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Chicken cathelicidin-2 (CATH-2) is a host defense peptide that exhibits immunomodulatory and antibacterial properties. Here we examined effects of CATH-2 in zebrafish embryos in the absence and presence of infection. Yolk-injection of 0.2-1.5 h post-fertilized (hpf) zebrafish embryos with 2.6 ng/kg CATH-2 increased proliferation of phagocytic cells at 48 hpf by 30%. A lethal infection model was developed to test the prophylactic protective effect of CATH-2 peptide. Embryos (0.2-1.5 hpf) were injected with 2.6 ng/kg CATH-2, challenged with a lethal dose of fluorescently labeled Salmonella enteritidis pGMDs3 at 28 hpf and monitored for survival. Prophylactic treatment with CATH-2 was found to delay infection starting at 22 h post-infection (hpi). At 18-20 hpi, significantly lower (2-fold) fluorescence intensity and decreased bacterial loads were detected in peptide-treated embryos. Thus prophylactic administration of low CATH-2 concentrations confer partial protection in zebrafish embryos by boosting the innate immune system., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Over the past decade the zebrafish (Danio rerio) has become an attractive new vertebrate model organism for studying mycobacterial pathogenesis. The combination of medium-throughput screening and real-time in vivo visualization has allowed new ways to dissect host pathogenic interaction in a vertebrate host. Furthermore, genetic screens on the host and bacterial sides have elucidated new mechanisms involved in the initiation of granuloma formation and the importance of a balanced immune response for control of mycobacterial pathogens. This article will highlight the unique features of the zebrafish-Mycobacterium marinum infection model and its added value for tuberculosis research., (Copyright © 2015 Cold Spring Harbor Laboratory Press; all rights reserved.)

In the last years, the zebrafish (Danio rerio) has become an important vertebrate animal model to study host-pathogen interactions, especially in its embryonic stage. The presence of a fully developed innate immune system in the first days of embryogenesis, the facility of obtaining and manipulating large numbers of embryos, the optical transparency of the embryos that allow the direct visualization of bacterial infections, a wide range of genetic tools, and extensive mutant resources and collections of transgenic reporter lines are important advantages of the zebrafish-embryo model. Pseudomonas aeruginosa is able to lethally infect zebrafish embryos when the amount of cells injected exceeds the phagocytic capacity of the embryo. Different studies have proved the suitability of zebrafish embryos as a model to analyze P. aeruginosa infection. Here we describe the detailed protocols to establish a P. aeruginosa infection in zebrafish embryos and to image the interaction of the bacterium with this host with fluorescent microscopy.

ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M. marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M. marinum to establish a moderate and persistent infection., (© 2012 Blackwell Publishing Ltd.)

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage. The site of micro-injection of bacteria into the embryo determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.

The hallmark of tuberculosis (TB) is the formation of granulomas, which are clusters of infected macrophages surrounded by additional macrophages, neutrophils and lymphocytes. Although it has long been thought that granulomas are beneficial for the host, there is evidence that mycobacteria also promote the formation of these structures. In this study, we aimed to identify new mycobacterial factors involved in the initial stages of granuloma formation. We exploited the zebrafish embryo Mycobacterium marinum infection model to study initiation of granuloma formation and developed an in vivo screen to select for random M. marinum mutants that were unable to induce granuloma formation efficiently. Upon screening 200 mutants, three mutants repeatedly initiated reduced granuloma formation. One of the mutants was found to be defective in the espL gene, which is located in the ESX-1 cluster. The ESX-1 cluster is disrupted in the Mycobacterium bovis BCG vaccine strain and encodes a specialized secretion system known to be important for granuloma formation and virulence. Although espL has not been implicated in protein secretion before, we observed a strong effect on the secretion of the ESX-1 substrates ESAT-6 and EspE. We conclude that our zebrafish embryo M. marinum screen is a useful tool to identify mycobacterial genes involved in the initial stages of granuloma formation and that we have identified a new component of the ESX-1 secretion system. We are confident that our approach will contribute to the knowledge of mycobacterial virulence and could be helpful for the development of new TB vaccines.

This paper reports the cloning and sequencing of interleukin (IL)-23 p19 subunit for the first time within a non-mammalian species, the zebrafish (Danio rerio), which was discovered using a synteny approach. In addition, amino acid sequences were for IL-23 p19 subunits were also predicted from the stickleback, Fugu and Tetraodon genomes and included in this investigation. The zebrafish IL-23 p19 cDNA consisted of a 66bp 5' UTR, a 249bp 3' UTR and a single open reading frame of 567bp giving a predicted 188 aa IL-23 p19 molecule. Multiple alignment of zebrafish IL-23 p19, with other known IL-23 p19 and IL-12 p35 amino acid sequences revealed areas of amino acid conservation, such as the presence of four predicted α-helixes, cysteines important for disulphide bond formation and the conservation of a tryptophan known to interact with the receptor. Amino acid homologies and phylogenetic analysis confirmed the relationship of the fish IL-23 p19 subunits with their mammalian homologues. All the teleost fish IL-23 p19 subunits were found to have 4 exons and 3 introns similar to that of human and mouse IL-23 p19 and a limited degree of synteny was found between the organisms for the regions containing the IL-23 p19 genes with only PAB-dependent poly(A)-specific ribonuclease subunit 2 (PAN2) and IL-23 p19 found in the same order on human chromosome 12 and all the fish genomes looked at. Lastly using real-time PCR, constitutive expression of IL-12 p40 and IL-23 p19 was observed in the kidney, liver, gut and muscle with IL-12 p40 expression higher than IL-23 p19. As soon as an hour after stimulation with LPS, there was an increase of IL-23 p19 in zebrafish leukocytes and an increase of IL-1β, IL-12 p40 and IL-23 p19 expression was found after infection of zebrafish for 1 or 6 days with Mycobaterium marinum strain E11., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The zebrafish genome contains ten genes that encode class II cytokine-like peptides, of which the two that are related most closely to mammalian interferon gamma (IFN-gamma) were named IFN-gamma1 and IFN-gamma2. Although the zebrafish has become a popular model system to study immune mechanisms, and although interferons are central regulators of immunity, which zebrafish cytokines correspond functionally to mammalian IFN-gamma has not been established. We used zebrafish embryos to assay the functions of IFN-gamma1 and IFN-gamma2, and have identified a subset of zebrafish homologs of the mammalian IFN-responsive genes as IFN-gamma targets in the zebrafish embryo: these genes are upregulated in response to raised levels of either IFN-gamma1 or IFN-gamma2. Infection studies using two different pathogens show that IFN-gamma signalling is required for resistance against bacterial infections in the young embryo and that the levels of IFN-gamma need to be regulated tightly: raising IFN-gamma levels sensitizes fish embryos against bacterial infection. Embryos injected with high doses of Escherichia coli are able to clear the bacteria within a day, and the gamma-interferons are necessary for this defence reaction. The protective response to Yersinia ruckeri, a natural fish pathogen that is lethal at low doses, also requires IFN-gamma. As in the induction of target genes, the two interferons act at least partly redundantly. Together with the previously demonstrated type III interferon response, these results show that the counterparts of the mammalian viral and bacterial interferon-dependent defence functions are in place in zebrafish embryos, and suggest that zebrafish IFN-gamma1 and IFN-gamma2 are functionally equivalent to mammalian IFN-gamma.

Basic research in pattern formation is concerned with the generation of phenotypes and tissues. It can therefore lead to new tools for medical research. These include phenotypic screening assays, applications in tissue engineering, as well as general advances in biomedical knowledge. Our aim here is to discuss this emerging field with special reference to tools based on zebrafish developmental biology. We describe phenotypic screening assays being developed in our own and other labs. Our assays involve: (i) systemic or local administration of a test compound or drug to zebrafish in vivo; (ii) the subsequent detection or "readout" of a defined phenotypic change. A positive readout may result from binding of the test compound to a molecular target involved in a developmental pathway. We present preliminary data on assays for compounds that modulate skeletal patterning, bone turnover, immune responses, inflammation and early-life stress. The assays use live zebrafish embryos and larvae as well as adult fish undergoing caudal fin regeneration. We describe proof-of-concept studies on the localised targeting of compounds into regeneration blastemas using microcarriers. Zebrafish are cheaper to maintain than rodents, produce large numbers of transparent eggs, and some zebrafish assays could be scaled-up into medium and high throughput screens. However, advances in automation and imaging are required. Zebrafish cannot replace mammalian models in the drug development pipeline. Nevertheless, they can provide a cost-effective bridge between cell-based assays and mammalian whole-organism models.

Innate immunity signaling mechanisms during vertebrate embryogenesis are largely unknown. To study Toll-like receptor (TLR) signaling function in the zebrafish embryo model, we designed an experimental setup for antisense morpholino knockdown under conditions of bacterial infection. Clearance of Salmonella enterica serovar Typhimurium Ra bacteria was significantly impaired after knockdown of myeloid differentiation factor 88 (MyD88), a common adaptor protein in TLR and interleukin-1 receptor signaling. Thereby, we demonstrate for the first time that the innate immune response of the developing embryo involves MyD88-dependent signaling, which further establishes the zebrafish embryo as a model for the study of vertebrate innate immunity.

The Mycobacterium marinum-zebrafish infection model was used in this study for analysis of a host transcriptome response to mycobacterium infection at the organismal level. RNA isolated from adult zebrafish that showed typical signs of fish tuberculosis due to a chronic progressive infection with M. marinum was compared with RNA from healthy fish in microarray analyses. Spotted oligonucleotide sets (designed by Sigma-Compugen and MWG) and Affymetrix GeneChips were used, in total comprising 45,465 zebrafish transcript annotations. Based on a detailed comparative analysis and quantitative reverse transcriptase-PCR analysis, we present a validated reference set of 159 genes whose regulation is strongly affected by mycobacterial infection in the three types of microarrays analyzed. Furthermore, we analyzed the separate datasets of the microarrays with special emphasis on the expression profiles of immune-related genes. Upregulated genes include many known components of the inflammatory response and several genes that have previously been implicated in the response to mycobacterial infections in cell cultures of other organisms. Different marker genes of the myeloid lineage that have been characterized in zebrafish also showed increased expression. Furthermore, the zebrafish homologs of many signal transduction genes with relationship to the immune response were induced by M. marinum infection. Future functional analysis of these genes may contribute to understanding the mechanisms of mycobacterial pathogenesis. Since a large group of genes linked to immune responses did not show altered expression in the infected animals, these results suggest specific responses in mycobacterium-induced disease.

Bacterial virulence is best studied in animal models. However, the lack of possibilities for real time analysis and the need for laborious and invasive sample analysis limit the use of experimental animals. In the present study 28 h-old zebrafish embryos were infected with DsRed-labelled cells of Salmonella typhimurium. Using multidimensional digital imaging microscopy we were able to determine the exact location and fate of these bacterial pathogens in a living vertebrate host during three days. A low dose of wild-type S. typhimurium resulted in a lethal infection with bacteria residing and multiplying both in macrophage-like cells and at the epithelium of blood vessels. Lipopolysaccharide (LPS) mutants of S. typhimurium, known to be attenuated in the murine model, proved to be non-pathogenic in the zebrafish embryos and were partially lysed in the bloodstream or degraded in macrophage-like cells. However, injection of LPS mutants in the yolk of the embryo resulted in uncontrolled bacterial proliferation. Heat-killed, wild-type bacteria were completely lysed extracellularly within minutes after injection, which shows that the blood of these zebrafish embryos does already contain lytic activity. In conclusion, the zebrafish embryo model allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host.

Receptor protein-tyrosine phosphatase alpha (RPTP alpha) is highly expressed in the developing retina of different species, but little is known about its function there. Here, we report that injection of antisense morpholinos in zebrafish embryos reduced RPTP alpha expression to almost nondetectable levels up to 3 days postfertilization (dpf). RPTP alpha was detectable again from 4 dpf onward. RPTP alpha knock-down resulted in smaller eyes. Examination of sections of the retina at different developmental stages demonstrated that already at 28 hours postfertilization (hpf) fewer cells were present in the retina of RPTP alpha-morpholino-injected embryos. At 3 dpf, the layered organization of the retina was absent. In addition, the morphology and labeling with an axon specific antibody, acetylated tubulin, demonstrated that most cells appeared to be undifferentiated. Strikingly, at 5 dpf the lamination of the retina was partially restored, concomitant with re-expression of RPTP alpha protein. Although cells in the retina were now differentiated, the layering of the retina remained disrupted and significant gaps were observed in the amacrine cell layer. Therefore, knock-down of RPTP alpha protein provides evidence that RPTP alpha is essential for normal retinal development., (Copyright 2002 Wiley‐Liss, Inc.)

Protein tyrosine phosphorylation is an important mechanism of eukaryotic cell signalling which is regulated by protein-tyrosine kinases and protein-tyrosine phosphatases. Here we report the molecular cloning of the first zebrafish protein-tyrosine phosphatase, zf-PTP-1B, the homologue of human PTP-1B. Zf-PTP-1B was catalytically active and localised to the endoplasmic reticulum, like human PTP-1B. Zf-PTP-1B was maternally expressed in zebrafish embryos, and low ubiquitous expression was detected up to day 7 of development. Microinjection of zf-PTP-1B RNA induced pleiotropic, but reproducible developmental defects. Evaluation of the live embryos at 24 h post fertilisation indicated that zf-PTP-1B induced defects in somite formation. The phenotype was dependent on protein-tyrosine phosphatase activity of zf-PTP-1B, since embryos injected with catalytically inactive zf-PTP-1B-C213S developed normally. Co-injection of wild type and inactive zf-PTP-1B led to a rescue of the zf-PTP-1B-induced phenotype, suggesting that zf-PTP-1B-C213S had dominant negative activity. The zf-PTP-1B-induced phenotype suggests that proper tyrosine phosphorylation of key proteins is essential for early development, most notably somitogenesis.