Zon Laboratory

Dr. Leonard Zon's laboratory focuses on the developmental biology of hematopoiesis and cancer. Over the past five years, we have collected over 30 mutants affecting the hematopoietic system. Some of the mutants represent excellent animal models of human disease. For instance, the isolation of the ferroportin iron transporter was based on a mutant zebrafish and subsequently was shown to be mutated in patients with iron overload disorders. The mutants also represent interesting key regulatory steps in the development of stem cells. Recently, a mutant was found that lacked blood stem cells and the mutated gene proved to be a caudal related homeoprotein called, CDX4. A Cdx-hox pathway was found to participate in early hematopoietic stem cell development and overexpression of CDX4 leads to ectopic blood development, within the zebrafish embryo and in mouse embryonic stem cells. We recently have developed hematopoietic cell transplantation for the zebrafish using blood cells labeled with green fluorescent protein and DSred. We were able to image the hematopoietic cells as they migrate to the marrow and to the thymus.

The laboratory has also developed zebrafish models of cancer. A screen for cell cycle mutants found 19 mutants. Some of these mutants get cancer at a very high rate as heterozygotes based on a carcinogenesis assay. The mutant genes appear to be new cancer genes and we have used small molecules in a chemical suppressor gene to find chemicals that bypass the mutant cell cycle problem. We also have generated a melanoma model in the zebrafish system using transgenics. Transgenic fish get nevi, and in a combination with a p53 mutant fish develop melanomas.



1.  Vertebrate Hematopoiesis and Zebrafish Development

The hematopoietic system is an excellent model for understanding tissue stem cells, their biology and cell differentiation regulation, and involvement in aging, disease, and oncogenesis. Hematopoiesis refers to the generation of hematopoietic stem cells and generation of specific blood cell lineages with distinct functions that are required to support life of a vertebrate.

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Hematopoietic stem cells are formed, migrated, and maintain in their niches during development and possess ability to self-renew and differentiate into all blood cell types. The process involves specific regulation of gene transcriptions and epigenetic modifications of genomes and includes controlling hematopoietic stem cell homeostasis, balancing of diverse differentiated cell populations, making different cell fate decisions, and maintaining differentiated cell states. Our better understandings of the regulation of hematopoietic stem cell biology and lineage differentiation will improve our diagnosis and treatment of human hematopoietic disorders and bone marrow transplantation therapies.

Hematopoiesis studies using the zebrafish genetic model system

Within the past two decades, the zebrafish (Danio rerio) has become an excellent model to study the development of hematopoietic stem cells (HSCs). All vertebrates including zebrafish have primitive and definitive waves of hematopoiesis, but self-renewing pluripotent HSCs are only produced by the definitive wave. The primitive wave occurs in two intraembryonic locations called the intermediate cell mass (ICM) and the anterior lateral mesoderm (ALM). Primitive erythropoiesis is in the ICM, whereas myelopoiesis initiates in the ALM. After circulation starts at 24 h post-fertilization, hematopoiesis shifts to the posterior blood island (PBI) for a brief period. The definitive wave starts in the aorta-gonadmesonephros (AGM). There are three different HSC migration and colonization events that begin 2 days post-fertilization: AGM progenitor cells migrate to (1) the caudal hematopoietic tissue (CHT), which is an intermediate site of blood development; (2) the thymus, which is a site of lymphocyte maturation; and (3) the developing kidney marrow, which is the larval and adult location for production of all hematopoietic cell types, and is comparable to the bone marrow of mammals. Many of the transcription factors and signaling pathways that regulate the formation of HSCs in a zebrafish are conserved with mammals. Large-scale forward and reverse genetic screens have identified zebrafish blood and HSC mutants that represent models for known human diseases. Along with the technological advancements in the field of zebrafish research, future HSC studies in zebrafish will help us illuminate the genetic network controlling the development and function of stem cells in all vertebrates.

Chemical Genetic Screens

Chemical genetic screening is a discovery approach in which chemicals are assayed for their effects on a defined biological system. The zebrafish, Danio rerio, is a well-characterized and genetically tractable vertebrate model organism that produces large numbers of rapidly developing embryos that develop externally. These characteristics allow for flexible, rapid, and scalable chemical screen design using the zebrafish by simply incubation of the organism in an aqueous environment that contains different compounds. Because screens are performed in the context of an intact, developing organism, this approach allows for a more comprehensive analysis of the range of a chemical’s effects than that provided by, for example, a cell culture-based or in vitro biochemical assay. Many available transgenic zebrafish lines that mark specific cell lineages, cell types, or cell states can be used to ask for cell specific effects upon exposure to different small compounds. Wildtype and genetic mutants can also be used to see if expression levels or patterns of expression of specific marker gene(s) are influenced by chemical exposure. Zebrafish has become an excellent system for in vivo chemical genetic screens that will score for small chemical compounds with therapeutic effects

2.  Tumorgenesis

Cancer is one of the most common diseases that affect human life. Zebrafish, a vertebrate genetic and developmental model system, posses the advantages of invertebrate organisms and those of mammalian models, such as large clutch sizes and relative transparency. Histology of normal tissue and cancers in zebrafish is highly similar to that of mouse and human samples. These properties facilitate identification of molecular genetic pathways involved in organ development and homeostasis. Zebrafish have been widely used to study cancer susceptibility and carcinogenesis and make contributions to human cancer biology and discovery of novel cancer targets and treatments.

Using the zebrafish, the lab was able to verify tumorgenesis ability of genes located in the cancer associated genomic regions from GWAS studies and genes whose expression regulations were associated with human cancers.  The system also allows the lab to analyze potential effects of small chemical compounds on cancer incidence, cancer proliferation, and tumor metastasis. This important vertebrate system will accelerate cancer biology research and improve our diagnosis and treatment of human cancer.