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Articles by Felix Ellett in JoVE
JoVE Immunology and Infection
Заражение эмбрионов данио рерио с внутриклеточные бактериальные патогены
1Department of Molecular Cell Biology, Institute of Biology, Leiden University, 2Department of Medical Microbiology and Infection Control, VU University Medical Center, 3Australian Regenerative Medicine Institute, Monash University
Прозрачный эмбрионов данио оказались полезными хостов модель для визуализации и функционально взаимодействия между врожденными исследование иммунных клеток и внутриклеточных бактериальных патогенов, таких как
Other articles by Felix Ellett on PubMed
The Role of the ETS Factor Erg in Zebrafish Vasculogenesis
Erg, a member of the ETS family of transcription factors, has been implicated by previous studies in endothelial and haematopoietic development. Deregulation of the human ERG locus is associated with acute myeloid leukaemia, prostate cancer and Ewing's sarcoma. To better understand the role of Erg during early development, we utilised the zebrafish as a model amenable to descriptive and functional studies in vivo. Zebrafish have a single erg gene that is expressed in mesoderm and its vascular derivatives during angioblast migration, vasculogenesis and early angiogenesis. Mutant and morphant expression analyses placed erg in a genetic pathway downstream of cloche, tal1/scl and etsrp during early angioblast migration. Furthermore, a combination of gain-of-function and loss-of-function studies suggested a redundant yet specific role for erg in both angioblast specification/proliferation and early angiogenesis, and a synergistic interaction with the critical ETS factor etsrp.
Zebrafish As a Model for Vertebrate Hematopoiesis
The zebrafish (Danio rerio) is a model organism making useful contributions in many areas of biological research. Zebrafish have proven particularly suitable for studying early development. The transparency and ex vivo development of zebrafish embryos means that early embryology can be easily visualized, especially using transgenic strains expressing fluorophores marking tissues of interest. High fecundity and tolerance of dense mutagenesis have made it a practical model for forward genetic screening and creation of mutagenized libraries from which stable mutant alleles can be recovered. Transient genetic manipulation by microinjection of mRNA (overexpression) or antisense morpholino oligonucleotides (knockdown) provide convenient methods for functionally assessing genetic regulatory pathways without the need for extended breeding strategies. A standout example of the utility of this model has been its application to modeling of the earliest stages of hematopoiesis. Zebrafish developmental hematopoiesis shows close correspondence to the development of the mammalian hematopoietic system and is regulated by conserved molecular pathways. This review highlights key recent studies that have used this model to provide insights into vertebrate hematopoietic development and innate immunity.
Mpeg1 Promoter Transgenes Direct Macrophage-lineage Expression in Zebrafish
Macrophages and neutrophils play important roles during the innate immune response, phagocytosing invading microbes and delivering antimicrobial compounds to the site of injury. Functional analyses of the cellular innate immune response in zebrafish infection/inflammation models have been aided by transgenic lines with fluorophore-marked neutrophils. However, it has not been possible to study macrophage behaviors and neutrophil/macrophage interactions in vivo directly because there has been no macrophage-only reporter line. To remove this roadblock, a macrophage-specific marker was identified (mpeg1) and its promoter used in mpeg1-driven transgenes. mpeg1-driven transgenes are expressed in macrophage-lineage cells that do not express neutrophil-marking transgenes. Using these lines, the different dynamic behaviors of neutrophils and macrophages after wounding were compared side-by-side in compound transgenics. Macrophage/neutrophil interactions, such as phagocytosis of senescent neutrophils, were readily observed in real time. These zebrafish transgenes provide a new resource that will contribute to the fields of inflammation, infection, and leukocyte biology.
In Vivo Visualization and Attenuation of Oxidized Lipid Accumulation in Hypercholesterolemic Zebrafish
Oxidative modification of LDL is an early pathological event in the development of atherosclerosis. Oxidation events such as malondialdehyde (MDA) formation may produce specific, immunogenic epitopes. Indeed, antibodies to MDA-derived epitopes are widely used in atherosclerosis research and have been demonstrated to enable cardiovascular imaging. In this study, we engineered a transgenic zebrafish with temperature-inducible expression of an EGFP-labeled single-chain human monoclonal antibody, IK17, which binds to MDA-LDL, and used optically transparent zebrafish larvae for imaging studies. Feeding a high-cholesterol diet (HCD) supplemented with a red fluorescent lipid marker to the transgenic zebrafish resulted in vascular lipid accumulation, quantified in live animals using confocal microscopy. After heat shock-induced expression of IK17-EGFP, we measured the time course of vascular accumulation of IK17-specific MDA epitopes. Treatment with either an antioxidant or a regression diet resulted in reduced IK17 binding to vascular lesions. Interestingly, homogenates of IK17-EGFP-expressing larvae bound to MDA-LDL and inhibited MDA-LDL binding to macrophages. Moreover, sustained expression of IK17-EGFP effectively prevented HCD-induced lipid accumulation in the vascular wall, suggesting that the antibody itself may have therapeutic effects. Thus, we conclude that HCD-fed zebrafish larvae with conditional expression of EGFP-labeled oxidation-specific antibodies afford an efficient method of testing dietary and/or other therapeutic antioxidant strategies that may ultimately be applied to humans.
Midbrain-hindbrain Boundary Patterning and Morphogenesis Are Regulated by Diverse Grainy Head-like 2-dependent Pathways
The isthmic organiser located at the midbrain-hindbrain boundary (MHB) is the crucial developmental signalling centre responsible for patterning mesencephalic and metencephalic regions of the vertebrate brain. Formation and maintenance of the MHB is characterised by a hierarchical program of gene expression initiated by fibroblast growth factor 8 (Fgf8), coupled with cellular morphogenesis, culminating in the formation of the tectal-isthmo-cerebellar structures. Here, we show in zebrafish that one orthologue of the transcription factor grainy head-like 2 (Grhl2), zebrafish grhl2b plays a central role in both MHB maintenance and folding by regulating two distinct, non-linear pathways. Loss of grhl2b expression induces neural apoptosis and extinction of MHB markers, which are rescued by re-expression of engrailed 2a (eng2a), an evolutionarily conserved target of the Grhl family. Co-injection of sub-phenotypic doses of grhl2b and eng2a morpholinos reproduces the apoptosis and MHB marker loss, but fails to substantially disrupt formation of the isthmic constriction. By contrast, a novel direct grhl2b target, spec1, identified by phylogenetic analysis and confirmed by ChIP, functionally cooperates with grhl2b to induce MHB morphogenesis, but plays no role in apoptosis or maintenance of MHB markers. Collectively, these data show that MHB maintenance and morphogenesis are dissociable events regulated by grhl2b through diverse transcriptional targets.
Computational Quantification of Fluorescent Leukocyte Numbers in Zebrafish Embryos
Fluorescent transgenes with leukocyte-restricted expression have been essential to recent studies using zebrafish models of inflammation and the innate immune response. Many of the experiments performed using these models involve quantifying changes in the number of fluorescent leukocytes. Here, we describe a tool for deriving a quantitative variable proportional to fluorescent leukocyte numbers from single-plane fluorescent digital images of whole live embryos. The parameter, called "Leukocyte units," provides reliable values linearly proportional to actual leukocyte numbers in the range 50-400 leukocytes/embryo, and its performance at higher leukocyte densities remains linear. Its usefulness is demonstrated by scoring changes in leukocyte numbers following perturbation of a characterized leukocyte specification pathway.
