| BackgroundHematopoiesis by definition is a complicated and dynamic process that produces all types of blood cells throughout the lifetime of the animal and is involved with a number of hematopoietic anatomical locations. Despite their functional diversities, all these different types of hematopoietic cells arise from a common ancestor known as hematopoietic stem cells (HSCs). During the process of HSCs maturing to blood cells, it experiences a phase containing hematopoietic progenitor cells and hematopoietic precursor cells, and finally differentiates to all the mature blood cells. Therefore, hematopoiesis embraces two aspects:the development of the first HSC from non-hematopoietic tissues in embryonic and fetal life, as well as its later specialization, proliferation and differentiation. Despite extensive studies in the past, the genetic programs governing the specification, migration, and survival of HSCs in these hematopoietic compartments and the mechanisms of hematopoietic development remain poorly understood.Although many critical concepts and valuable insights have been derived from the study of nematodes, fruit flies in the past, the usage of these conventional systems in dissecting hematopoietic program is limited by their own inherited attributes. Although the development of hematopoiesis has been best characterized in the mouse model, the slow reproduction and relatively large size of mouse, and its embryonic development in uterus are not suitable for the early hematopoietic phenotype observation, especially for the forward genetic analysis. To complement these traditional models, zebrafish, a fresh water tropical fish that is noted to integrate the features suitable for both ENU mutagenesis and large-scale forward genetic screening, has recently been emerged as an excellent vertebrate system to study developmental mechanisms of hematopoietic system. A large number of zebrafish could be maintained in a relatively small space and hundreds of progenies could be collected at weekly intervals. The rapid growth and external development of zebrafish embryos also make it well suitable for embryological observation and manipulation. As hematopoietic program is highly conserved between fish and mammals, the study on zebrafish hematopoiesis would be also contributed to our understanding of this process in higher organisms.Similarly to mammals, zebrafish hematopoiesis also consists of primitive and definitive programs, and generates differentiated cells analogous to most of the mature blood lineages found in mammals. Zebrafish primitive erythropoiesis originates from the posterior lateral mesoderm (PLM) as a pair of bilateral stripes at 5-somite stage. These stripes subsequently extend anteriorly and posteriorly, and converge in the midline at 20-somite stage to form the main structure of the intermediate cell mass (ICM) where the erythroid progenitors further proliferate and differentiate, enter the blood circulation, and finally mature at around 5-7 days post fertilization (dpf). Zebrafish primitive myelopoiesis arises from the anterior lateral mesoderm (ALM) at 10-somite stage and produces mainly macrophages and neutrophils. HSCs in zebrafish are believed to initiate at 26—30 hours postfertilization (hpf) from the ventral wall of DA (VDA), an equivalence of the mouse AGM. By 2 days postfertilization (dpf), these HSCs in the ventral wall of DA migrate to the posterior blood island (PBI) (also referred to as caudal hematopietic tissue) located between caudal artery and caudal vein, and finally home to kidney, the adult hematopoietic organ in zebrafish, by 5dpf. Thus function analogies of AGM, FL, and BM in zebrafish are very likely to be represented by the ventral wall of DA, PBI, and kidney respectively.Zebrafish vascular system originates from the ventral mesoderm (VM) as bi-potential cells-hemangioblast at 6hpf stage in gastrulation. Then by 12hpf these cells migrate to lateral plate mesoderm (LPM) where they differentiate into angioblast. By 16hpf, these angioblasts in the LPM converge in the midline of vascular cord located between dorsal ectoderm and notochord. Some complex processes lead to the formation of a functional vascular network, such as sprouting, branching, artery and vein differentiation, lumen formation. The latter remodelling process requires the recruitment of supporting cells, such as smooth muscle cells and pericytes, and the formation of cell-cell and cell-extracellular matrix junctions.The chemical mutagen N-ethyl-N-nitrosourea (ENU) is used to treat healthy wild type male fish (AB stain, FO). Subsequently the surviving ENU-treated male fish is mated with wild type female fish to generate F1, and further F2. Once the F2 fish mature, they are ready to use for forward genetic screen. In this study, we carried out the forward genetic screen to search forβel-deficient zebrafish mutants. The adult F2 fish will intercross within each F2 family and the resulting F3 embryos from each crossing will be collected at 5dpf and subjected to whole mount in situ hybridization (WISH) with theβel probe. We have got fourβel-deficient fishes which we called 336-2,350-1,373-6 and 483-2 mutants respectively were given for further detailed characterizations of hematopoiesis process. The analysis of the function ofβel gene in hematopoiesis will help us to understand the molecular regulation of hematopoiesis. On the other hand, from the clinical point of view, a basic understanding of molecular regulation mechanisms of hematopoiesis is essential for finding etiopathogenisis, clinical diagnosis and developing new therapeutic approaches for treatment of hematopoietic diseases.There are two chapters in this study. Chapter one, ENU mutagenesis and generate F1, F2 family. Chapter two, forward genetic screen forβel -deficient mutant and these putative mutants were characterized with different hematopoiesis markers.Chapter one ENU mutagenesis and generate F1, F2 familyObjective:To use chemical mutagen ENU to treat AB stain male fish and generate F1, F2 familyMethod:The mutagenesis process was carried out with chemical mutagen ENU according to Dr. Zilong Wen, HKUST. Subsequently, surviving ENU-treated male fish was used to generate F1, F2 family. The genetic screen was carried out between F2 families to search forβel-deficient zebrafish mutant by WISH.Results:16 surviving ENU-treated male fishes were used to generate 2000 F1 family. Finally we selected fourβel-deficient zebrafish by WISH. And we have already found 4 putative mutantsChapter two, forward genetic screen forβel-deficient mutant and these putative mutants were characterized with different hematopoiesis markers.Objective:To search forβel-deficient mutants and isolate these mutants for further studyMethod:From theβel-deficient mutants we isolated, I gave these mutants for further study. The WISH,O-dianisidine staining of hemoglobin, Neutral Red staining for macrophage and Sudan Black B for granulocyte were performed to analyze the putative mutants.Results:There are no morphological changes between wild type embryos and putative mutants except for the abolished expression ofβel gene. And then four different hematopoiesis markers and three stainings were performed on the fourβel-deficient mutants.The results show that these mutants had not affected in primitive hematopoietic stage, hematopoietic stem cell and definitive myelopoiesis. However, there are no expression of ragl which is specific marker of T lymphocyte in thymus at 5dpf in 336-2 and 350-1 mutants, and 373-6 and 483-2 mutants have similar ragl signal with wild type.Conclusion:1 The chemical mutagen ENU based on mutagenesis is an excellent method for screening hematopoiesis-deficent mutant and erythroid lineage markerβel globin serve as a reliable marker2 We have isolated fourβel-deficient mutants by WISH. The two of four mutants, 336-2 and 350-1, show erythroid defects during definitive hematopoiesis, whereas the others,373-6 and 483-2, present defective both in erythriod lineage and lymphocytes development...
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