The long-term goal of this project is to determine molecular mechanisms that regulate embryonic red blood cell development. Hematopoiesis is controlled by a large number of cytokine signaling molecules that activate, within a stem cell population, specific cell differentiation programs. Failure to regulate from a stem cell compartment the regeneration of properly differentiated erythroid cells is implicated in human diseases including erythroleukemias, aplastic anemia and pancytopenia. In additiOn, the process Of lineage-speCifiC differentiation of the hematopoietiC stem cell is central to the maintenanCe of normal blood cell counts, and to the reconstitution of blood counts following bone marrow insults, such as infections, radiation and chemotherapy. Therefore, an increased understanding of the molecular mechanisms underlying lineage-speCifiC differentiation processes could enhance our ability to combat selective leukemias and cytopenias, and facilitate bone marrow reconstitution following transplantation. Progress toward these goals is hampered by difficulties in working with purified stem cells. While differentiation programs are likely to be conserved among vertebrates, the Xenopus embryological system provides important advantages for studying blood formation. In Xenopus, the erythroid progenitors are isolated and cultured to differentiate in vitro, and gene expression modulated easily by injection of RNA and DNA. Using this system, it is shown that the growth factor BMP-4 is necessary during gastrulation to activate the erythroid-specific program of gene expression. The genes encoding transcriptional regulators GATA-1 and GATA- 2 represent downstream components of a serine/threonine kinase signal transduction pathway initiated by BMPs. However, little is known in any system regarding how a BMP signal leads to activation of the transcriptional program. Experiments are designed to exploit Xenopus embryology to understand how BMP signaling results in differentiation of embryonic red blood cells. Using explant assays, it will be determined whether BMP-4 activates differentiation directly. Recently identified components of the BMP pathway will be tested for function during erythropoiesis. DNA regulatory elements will be identified that mediate the BMP response. Finally, novel genes that are regulated in blood progenitors by BMP-4 will be identified. It is anticipated that these experiments will yield insight into growth factor mediated regulation of a blood stem cell population.
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