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 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.
The Genetics of Transplantation
A new project was started to understand how stem cell populations home to the correct organs, engraft, and ultimately self-renew. The visualization of transplants of fluorescent-based marrow into zebrafish has been hampered by the pigmentation cells on the skin. We recently developed a transparent adult zebrafish, called casper, using two pigment mutants. Transplantation of tissue into casper allows the complete visualization of the engraftment of all organs. By transplanting fluorescent cells into the casper fish, it is possible to image the fish and study single cells. Competitive transplantation between red and green fluorescent blood stem cell populations has been done. Chemical and genetic screens are being used to evaluate the biology of homing, engraftment, and self-renewal.
