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Developmental Regulation of Hematopoiesis

Research 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. It is possible to evaluate pathways important for vertebrate hematopoiesis using zebrafish genetics or chemical biology.

Our laboratory focuses on the developmental biology of hematopoiesis. Hematopoietic stem cells are derived early in vertebrate embryogenesis. We use the zebrafish as an alternative model system to study embryonic hematopoiesis. The zebrafish embryos are transparent, and all organs can be visualized under a microscope, 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 marrow smears were examined in the mutant embryos or 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 by using whole-embryo in situ hybridization to determine the spatial localization of the hematopoietic program during zebrafish ontogeny. Our recent work has focused on the generation of adult blood stem cells in the developing embryo. We used in situ techniques to show that the beginning of the adult blood program correlates to the onset of circulation and involves the notch pathway and nitric oxide biology. 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 an increase in stem cell number. PGE2 also increases engraftment of human cells into immunodeficient mice. A clinical trial with 12 patients with leukemia who are receiving cord blood transplants suggests that PGE2 enhances engraftment of cord blood, and a phase II clinical trial is under way. Our work on PGE2 has established a basic mechanism of stem cell migration and self-renewal and could lead to a new therapy for patients with cancer or immunodeficiency.

Two zebrafish blood stem cells (green) as they crawl into their vascular hematopoietic niche.

The imaging in the zebrafish is exquisite. We have created a transgenic fish in which blood stem cells express fluorescent colors. Using these fish, we can witness the engraftment process of endogenous stem cells. By examining the interactions with endothelial cells and stromal cells, we can study the sequential steps of engraftment. Blood stem cells attach to the vessel wall in the hematopoietic site and then transmigrate to an extravascular niche. The endothelial cells in niche respond to the arriving stem cell, and "cuddle" the stem cell, and stromal cells physically attach to the stem cell. Using genetics and chemical biology, we are developing a basic understanding of how blood stem cells home to the marrow, engraft, and self-renew.

The Genetics of Transplantation
Our new work has studied the visualization of fluorescent marrow in adult zebrafish. We recently developed a transparent adult zebrafish, called casper, from 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. We have carried out competitive transplantation between red and green fluorescent blood stem cell populations. Chemical and genetic screens are being used to evaluate the biology of homing, engraftment, and self-renewal.

Zebrafish Cancer Work
As we were studying blood development, it became possible to study cancer in the zebrafish. We have created models of leukemia, lymphoma, muscle tumors, and melanoma. We developed novel transgenic approaches to overexpress genes to evaluate their roles as oncogenes. In a region amplified in 30 percent of human melanomas, we found an epigenetic regulator, SETDB1, that drives melanoma. We also found that an arthritis drug called leflunomide, which blocks neural crest development, is also toxic to human melanoma. This chemical is now in a clinical trial in combination with a BRAF and MEK inhibitor. We recently developed technology to study tumor suppressors in the zebrafish melanoma model. Our research has linked studies of development and cancer.

This work is supported in part by grants from the National Institutes of Health.

As of November 3, 2014

Scientist Profile

Boston Children's Hospital
Developmental Biology, Genetics