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Developmental Regulation of Hematopoiesis
Summary: Leonard Zon's laboratory uses the zebrafish as a model system for understanding vertebrate blood development. Zebrafish blood formation is similar to that of humans, and several mutants have disorders resembling human disease. Thus, it is possible to evaluate in the zebrafish genetic pathways important for vertebrate hematopoiesis.
Our laboratory focuses on the developmental biology of hematopoiesis. Hematopoietic stem cells are derived early in vertebrate embryogenesis, during gastrula stages. The mammalian embryo is difficult to evaluate at this particular time due to its development in utero. We have used the zebrafish, which is externally fertilized, as an alternative model system to study embryonic hematopoiesis. The zebrafish embryo is completely clear, and all organs can be visualized, including circulating blood. In addition, a large number of animals can be kept in a relatively small space, and each mother lays 200–300 eggs weekly. These qualities enable a forward genetic approach to vertebrate blood formation. Our studies demonstrate that the zebrafish can provide information about the hematopoietic program that is relevant to vertebrate biology and to clinical disorders of blood formation.
Mutant Zebrafish
We have collected more than 40 complementation groups of mutants with hematopoietic problems, and we have developed several techniques to study the phenotype of the mutants. First, peripheral blood and hematopoietic kidney smears were examined in the mutants or in the adults, providing a potential mechanism of the anemia. We developed technology to do hematopoietic progenitor assays and stem cell transplantation. This analysis demonstrates that there are mutants that lack blood and blood vessels, mutants that have no hematopoietic stem cells, and mutants with proliferation difficulties and differentiation problems. A number of mutants have defects in hemoglobin production, and eight new mutants have no T cells.
Stem Cell Biology
The zebrafish embryo can live without blood for up to 10 days, even though blood initially forms within 24 hours after fertilization. We have studied gene expression using whole-embryo in situ hybridization to determine the spatial localization of the hematopoietic program during zebrafish ontogeny. Using cDNAs encoding the GATA-binding proteins as probes, we have defined the region of the intermediate cell mass as the equivalent of the yolk sac hematopoiesis. We are currently studying the next site of blood stem cell development in the aorta. The Notch pathway is necessary and sufficient to generate the definitive blood stem cells. We are doing a genetic screen and a chemical screen to find pathways affecting adult stem cell homeostasis.
We recently isolated the mutant gene kugelig. This zebrafish mutant has a decreased number of stem cells. We isolated the gene cdx4, which activates homeobox genes and establishes the mesoderm's competency to make blood. Overexpression of cdx4 leads to ectopic blood formation in the middle of the embryo. We also demonstrated that cdx4 can increase multipotential hematopoietic progenitors in mouse embryonic stem cells. The cdx4-hox pathway thus regulates hematopoietic stem cell production by the embryo.
Recently, we undertook a chemical genetic screen in zebrafish and found that prostaglandins stimulate blood stem cell production. In particular, prostaglandin E2 can increase blood stem cells during embryogenesis and in adulthood in zebrafish and mice. A treatment of prostaglandin to mouse bone marrow leads to a threefold increase in stem cell number. We are currently investigating clinical trials to treat cord blood samples with prostaglandin E2, in an effort to increase the number of stem cells in that sample.
Relevance of Zebrafish: The Hypochromic Mutants
There are five complementation groups of mutants with a hypochromic microcytic anemia. This is characteristic of human patients with thalassemia or a defect in hemoglobin production. We initiated a positional cloning approach to isolate the sauternes gene and demonstrated that it is a defect in ALAS-2. The ALAS-2 gene is responsible for heme biosynthesis, and defects in this gene in humans lead to a congenital sideroblastic anemia. The fish disease and the human disease are similar, and sauternes is the first animal model of this human disease.
In the zebrafish mutant weissherbst, maternal stores of iron in the yolk are not exported into the embryonic circulation. The positional cloning of this gene proved that it encodes a novel iron transporter, ferroportin. In humans, ferroportin is expressed highly in the placenta. It is likely that iron is transported into the circulation from the mother to the fetus through ferroportin. In the human duodenum, iron is transported from the enterocyte into the circulation, again through ferroportin. Ferroportin is thus a critical regulator of iron biology. Recently, ferroportin mutations have been detected in patients with hemochromatosis type IV. By isolating the shiraz gene, we found a new pathway involving hemoglobin production that requires iron-sulfur clusters. This conservation of genetics and biochemistry illustrates how valuable the zebrafish is as a tool for understanding human biology and disease.
Last updated: September 27, 2007
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