Disorders of the hematopoietic system are a common cause of morbidity and mortality in the United States. In vertebrates, definitive hematopoietic stem cells (HSCs) produce each of the mature blood cell lineages of an individual throughout its lifetime. Defects in HSC formation or differentiation can lead to devastating diseases in childhood or to leukemia. The first HSCs arise in the embryo from an area encompassing the dorsal aorta, termed the aorta-gonad mesonephros (AGM) region. Most genes involved in HSC formation are highly conserved across vertebrates, and continue to regulate HSC self-renewal and differentiation in the adult. The transcription factor Runx1, a frequent target of chromosomal mutation in human leukemia, is absolutely required for HSC specification in mammals;runx1 expression is conserved in zebrafish, appearing robustly by 36 hours post fertilization (hpf) in the AGM. Through a zebrafish chemical screen for modifiers of runx1 expression, we have found that compounds related to eicosanoid production and signaling modulate HSC number. The eicosanoid prostaglandin E2 (PGE2) can regulate embryonic HSC formation and adult homeostasis across vertebrate species. In contrast, the function of related eicosanoids, cannabinoids (CB), in HSC development and PGE2-mediated repair has not been addressed. Our long-term goal is to understand the molecular and cellular mechanisms by which various factors interact to affect hematopoiesis. Our objective here is to characterize the functional implications of eicosanoid cross-regulation on HSC formation, proliferation and migration during development and recovery after injury, in both zebrafish and mice. Our central hypothesis is that two related eicosanoid subclasses, PGE2 and CBs, act individually and in concert to exert specific effects on HSCs during embryogenesis and regeneration. This hypothesis has been derived from our own screening results and subsequent preliminary data as well as previously published studies. The rationale for our work is that a detailed understanding of the impact of CB ligands on PGE2-mediated HSC growth and proliferation will enable targeted therapeutic approaches for the treatment of bone marrow failure states, recovery from chemotherapy, or after HSC transplant. In Specfic Aim 1, we plan to investigate the effects of CB agonists/antagonists on HSC specification and proliferation during hematopoietic development in zebrafish. We will examine the conservation of this activity during and after marrow injury.
In Specific Aim 2, we plan to analyze the combined effects of CB agonists and PGE2 on HSCs. We postulate that PGE2 and CB-agonists together can increase HSC number in the AGM to a higher level than each of these compounds alone. We will then examine if the interaction between eicosanoids is relevant in directing enhanced marrow recovery after injury in zebrafish and murine transplantation models and in assays with human cord blood. These results are expected to reveal deeper insight into interactive roles of inflammatory mediators in marrow regeneration and have relevance for combinatorial targeting to direct clinical outcome in transfusion biology.
Hematopoietic stem cells form the foundation of our blood and immune system;the formation and function of these cells are carefully controlled in the body. The proposed research will help to identify mechanisms that regulate the birth and propagation of these stem cells. This work has great relevance for the development of novel agents to regulate leukemia, and for recovery from chemotherapy and bone marrow transplant.
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