Forward Genetic Screen And Preliminary Characterization Of Zebrafish Mutants With Defects In Myelopoiesis

This thesis comprises three parts:Ⅰ) Zebrafish as a powerful model for studying hematopoiesis;Ⅱ) Large-scale forward genetic screening for zebrafish mutants defective in myelopoiesis;Ⅲ) Preliminary characterization of zebrafish mutants with defects in Sudan black B.To recognize the many advantages of the zebrafish as a vertebrate genetic model for studying blood developmental processes The tropical fresh water fish, Danio rerio, provides a powerful model organism for studying hematopoiesis.1) due to its inherent advantages as a developmental model, including developing very rapidly and a single female can lay up to 200-300 eggs per week. Moreover, the small size of adult fish permits large numbers of animals to be maintained in relatively small facilities, a practical requirement for forward genetic screens; 2) The transparency of the zebrafish's chorion and the translucency of its embryos and early larval stages allow the easy visualization of hematopoiesis.3) Zebrafish hematopoiesis has been shown to be very similar to that of higher vertebrates, and homologues of a large number of genes involved in mammalian hematopoiesis have been identified in the zebrafish. Therefore, zebrafish-related research has great application value.4) It had been identified and recovered of many mutant lines exhibiting defects in all aspects of embryonic development by large-scale N-ethyl-N-nitro-sourea (ENU) mutagenesis screens. By studying the phenotypes of these mutants together with the identification of the mutated genes, molecular pathways are being defined that are essential to hematopoietic lineage commitment and differentiation.5) New insights into vertebrate hematopoiesis provided by new technology and genomic science highlight the power of the zebrafish model for identifying new and unexpected genes that regulate hematopoiesis.Hematopoiesis in the zebrafish Primitive hematopoietic cells in the zebrafish embryo are also derived from lateral mesoderm but, by contrast,arise in two regions:one in the posterior of the animal between the notochord and endoderm of the trunk,called the intermediatecell mass(ICM);and the second in a more anterior location under the head called the anterior lateral plate mesoderm(ALPM). The ICM is further divided into two regions, the anterior-trunk domain and the posterior blood islands, which form slightly later in the ventral region of the tail. Erythrocytes develop exclusively in the ICM. While the ALPM is the major site of origin for early macrophage precursors and other myeloid cell types. By 32hpf c-myb and runxl are expressed in the cells of the ventral wall of the dorsal aorta, which are likely to represent definitive hematopoietic stem cells, as they do in other vertebrates. By 4 to 5 days post fertilization(dpf), hematopoiesis occurs in the kidney marrow, which remains as the major site of this process throughout adulthood.To study zebrafish myelopoiesis In the zebrafish, there are two myeloid lineages:granulocyte and monocyte/macrophages. Granulocyte has two lineages, one resembling mammalian neutrophils and the other possessing features of both mammalian eosinophils and basophils. In recent years, it has been identified molecular markers with the myeloid cells development and its progenitors, such as genes expressed in hematopoietic stem cells (scl,lmo2) and early progenitors (pu.1,c/ebp1) and neutrophil (mpo) and monocyte/macrophages(L-plastin,fms). Monocyte/macrophages have also been identified in zebrafish embryos. These cells originate from the APM and migrate over the yolk sac as early as 16hpf. After blood circulation beginning, monocyte/macrophages was evident over the anterior yolk, in the posterior ICM, and in the posterior blood island they were distributed throughout the embryo. In the early embryonic development, zebrafish macrophage has phagocytosis cell activity and involvement in acute inflammation with neutrophils together.ENU mutagenesis and large-scale forward genetic screening One of the most widely used substances to induce mutations is the chemical N-ethyl-N-nitrosourea(ENU). ENU is a potent mutagen, which has been used to induce mutations in various organisms. ENU mutagenesis has high efficiency, and the mutant gene can be identified by positioning cloning. Male zebrafish were mutagenized with N-ethyl N-nitrosourea to induce mutations in spermatogonial cells to generate founders, and founders were outcrossed with AB to raise Fl fish. F1 fish from different founder were mated to generate F2 families. F3 embryos from F2 sibling crosses were screened by Sudan black B staining and neutral red staining.350 F2 families from F1 sibling crosses were screened. A total of 1424 F2 crosses were analyzed.We identified 6 mutations resulted in abnormal Sudan black B staining and neutral red staining. They indicated involvement of the neutrophils deficiency or macrophage abnormalities.Preliminary characterization of zebrafish mutants with defects in myelopoiesis The whole mount in situ hybridization (WISH) technique allows the sites of expression of particular genes to be detected. It has several important applications:It allows gene expression profiles in cells and tissues to be determined during the time course of embryo differentiation. Collections of tissue-and cell-specific markers at distinct developmental stages, WISH can be used for phenotypic analysis after chemical mutagenesis. We carried out whole mount mRNA in situ hybridization assay for fms, mpo, Lysozyme C, ragl, globinβel which are specific for myeloipoisis and identified 350 mutant with deficts in Sudan black B staining. Our results suggested that markers such as fms, mpo, Lysozyme C, ragl, globinβel expressed normaly. Further analysis showed granules of granulocytes were defected, which led to no signals when the mutants staining with Sudan black B.