces hemolysis, and their RBCs were osmotically fragile. Importantly, the hemolytic response to PHZ was partially restored by the simultaneous expression of wild-type GATA-1 with the ∆ ∆ ∆ ∆ NF mutant, supporting our contention that ∆ ∆ ∆ ∆ NF protein competitively inhibits the function of endogenous GATA-1. These data provide the first in vivo evidence that the NF domain contributes to the gene regulation that is critical for differentiation and survival of mature RBCs in postnatal erythropoiesis.