Hematopoietic stem cells (HSC) are derived during embryogenesis in distinct developmental waves. The first wave, called primitive, occurs on the yolk sac and is mostly confined to erythropoiesis. Subsequent waves, called definitive, include the aorta-gonads-mesonephros (AGM) region, fetal liver and bone marrow hematopoiesis. We undertook a chemical genetic screen for inducers of hematopoietic stem cells in the AGM region of the zebrafish. After screening about 2500 chemicals, several that increase prostaglandin E2 were found to increase HSCs, and compounds that reduce prostaglandin E2 expression decreased stem cell number. We have demonstrated that prostaglandin E2 improves marrow recovery in sublethally irradiated adult zebrafish, increases CFU-S activity from purified mouse HSCs, and causes a 4 fold increase in the number of long-term repopulation units from mouse marrow (based on limit dilution competitive transplantation experiments). Using morpholinos and specific inhibitors of the PG receptors, we will identify the receptor(s) responsible for the PGE2 effect. The effect of PGE2 on mouse HSCs in the AGM will be examined. We also demonstrated that induction of wnt8 leads to enhanced stem cells in the zebrafish AGM. A genetic interaction for HSC formation has been found between the wnt pathway and prostaglandins. This interaction will be further characterized using mutants, morpholinos, and chemicals that manipulate specific pathway components. The chemical genetic screen also demonstrated that vasodilators that increase PGE2 levels led to enhanced stem cell production and vasoconstrictors that reduced PGE2 decreased stem cell number. This grant will test the hypothesis that alterations in circulation by prostaglandin trigger the onset of definitive hematopoiesis during embryogenesis. In an effort to better understand the HSC development in the AGM, we have undertaken a large-scale genetic screen for mutants affecting the process. To date, 10 individual mutants have been isolated using insertional mutagenesis, and an END mutagenesis will be undertaken to find up to 40-50 mutants that affect AGM stem cell production. The mutant genes will be examined for genetic interactions with other stimulators of HSC formation such as notch, wnt, and prostaglandins. These studies should provide critical insight into stem cell homeostasis and may lead to potential new therapies of anemia and approaches in bone marrow transplantation. ? ? ?
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