An Early Block to Erythro-Megakaryocytic Development Conferred by Loss of Transcription Factor GATA-1.

Transcription factor GATA-1 is essential at multiple stages of hematopoiesis. Murine gene targeting and analysis of naturally occurring human mutations demonstrate that GATA-1 is required for the maturation of committed erythroid precursors and megakaryocytes. Prior studies also suggest additional, poorly defined, roles for GATA-1 at earlier stages of erythro-megakaryocytic development. To investigate these functions further, we studied hematopoietic differentiation of Gata1⁻ murine embryonic stem cells on OP9 stroma with the cytokine thrombopoietin (TPO) present. Initially, the Gata1⁻ cultures generated a wave of mutant megakaryocytes, but these were rapidly overgrown by a unique population of TPO-dependent blasts that continued to proliferate for more than 6 months in culture. These immature Gata1⁻ cells arose reproducibly in culture without growth lag or crisis, indicating that they derive directly from loss of GATA-1 and not from random genetic events acquired during cell culture. The cells express transcription factors GATA-2, FOG-1 and PU.1 and exhibit the surface phenotype Lin⁻, Sca1⁻, IL7R⁻, CD41⁺, cKit⁺, CD9⁺, and GPIb^low. Importantly, upon restoration of GATA-1 function, these cells undergo both erythroid and megakaryocytic differentiation, as assessed by morphology, ultrastructural analysis and the induction of lineage-specific markers. Clonal analysis shows that individual cells maintain the capacity for erythro-megakaryocytic differentiation. Hence, we term this unique population G1ME for Gata1⁻ -Megakaryocyte-Erythroid. To determine if G1ME cells are present in vivo, we analyzed E13.5 fetal livers of Gata1⁻/Gata1^wild-type chimeric embryos. Flow cytometry analysis demonstrates an expanded population of cells expressing the G1ME surface phenotype. Individual cells within this population also exhibit TPO-dependency, extensive proliferative capacity and GATA-1-dependent biphenotypic erythro-megakaryocytic maturation in vitro. Our findings indicate that the loss of GATA-1 impairs the maturation of a specific megakaryocyte-erythroid progenitor. This defines a new role for GATA-1 at a relatively early stage of hematopoiesis and provides potential insight into recent discoveries that human GATA1 mutations promote acute megakaryoblastic leukemia (AMKL), a clonal malignancy with features of both erythroid and megakaryocyte maturation.