| BackgroundZebrafish has emerged as a model organism since 1970s. The zebrafish model shows many unique biological advantages such as its small size, large number of progeny, short life cycle, external fertilization and development, transparent embryos, as well as sharing almost 87% homology with human genome, thus become increasingly popular in the recently years. Various study models of zebrafish has been gradually established, such as drug screening and safety assessment model, immune system model, microorganism infection model, cardiovascular development and disease model, leukemia model and tissue regeneration model. These models greatly improve our understanding of the mechanisms of related disease.The definition of hematopoiesis is the formation of blood cellular components, which occurs during embryonic development and throughout the whole lifetime to produce and replenish the blood system. Hematopoietic cells consist of erythrocytes, monocytes, neutrophils, basophils, eosinophils, mast cells, magakaryocytes, nature killer (NK) cells, T and B lymphocytes. All kinds of blood cells play important roles to maintain the normal physiological function. For example, erythrocytes deliver oxygen to the body tissues via the blood flow through the circulatory system; leukocytes are involved in defending the body against both infectious disease and foreign materials; lymphocytes are the major cells mediated the immune response. Many malignant diseases, such as anemia, leukemia and malignant lymphoma, are closely related to the defect of blood cells formation. Thus, studying hematopoiesis can help us to further understand the processes behind these blood disorders and cancers.Hematopoiesis development in vertebrates involves two waves:the primitive wave and the definitive wave. The primary purpose of the primitive wave is to produce erythrocytes that can provide tissue oxygen as the embryo undergoes rapid growth. Definitive wave occurs later in development, notably at different time points in different species. Definitive hematopoiesis produces hematopoietic stem cells (HSCs), which are multipotent and can differentiate into all blood lineages of the adult organism. In vertebrates, definitive HSCs originate from the aorta-gonad-mesonephros (AGM) region. Later they migrate to the fetal liver and then to the bone marrow.The process of hematopoiesis development in zebrafish is similar to mammals, involving the primitive wave and the definitive wave.Primitive wave of hematopoiesis begins in zebrafish embryos at 12 h post-fertilization (hpf) in the lateral plate mesoderm, anatomically separating specification of myeloid and erythroid progenitors in the anterior and posterior mesoderm, respectively. From approximately 30 hpf, HSCs differentiate from the ventral wall of the dorsal aorta (VDA) region. These HSCs migrate to the caudal hematopoietic tissue (CHT) in the posterior region of the tail. By 3 days post-fertilization (dpf), lymphopoiesis occurs in the thymus and one day later the HSCs migrate to the kidney marrow, which is the location for HSCs in adult zebrafish.Hematopoiesis development is a complicated and dynamic process that tightly regulated by multiple transcription factors. Primitive hematopoiesis is mainly regulated by two transcription factors, Gatal and Pu.l, which exhibit a cross-inhibitory relationship to determine further specification towards erythroid or myeloid fates, respectively. Runx1 and Cmyb, which represent the marker genes of HSCs, are the main transcription factors during the process of definitive hematopoiesis. Besides, there are various genes which mark the different blood lineages, including αe1 and βe1 which mark the definitive erythroid lineage, lcp1 which mark the definitive myeloid lineage, mpx and lyz which mark neutrophils, mfap4 and mpegl which mark macrophages, ikaros and ragl which mark the lymphoid lineage.Myeloperoxidase (MPO) was first reported in 1940s. After the initial description, it took almost 30 years before the functional role of MPO was elucidated. MPO belongs to the mammalian heme peroxidase enzyme family, and is a key component of the oxygen-dependent microbicidal activity of neutrophils. MPO is mainly stored in the primary granules of neutrophils, and is released during inflammatory response. For a long time, MPO has been considered to be a bactericidal enzyme whose main function is to generate reactive oxygen species (ROS) that contribute to the killing of invading pathogens. Indeed, as the result of numerous studies, the antimicrobial action of MPO has been attributed to its unique capacity to produce hypochlorous acid and other toxic agents that create an environment within the phagolysosome of neutrophils that inhibits or kills ingested microbes. However, recent evidence has extended this view by demonstrating that MPO is also involved in cellular homeostasis, and also is an important factor in the initiation and progression of various inflammatory diseases.MPO has been considered to be closely related with various human diseases. A number of studies of MPO-deficient individuals have suggested that hereditary MPO deficiency is associated with increased susceptibility to infection. Many clinical studies have implicated MPO in a broad range of aspects of cardiovascular disease (CVD) in human patients, such as atherosclerosis. Besides, excessive generation of oxidants by MPO has been linked to tissue damage in many diseases which characterized by acute or chronic inflammation.In zebrafish, myeloperoxidase (mpx) gene is usually utilized as a specific marker of neutrophils. Howerver, the biological function of Mpx in zebrafish remains poorly understood.In this study, the chemical mutagen N-ethyl-N-nitrosourea (ENU) is used to treat healthy wild type adult male zebrafish (AB stain, Fo). The surviving ENU-treated male fish are mated with wild type female fish to generate F1, and then F2. The F3 embryos generated from F2 inter-cross are used for the forward genetic screen. We performed a large-scale screening, in order to isolate novel zebrafish mutants with hematopoietic defects. Subsequently, we carried out positional cloning to locate the mutant gene of one of the mutants we obtained. Then, we further characterized this mutant, smu681, with multiple functional assay and gene expression detection. Finally, we performed Candida albicans infection to analyze the microbicidal activity of zebrafish myeloperoxidase.This study is divided into three parts:part one, large-scale screening of zebrafish mutants with hematopoietic defect; part two, positional cloning and characterization of smu681; part three, Candida albicans infection of smu681.Part one:Large-Scale Screening of Zebrafish Mutants with Hematopoietic DefectObjective:We used chemical mutagen ENU to treat wild type adult male zebrafish, in order to isolate novel zebrafish mutants with hematopoietic defects by large-scale screening.Method:The ENU mutagenic treatment was carried out according to the ordinary procedure. Surviving ENU-treated male fish were used to generate F1, and then F2 family. The phenotypes of F3 embryos, which were obtained by random crossing between siblings from F2 families, were analyzed by three different approaches. Neutral red staining, sudan black B staining and whole-mount in situ hybridization were utilized to identify the defect of macrophage development, granulopoiesis and definitive hematopoiesis, respectively. Results:1) Forty wild-type adult male zebrafish were treated with ENU four times at weekly intervals. Only 20 males survived after all four sessions of treatment. The surviving males were crossed with wild-type females. About 2000 F1 progeny and 1564 F2 families were raised.2) A total of 52 candidate mutants were identified by scoring from 1383 F2 families, and 25 individual mutants were obtained after the complementation assay. These mutants were then classified into four types based on specific phenotypic traits: type I, defective macrophage development; type II, defective granulocyte development; type III, defective embryonic myelopoiesis; type IV, defective definitve hematopoiesis.Part two:Positional Cloning and Characterization of smu681Objective:Positional cloning was performed in order to locate the mutant gene of smu681, and multiple functional assay and gene expression detection were employed to further characterize smu681. Rescue experiment was carried out to confirm the correspondence between the phenotype and mutant gene of smu681.Method:Mapping families were generated by mating mutants with WIK strains. After genomic DNA was prepared from the embryos, initial mapping and fine mapping were performed as described in the Zon Lab guide to positional cloning in zebrafish. Genotyping method was designed accroding to the sequence signature of the mutant gene, in order to distinguish the different genotype of smu681.Three independent assay, including sudan black B staining, diaminobenzidine staining and tyramine signal amplification staining, were performed to detect endogenous peroxidase activity of smu681. Whole-mount in situ hybridization and quantitative fluorescence polymerase chain reaction (QF-PCR) were utilized to detect gene expression of smu681. Rescue experiment was carried out by using either mRNA or stable transgenic line. Results:1) Initial mapping located the mutant gene on the chromosome 10 of zebrafish, and fine mapping revealed that the mutational site was located in mpx gene. Sequencing chromatograms showed a three nucleotides deletion in the exon 13 of mpx gene, introducing a premature stop codon.2) Three independent assay of peroxidase activity revealed complete Mpx-deficiency in the neutrophils of smu681.3) The mRNA level of mpx in smu681 embryos is much less than which in wild-type embryos at 24 hours postfertilization (hpf),36 hpf,48 hpf and 72 hpf. Normal expression of green fluorescence in the transgenic line Tg(mpx:GFP) indicated that the transcriptional activity of mpx was intact in smu681.4) Normal expression of multiple hematopoietic genes, such as pu.l, gatal, c-myb, lyz, mfap4, βe1 and ragl, showed other hematopoietic development in smu681 was unaffected. Particularly, normal expression of the neutrophil specific gene, npsn and srgn, confirmed the granulopoiesis was intact in smu681.5) Direct injection with mpx mRNA didn’t seem work in rescue experiment of smu681. On the contrary, stable transgenic line in which mpx expression was driven by coroninla promoter could partly restore the phenotype of smu681.Part three:Candida albicans Infection of smu681 Objective:We carried out Candida albicans infection to assay the microbicidal activity of zebrafish myeloperoxidase. Method:Zebrafish embryos were microinjected with Candida albicans in the hindbrain at 2 dpf. Three levels of fungus dosage were applied to evaluate the effect of infection by monitoring the growth condition of Candida albicans inside the embryos using confocal microscope. The survival analysis was employed to compare the difference between wild-type and smu681.Infected embryos were homogenized with sterile syringe needle, and the homogenate was plated in triplicate onto YPD agar. Plates were incubated for 24 h at 37℃. Viable colonies were counted, and fungal burdens were calculated and graphed as CFU per fish.The migration and accumulation of neutrophils in the infected embryos were monitored and calculated by utilizing the transgenic line Tg(lyz:DsRED2). The expression of inflammatory factors, IL-1β, myd88 and IL-8, were examined by QF-PCR.Results:1) High doses injection of Candida albicans leaded to all embryos death within 30 hours post-injection. Moderate doses injection could also lead to serious infection and death of most embryos. While low doses injection would not cause any infection, and all embryos could survive more than 4 days after injection. The survival rate of smu681 is comparable with which of wild type after infection.2) The fungi burdens of smu681 is heavier than which of wild type at 24hpi and 48hpi. Howerver, there is no significant difference between the two genotypes at 72hpi.3) There are more neutrophils accumulate in the hindbrain of infected smu681 embryos, indicating more severe inflammation inside smu681.4) The increased expression of IL-1β in the infected smu681 embryos lead to up-regulation of its downstream factor IL-8. Howerver, the affection on myd88 is relatively unconspicuous. This result shows that Mpx deficiency could lead to overexpression of both IL-1β and IL-8 after infection.Conclusions:1) We obtained 25 zebrafish mutants with hematopoietic defect by large-scale screening based on ENU mutagenesis. These mutants were then classified into four types based on specific phenotypic traits. Further work is ongoing to categorize these mutants in the hope of improving our understanding of the mechanisms of hematopoiesis, and probably providing opportunities to establish models of hematopoietic diseases.2) Positional cloning indicated that the mutated gene of smu681 was mpx. Histochemistry assay revealed that Mpx activity was totally lost in smu681. Howerver, hematopoiesis of smu681 was intact, and the embryos could develop normally, suggesting that smu681 could serve as an excellent model for studying the function of myeloperoxidase.3) The infection experiment of Candida albicans revealed equally survival rate of infected embryos of both genotype. However, the cell number of C. albicans in smu681 embryos was significantly more than that in wild type embryos according to the enumeration of fungal burdens. We also found more neutrophils accumulating in smu681 embryos after infection, as well as higher expression of inflammatory factors, implying that increased inflammatory response would occur during infection in the condition of losing myeloperoxidase activity. |
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