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Lymphatic vessels are known to be derived from veins; however, recent lineage-tracing experiments propose that specific lymphatic networks may originate from both venous and non-venous sources. Despite this, direct evidence of a non-venous lymphatic progenitor is missing. Here, we show that the zebrafish facial lymphatic network is derived from three distinct progenitor populations that add sequentially to the developing facial lymphatic through a relay-like mechanism. We show that while two facial lymphatic progenitor populations are venous in origin, the third population, termed the ventral aorta lymphangioblast (VA-L), does not sprout from a vessel; instead, it arises from a migratory angioblast cell near the ventral aorta that initially lacks both venous and lymphatic markers, and contributes to the facial lymphatics and the hypobranchial artery. We propose that sequential addition of venous and non-venous progenitors allows the facial lymphatics to form in an area that is relatively devoid of veins. Overall, this study provides conclusive, live imaging-based evidence of a non-venous lymphatic progenitor and demonstrates that the origin and development of lymphatic vessels is context-dependent., (© 2019 The Authors.)

Chemical interventions are regularly used to examine and manipulate macrophage function in larval zebrafish. Given chemicals are typically administered by simple immersion or injection, it is not possible to resolve whether their impact on macrophage function is direct or indirect. Liposomes provide an attractive strategy to target drugs to specific cellular compartments, including macrophages. As an example, injecting liposomal clodronate into animal models, including zebrafish, is routinely used to deliver toxic levels of clodronate specifically to macrophages for targeted cell ablation. Here we show that liposomes can also target the delivery of drugs to zebrafish macrophages to selectively manipulate their function. We utilized the drugs etomoxir (a fatty acid oxidation inhibitor) and MitoTEMPO (a scavenger of mitochondrial reactive oxygen species [mROS]), that we have previously shown, through free drug delivery, suppress monosodium urate (MSU) crystal-driven macrophage activation. We generated poloxamer 188 modified liposomes that were readily phagocytosed by macrophages, but not by neutrophils. Loading these liposomes with etomoxir or MitoTEMPO and injecting into larvae suppressed macrophage activation in response to MSU crystals, as evidenced by proinflammatory cytokine expression and macrophage-driven neutrophil recruitment. This work reveals the utility of packaging drugs into liposomes as a strategy to selectively manipulate macrophage function.

Tumour angiogenesis has long been a focus of anti-cancer therapy; however, anti-angiogenic cancer treatment strategies have had limited clinical success. Tumour-associated myeloid cells are believed to play a role in the resistance of cancer towards anti-angiogenesis therapy, but the mechanisms by which they do this are unclear. An embryonic zebrafish xenograft model has been developed to investigate the mechanisms of tumour angiogenesis and as an assay to screen anti-angiogenic compounds. In this study, we used cell ablation techniques to remove either macrophages or neutrophils and assessed their contribution towards zebrafish xenograft angiogenesis by quantitating levels of graft vascularisation. The ablation of macrophages, but not neutrophils, caused a strong reduction in tumour xenograft vascularisation and time-lapse imaging demonstrated that tumour xenograft macrophages directly associated with the migrating tip of developing tumour blood vessels. Finally, we found that, although macrophages are required for vascularisation in xenografts that either secrete VEGFA or overexpress zebrafish vegfaa , they are not required for the vascularisation of grafts with low levels of VEGFA, suggesting that zebrafish macrophages can enhance Vegfa-driven tumour angiogenesis. The importance of macrophages to this angiogenic response suggests that this model could be used to further investigate the interplay between myeloid cells and tumour vascularisation., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2018. Published by The Company of Biologists Ltd.)

The circadian clock, which evolved to help organisms harmonize physiological responses to external conditions (such as the light/dark cycle, LD), is emerging as an important regulator of the immune response to infection. Gaining a complete understanding of how the circadian clock influences the immune cell response requires animal models that permit direct observation of these processes within an intact host. Here, we investigated the use of larval zebrafish, a powerful live imaging system, as a new model to study the impact of a fundamental zeitgeber, light, on the innate immune cell response to infection. Larvae infected during the light phase of the LD cycle and in constant light condition (LL) demonstrated enhanced survival and bacterial clearance when compared with larvae infected during the dark phase of the LD cycle and in constant dark condition (DD). This increased survival was associated with elevated expression of the zebrafish orthologues of the mammalian pro-inflammatory cytokine genes, Tumour necrosis factor-α, Interleukin-8 and Interferon-γ, and increased neutrophil and macrophage recruitment. This study demonstrates for the first time that the larval zebrafish innate immune response to infection is enhanced during light exposure, suggesting that, similar to mammalian systems, the larval zebrafish response to infection is light-regulated.

Cellular responses to injury are crucial for complete tissue regeneration, but their underlying processes remain incompletely elucidated. We have previously reported that myeloid-defective zebrafish mutants display apoptosis of regenerative cells during fin fold regeneration. Here, we found that the apoptosis phenotype is induced by prolonged expression of interleukin 1 beta ( il1b ). Myeloid cells are considered to be the principal source of Il1b, but we show that epithelial cells express il1b in response to tissue injury and initiate the inflammatory response, and that its resolution by macrophages is necessary for survival of regenerative cells. We further show that Il1b plays an essential role in normal fin fold regeneration by regulating expression of regeneration-induced genes. Our study reveals that proper levels of Il1b signaling and tissue inflammation, which are tuned by macrophages, play a crucial role in tissue regeneration.

Objectives: Mycobacterium tuberculosis is a deadly human pathogen that causes the lung disease TB. M. tuberculosis latently infects a third of the world's population, resulting in ∼1.5 million deaths per year. Due to the difficulties and expense of carrying out animal drug trials using M. tuberculosis and rodents, infections of the zebrafish Danio rerio with Mycobacterium marinum have become a useful surrogate. However, the infection methods described to date require specialized equipment and a high level of operator expertise., Methods: We investigated whether zebrafish larvae could be naturally infected with bioluminescently labelled M. marinum by immersion, and whether infected larvae could be used for rapid screening of anti-mycobacterial compounds using bioluminescence. We used rifampicin and a variety of nitroimidazole-based next-generation and experimental anti-mycobacterial drugs, selected for their wide range of potencies against M. tuberculosis, to validate this model for anti-mycobacterial drug discovery., Results: We observed that five of the six treatments (rifampicin, pretomanid, delamanid, SN30488 and SN30527) significantly reduced the bioluminescent signal from M. marinum within naturally infected zebrafish larvae. Importantly, these same five treatments also retarded the growth of M. tuberculosis in vitro. In contrast, only three of the six treatments tested (rifampicin, delamanid and SN30527) retarded the growth of M. marinum in vitro., Conclusions: We have demonstrated that zebrafish larvae naturally infected with bioluminescent M. marinum M can be used for the rapid screening of anti-mycobacterial compounds with readily available equipment and limited expertise. The result is an assay that can be carried out by a wide variety of laboratories for minimal cost and without high levels of zebrafish expertise., (© The Author 2016. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.)

The physical attributes of the zebrafish, including optical transparency during embryogenesis, large clutch sizes, external development, and rapid organogenesis were features that initially attracted developmental biologists to use this vertebrate as an experimental model system. With the progressive development of an extensive genetic "tool kit" and an ever-growing number of transgenic reporter lines, the zebrafish model has evolved into an informative system in which to mimic and study aspects of human disease, including those associated with bacterial infections. This chapter provides detailed protocols for microinjection of bacterial strains into zebrafish larvae and subsequent experiments to investigate single-larva bacterial burdens, live imaging of specific neutrophil and macrophage bactericidal functions, and how these protocols may be applied to drug discovery approaches to uncover novel immunomodulatory drugs., (Copyright © 2017 Elsevier Inc. All rights reserved.)

3D printing has emerged as a rapid and cost-efficient manufacturing technique to enable the fabrication of bespoke, complex prototypes. If the technology is to have a significant impact in biomedical applications, such as drug discovery and molecular diagnostics, the devices produced must be biologically compatible to enable their use with established reference assays and protocols. In this work we demonstrate that we can adapt the Fish Embryo Test (FET) as a new method to quantify the toxicity of 3D printed microfluidic devices. We assessed the biocompatibility of four commercially available 3D printing polymers (VisiJetCrystal EX200, Watershed 11122XC, Fototec SLA 7150 Clear and ABSplus P-430), through the observation of key developmental markers in the developing zebrafish embryos. Results show all of the photopolymers to be highly toxic to the embryos, resulting in fatality, although we do demonstrate that post-printing treatment of Fototec 7150 makes it suitable for zebrafish culture within the FET.

Macrophages are the most functionally heterogenous cells of the hematopoietic system. Given many diseases are underpinned by inappropriate macrophage activation, macrophages have emerged as a therapeutic target to treat disease. A thorough understanding of what controls macrophage activation will likely reveal new pathways that can be manipulated for therapeutic benefit. Live imaging fluorescent macrophages within transgenic zebrafish larvae has provided a valuable window to investigate macrophage behavior in vivo. Here we describe the first transgenic zebrafish line that reports macrophage activation, as evidenced by induced expression of an immunoresponsive gene 1(irg1):EGFP transgene. When combined with existing reporter lines that constitutively mark macrophages, we reveal this unique transgenic line can be used to live image macrophage activation in response to the bacterial endotoxin lipopolysaccharide and xenografted human cancer cells. We anticipate the Tg(irg1:EGFP) line will provide a valuable tool to explore macrophage activation and plasticity in the context of different disease models., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

To spearhead deployment of zebrafish embryo biotests in large-scale drug discovery studies, automated platforms are needed to integrate embryo in-test positioning and immobilization (suitable for high-content imaging) with fluidic modules for continuous drug and medium delivery under microperfusion to developing embryos. In this work, we present an innovative design of a high-throughput three-dimensional (3D) microfluidic chip-based device for automated immobilization and culture and time-lapse imaging of developing zebrafish embryos under continuous microperfusion. The 3D Lab-on-a-Chip array was fabricated in poly(methyl methacrylate) (PMMA) transparent thermoplastic using infrared laser micromachining, while the off-chip interfaces were fabricated using additive manufacturing processes (fused deposition modelling and stereolithography). The system's design facilitated rapid loading and immobilization of a large number of embryos in predefined clusters of traps during continuous microperfusion of drugs/toxins. It was conceptually designed to seamlessly interface with both upright and inverted fluorescent imaging systems and also to directly interface with conventional microtiter plate readers that accept 96-well plates. Compared with the conventional Petri dish assays, the chip-based bioassay was much more convenient and efficient as only small amounts of drug solutions were required for the whole perfusion system running continuously over 72 h. Embryos were spatially separated in the traps that assisted tracing single embryos, preventing interembryo contamination and improving imaging accessibility.

Pathogenic mycobacteria induce the formation of complex cellular aggregates called granulomas that are the hallmark of tuberculosis. Here we examine the development and consequences of vascularization of the tuberculous granuloma in the zebrafish-Mycobacterium marinum infection model, which is characterized by organized granulomas with necrotic cores that bear striking resemblance to those of human tuberculosis. Using intravital microscopy in the transparent larval zebrafish, we show that granuloma formation is intimately associated with angiogenesis. The initiation of angiogenesis in turn coincides with the generation of local hypoxia and transcriptional induction of the canonical pro-angiogenic molecule Vegfaa. Pharmacological inhibition of the Vegf pathway suppresses granuloma-associated angiogenesis, reduces infection burden and limits dissemination. Moreover, anti-angiogenic therapies synergize with the first-line anti-tubercular antibiotic rifampicin, as well as with the antibiotic metronidazole, which targets hypoxic bacterial populations. Our data indicate that mycobacteria induce granuloma-associated angiogenesis, which promotes mycobacterial growth and increases spread of infection to new tissue sites. We propose the use of anti-angiogenic agents, now being used in cancer regimens, as a host-targeting tuberculosis therapy, particularly in extensively drug-resistant disease for which current antibiotic regimens are largely ineffective.

Biotests performed on small vertebrate model organisms provide significant investigative advantages as compared with bioassays that employ cell lines, isolated primary cells, or tissue samples. The main advantage offered by whole-organism approaches is that the effects under study occur in the context of intact physiological milieu, with all its intercellular and multisystem interactions. The gap between the high-throughput cell-based in vitro assays and low-throughput, disproportionally expensive and ethically controversial mammal in vivo tests can be closed by small model organisms such as zebrafish or Xenopus. The optical transparency of their tissues, the ease of genetic manipulation and straightforward husbandry, explain the growing popularity of these model organisms. Nevertheless, despite the potential for miniaturization, automation and subsequent increase in throughput of experimental setups, the manipulation, dispensing and analysis of living fish and frog embryos remain labor-intensive. Recently, a new generation of miniaturized chip-based devices have been developed for zebrafish and Xenopus embryo on-chip culture and experimentation. In this work, we review the critical developments in the field of Lab-on-a-Chip devices designed to alleviate the limits of traditional platforms for studies on zebrafish and clawed frog embryo and larvae. © 2014 International Society for Advancement of Cytometry., (© 2014 International Society for Advancement of Cytometry.)

Lymphangiogenesis is a dynamic process that involves the sprouting of lymphatic endothelial cells (LECs) from veins to form lymphatic vessels. Vegfr3 signalling, through its ligand Vegfc and the extracellular protein Ccbe1, is essential for the sprouting of LECs to form the trunk lymphatic network. In this study we determined whether Vegfr3, Vegfc and Ccbe1 are also required for development of the facial and intestinal lymphatic networks in the zebrafish embryo. Whereas Vegfr3 and Ccbe1 are required for the development of all lymphatic vessels, Vegfc is dispensable for facial lymphatic sprouting but not for the complete development of the facial lymphatic network. We show that zebrafish vegfd is expressed in the head, genetically interacts with ccbe1 and can rescue the lymphatic defects observed following the loss of vegfc. Finally, whereas knockdown of vegfd has no phenotype, double knockdown of both vegfc and vegfd is required to prevent facial lymphatic sprouting, suggesting that Vegfc is not essential for all lymphatic sprouting and that Vegfd can compensate for loss of Vegfc during lymphatic development in the zebrafish head., (© 2014. Published by The Company of Biologists Ltd.)

Transgenic zebrafish (Danio rerio) models of human diseases have recently emerged as innovative experimental systems in drug discovery and molecular pathology. None of the currently available technologies, however, allow for automated immobilization and treatment of large numbers of spatially encoded transgenic embryos during real-time developmental analysis. This work describes the proof-of-concept design and validation of an integrated 3D microfluidic chip-based system fabricated directly in the poly(methyl methacrylate) transparent thermoplastic using infrared laser micromachining. At its core, the device utilizes an array of 3D micromechanical traps to actively capture and immobilize single embryos using a low-pressure suction. It also features built-in piezoelectric microdiaphragm pumps, embryo-trapping suction manifold, drug delivery manifold, and optically transparent indium tin oxide heating element to provide optimal temperature during embryo development. Furthermore, we present design of the proof-of-concept off-chip electronic interface equipped with robotic servo actuator driven stage, innovative servomotor-actuated pinch valves, and embedded miniaturized fluorescent USB microscope. Our results showed that the innovative device has 100% embryo-trapping efficiency while supporting normal embryo development for up to 72 hr in a confined microfluidic environment. We also showed data that this microfluidic system can be readily applied to kinetic analysis of a panel of investigational antiangiogenic agents in transgenic zebrafish lines. The optical transparency and embryo immobilization allow for convenient visualization of developing vasculature patterns in response to drug treatment without the need for specimen re-positioning. The integrated electronic interfaces bring the lab-on-a-chip systems a step closer to realization of complete analytical automation., (© 2014 International Society for Advancement of Cytometry.)

Zebrafish (Danio rerio) embryo assays have recently come into the spotlight as convenient experimental models in both biomedicine and ecotoxicology. As a small aquatic model organism, zebrafish embryo assays allow for rapid physiological, embryo-, and genotoxic tests of drugs and environmental toxins that can be simply dissolved in water. This protocol describes prototyping and application of an innovative, miniaturized, and polymeric chip-based device capable of immobilizing a large number of living fish embryos for real-time and/or time-lapse microscopic examination. The device provides a physical address designation to each embryo during analysis, continuous perfusion of medium, and post-analysis specimen recovery. Miniaturized embryo array is a new concept of immobilization and real-time drug perfusion of multiple individual and developing zebrafish embryos inside the mesofluidic device. The OpenSource device presented in this protocol is particularly suitable to perform accelerated fish embryo biotests in ecotoxicology and phenotype-based pharmaceutical screening., (Copyright © 2014 John Wiley & Sons, Inc.)

Lab-on-a-Chip (LOC) biomicrofluidic technologies are rapidly emerging bioanalytical tools that can miniaturize and revolutionize in situ research on embryos of small vertebrate model organisms such as zebrafish (Danio rerio) and clawed African frog (Xenopus laevis). Despite considerable progress being made in fabrication techniques of chip-based devices, they usually still require excessive and manual actuation and data acquisition that significantly reduce throughput and introduce operator-related analytical bias. This work describes the development of a proof-of-concept embedded platform that integrates an innovative LOC zebrafish embryo array technology with an electronic interface to provide higher levels of laboratory automation for in situ biotests. The integrated platform was designed to perform automatic immobilization, culture and treatment of developing zebrafish embryos during fish embryo toxicity (FET) biotests. The system was equipped with a stepper motor driven stage, solenoid-actuated pinch valves, miniaturized peristaltic pumps as well as Peltier heating module. Furthermore, a Field Programmable Gate Array (FPGA) was used to implement an embedded hardware/software solution and interface to enable real-time control over embryo loading and immobilization; accurate microfluidic flow control; temperature stabilization and also automatic time-resolved image acquisition of developing zebrafish embryos. This work presents evidence that integration of embedded electronic interfaces with microfluidic chip-based technologies can bring the Lab-on-a-Chip a step closer to fully automated analytical systems., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Several intestinal damage models have been developed using zebrafish, with the aim of recapitulating aspects of human inflammatory bowel disease (IBD). These experimentally induced inflammation models have utilized immersion exposure to an array of colitogenic agents (including live bacteria, bacterial products, and chemicals) to induce varying severity of inflammation. This technical report describes methods used to generate two chemically induced intestinal damage models using either dextran sodium sulfate (DSS) or trinitrobenzene sulfonic acid (TNBS). Methods to monitor intestinal damage and inflammatory processes, and chemical-genetic methods to manipulate the host response to injury are also described.

Prompt neutrophil arrival is critical for host defense immediately after injury [1-3]. Following wounding, a hydrogen peroxide (H(2)O(2)) burst generated in injured tissues is the earliest known leukocyte chemoattractant [4]. Generating this tissue-scale H(2)O(2) gradient uses dual oxidase [4] and neutrophils sense H(2)O(2) by a mechanism involving the LYN Src-family kinase [5], but the molecular mechanisms responsible for H(2)O(2) clearance are unknown [6]. Neutrophils carry abundant amounts of myeloperoxidase, an enzyme catalyzing an H(2)O(2)-consuming reaction [7, 8]. We hypothesized that this neutrophil-delivered myeloperoxidase downregulates the high tissue H(2)O(2) concentrations that follow wounding. This was tested in zebrafish using simultaneous fluorophore-based imaging of H(2)O(2) concentrations and leukocytes [4, 9-11] and a new neutrophil-replete but myeloperoxidase-deficient mutant (durif). Leukocyte-depleted zebrafish had an abnormally sustained wound H(2)O(2) burst, indicating that leukocytes themselves were required for H(2)O(2) downregulation. Myeloperoxidase-deficient zebrafish also had abnormally sustained high wound H(2)O(2) concentrations despite similar numbers of arriving neutrophils. A local H(2)O(2)/myeloperoxidase interaction within wound-recruited neutrophils was demonstrated. These data demonstrate that leukocyte-delivered myeloperoxidase cell-autonomously downregulates tissue-generated wound H(2)O(2) gradients in vivo, defining a new requirement for myeloperoxidase during inflammation. Durif provides a new animal model of myeloperoxidase deficiency closely phenocopying the prevalent human disorder [7, 12, 13], offering unique possibilities for investigating its clinical consequences., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

We have generated novel transgenic lines that brightly mark the lymphatic system of zebrafish using the lyve1 promoter. Facilitated by these new transgenic lines, we generated a map of zebrafish lymphatic development up to 15 days post-fertilisation and discovered three previously uncharacterised lymphatic vessel networks: the facial lymphatics, the lateral lymphatics and the intestinal lymphatics. We show that a facial lymphatic vessel, termed the lateral facial lymphatic, develops through a novel developmental mechanism, which initially involves vessel growth through a single vascular sprout followed by the recruitment of lymphangioblasts to the vascular tip. Unlike the lymphangioblasts that form the thoracic duct, the lymphangioblasts that contribute to the lateral facial lymphatic vessel originate from a number of different blood vessels. Our work highlights the additional complexity of lymphatic vessel development in the zebrafish that may increase its versatility as a model of lymphangiogenesis.

Zebrafish (Danio rerio) has recently emerged as a powerful experimental model in drug discovery and environmental toxicology. Drug discovery screens performed on zebrafish embryos mirror with a high level of accuracy the tests usually performed on mammalian animal models, and fish embryo toxicity assay (FET) is one of the most promising alternative approaches to acute ecotoxicity testing with adult fish. Notwithstanding this, automated in-situ analysis of zebrafish embryos is still deeply in its infancy. This is mostly due to the inherent limitations of conventional techniques and the fact that metazoan organisms are not easily susceptible to laboratory automation. In this work, we describe the development of an innovative miniaturized chip-based device for the in-situ analysis of zebrafish embryos. We present evidence that automatic, hydrodynamic positioning, trapping and long-term immobilization of single embryos inside the microfluidic chips can be combined with time-lapse imaging to provide real-time developmental analysis. Our platform, fabricated using biocompatible polymer molding technology, enables rapid trapping of embryos in low shear stress zones, uniform drug microperfusion and high-resolution imaging without the need of manual embryo handling at various developmental stages. The device provides a highly controllable fluidic microenvironment and post-analysis eleuthero-embryo stage recovery. Throughout the incubation, the position of individual embryos is registered. Importantly, we also for first time show that microfluidic embryo array technology can be effectively used for the analysis of anti-angiogenic compounds using transgenic zebrafish line (fli1a:EGFP). The work provides a new rationale for rapid and automated manipulation and analysis of developing zebrafish embryos at a large scale.

Inflammatory bowel disease (IBD), in the form of Crohn's disease (CD) or ulcerative colitis (UC), is a debilitating chronic immune disorder of the intestine. A complex etiology resulting from dysfunctional interactions between the intestinal immune system and its microflora, influenced by host genetic susceptibility, makes disease modeling challenging. Mutations in NOD2 have the highest disease-specific risk association for CD, and a related gene, NOD1, is associated with UC. NOD1 and NOD2 encode intracellular bacterial sensor proteins acting as innate immune triggers, and represent promising therapeutic targets. The zebrafish has the potential to aid in modeling genetic and environmental aspects of IBD pathogenesis. Here, we report the characterization of the Nod signaling components in the zebrafish larval intestine. The nod1 and nod2 genes are expressed in intestinal epithelial cells and neutrophils together with the Nod signaling pathway genes ripk2, a20, aamp, cd147, centaurin b1, erbin and grim-19. Using a zebrafish embryo Salmonella infection model, morpholino-mediated depletion of Nod1 or Nod2 reduced the ability of embryos to control systemic infection. Depletion of Nod1 or Nod2 decreased expression of dual oxidase in the intestinal epithelium and impaired the ability of larvae to reduce intracellular bacterial burden. This work highlights the potential use of zebrafish larvae in the study of components of IBD pathogenesis.

The zebrafish is increasingly being utilized to study aspects of the conserved innate intestinal immunity of vertebrates. In mammals, some antimicrobial proteins are synthesised by specialised immune cells that appear to have no equivalent in zebrafish. To delineate foci of antimicrobial protein production along the zebrafish intestine, we examined the antero-posterior expression gradients of antimicrobial genes. Quantitative PCR revealed distinct expression gradient profiles, with the mid-intestine exhibiting elevated expression of several genes such as dual oxidase and the defensin beta-like and peptidoglycan recognition protein families. This region also presented with the most numbers of leukocytes and endocytic cells, supporting a specialised immunological role. Conversely, expression of the Dr-RNase family was prominent in the anterior intestine. Expression of the zebrafish β-defensin family was examined in adult zebrafish tissues. Strong expression of defensin beta-like 1 was detected in the swim bladder of zebrafish from the larval stage of development through to adults., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Inflammatory bowel disease (IBD) results from dysfunctional interactions between the intestinal immune system and microbiota, influenced by host genetic susceptibility. Because a key feature of the pathology is intestinal epithelial damage, potential disease factors have been traditionally analyzed within the background of chemical colitis models in mice. The zebrafish has greatly complemented the mouse for modeling aspects of disease processes, with an advantage for high content drug screens. Larval zebrafish exposed to the haptenizing agent trinitrobenzene sulfonic acid (TNBS) displayed impaired intestinal homeostasis and inflammation reminiscent of human IBD. There was a marked induction of pro-inflammatory cytokines, the degradative enzyme mmp9 and leukocytosis. Enterocolitis was dependent on microbiota and Toll-like receptor signaling, that can be ameliorated by antibiotic and anti-inflammatory drug treatments. This system will be useful to rapidly interrogate in vivo the biological significance of the IBD candidate genes so far identified and to carry out pharmacological modifier screens., (© 2010 Wiley-Liss, Inc.)

Reactive oxygen species (ROS) function in a range of physiological processes such as growth, metabolism and signaling, and also have a pathological role. Recent research highlighted the requirement for ROS generated by dual oxidase (DUOX) in host-defence responses in innate immunity and inflammatory disorders such as inflammatory bowel disease (IBD), but in vivo evidence to support this has, to date, been lacking. In order to investigate the involvement of Duox in gut immunity, we characterized the zebrafish ortholog of the human DUOX genes. Zebrafish duox is highly expressed in intestinal epithelial cells. Knockdown of Duox impaired larval capacity to control enteric Salmonella infection., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Contact between sister chromatids from S phase to anaphase depends on cohesin, a large multi-subunit protein complex. Mutations in sister chromatid cohesion proteins underlie the human developmental condition, Cornelia de Lange syndrome. Roles for cohesin in regulating gene expression, sometimes in combination with CCCTC-binding factor (CTCF), have emerged. We analyzed zebrafish embryos null for cohesin subunit rad21 using microarrays to determine global effects of cohesin on gene expression during embryogenesis. This identified Rad21-associated gene networks that included myca (zebrafish c-myc), p53 and mdm2. In zebrafish, cohesin binds to the transcription start sites of p53 and mdm2, and depletion of either Rad21 or CTCF increased their transcription. In contrast, myca expression was strongly downregulated upon loss of Rad21 while depletion of CTCF had little effect. Depletion of Rad21 or the cohesin-loading factor Nipped-B in Drosophila cells also reduced expression of myc and Myc target genes. Cohesin bound the transcription start site plus an upstream predicted CTCF binding site at zebrafish myca. Binding and positive regulation of the c-Myc gene by cohesin is conserved through evolution, indicating that this regulation is likely to be direct. The exact mechanism of regulation is unknown, but local changes in histone modification associated with transcription repression at the myca gene were observed in rad21 mutants., (Copyright 2010 Elsevier Inc. All rights reserved.)

Blood cells of an adult vertebrate are continuously generated by hematopoietic stem cells (HSCs) that originate during embryonic life within the aorta-gonad-mesonephros region. There is now compelling in vivo evidence that HSCs are generated from aortic endothelial cells and that this process is critically regulated by the transcription factor Runx1. By time-lapse microscopy of Runx1-enhanced green fluorescent protein transgenic zebrafish embryos, we were able to capture a subset of cells within the ventral endothelium of the dorsal aorta, as they acquire hemogenic properties and directly emerge as presumptive HSCs. These nascent hematopoietic cells assume a rounded morphology, transiently occupy the subaortic space, and eventually enter the circulation via the caudal vein. Cell tracing showed that these cells subsequently populated the sites of definitive hematopoiesis (thymus and kidney), consistent with an HSC identity. HSC numbers depended on activity of the transcription factor Runx1, on blood flow, and on proper development of the dorsal aorta (features in common with mammals). This study captures the earliest events of the transition of endothelial cells to a hemogenic endothelium and demonstrates that embryonic hematopoietic progenitors directly differentiate from endothelial cells within a living organism.

Lymphangiogenesis induced during tumor growth contributes to metastasis. Genetic and chemical screens using the zebrafish model have the potential to enhance our understanding of lymphangiogenesis, and lead to the discovery of pharmacological agents with activity in the lymphatic system. Large-scale screening of lymphatic development in the whole zebrafish embryo requires a specific lymphatic endothelial cell marker. We isolated the zebrafish ortholog of Lyve1, and analyzed its expression pattern during embryogenesis, and under conditions where key regulators of lymphangiogenesis such as Prox1 and VegfC were depleted. Like humans, zebrafish embryos form lymph sacs, lymphangioblasts arise from venous endothelia, and they form asymmetric left and right collecting ducts. By monitoring the earliest lymphatic sprouting in the head, a pilot drug assay was performed showing rapamycin, an inhibitor of mammalian lymphangiogenesis, can also suppress zebrafish lymphangiogenesis. This work opens up a novel opportunity to further the understanding of, and potentially manipulate, human lymphangiogenesis., ((c) 2010 Wiley-Liss, Inc.)

Cxcl8 is a pro-inflammatory chemokine, best known for its role in neutrophil chemotaxis. Signalling through its receptors, Cxcr1 and Cxcr2, is induced by inflammatory stimuli evoked by microbial, chemical or environmental stress, and hormonal signals. While it is recognised that Cxcl8 signalling is active in the gut mucosa, this is not as well understood as its role in leukocyte trafficking. Here, we report the characterisation of genes encoding the zebrafish Cxcl8, Cxcr1 and Cxcr2. By a combination of genomic, expression and functional analyses, we show that the Cxcl8 signalling pathway is conserved in zebrafish. As in humans, cxcl8 is expressed in zebrafish leukocytes. Transcripts were also detected in intestinal epithelial cells, and this expression is upregulated under inflammatory conditions caused by bacterial or chemical insult. Expression of cxcr1 and cxcr2 is robust within the developing gut. This work provides a model for the study of Cxcl8 signalling during gut inflammation., (Copyright 2009 Elsevier Ltd. All rights reserved.)

Pseudomonas aeruginosa is an opportunistic human pathogen that can cause serious infection in those with deficient or impaired phagocytes. We have developed the optically transparent and genetically tractable zebrafish embryo as a model for systemic P. aeruginosa infection. Despite lacking adaptive immunity at this developmental stage, zebrafish embryos were highly resistant to P. aeruginosa infection, but as in humans, phagocyte depletion dramatically increased their susceptibility. The virulence of an attenuated P. aeruginosa strain lacking a functional Type III secretion system was restored upon phagocyte depletion, suggesting that this system influences virulence through its effects on phagocytes. Intravital imaging revealed bacterial interactions with multiple blood cell types. Neutrophils and macrophages rapidly phagocytosed and killed P. aeruginosa, suggesting that both cell types play a role in protection against infection. Intravascular aggregation of erythrocytes and other blood cells with resultant circulatory blockage was observed immediately upon infection, which may be relevant to the pathogenesis of thrombotic complications of human P. aeruginosa infections. The real-time visualization capabilities and genetic tractability of the zebrafish infection model should enable elucidation of molecular and cellular details of P. aeruginosa pathogenesis in conditions associated with neutropenia or impaired phagocyte function., (© 2009 Blackwell Publishing Ltd.)

Runx transcription factors determine cell fate in many lineages. Maintaining balanced levels of Runx proteins is crucial, as deregulated expression leads to cancers and developmental disorders. We conducted a forward genetic screen in zebrafish for positive regulators of runx1 that yielded the cohesin subunit rad21. Zebrafish embryos lacking Rad21, or cohesin subunit Smc3, fail to express runx3 and lose hematopoietic runx1 expression in early embryonic development. Failure to develop differentiated blood cells in rad21 mutants is partially rescued by microinjection of runx1 mRNA. Significantly, monoallelic loss of rad21 caused a reduction in the transcription of runx1 and of the proneural genes ascl1a and ascl1b, indicating that downstream genes are sensitive to Rad21 dose. Changes in gene expression were observed in a reduced cohesin background in which cell division was able to proceed, indicating that cohesin might have a function in transcription that is separable from its mitotic role. Cohesin is a protein complex essential for sister chromatid cohesion and DNA repair that also appears to be essential for normal development through as yet unknown mechanisms. Our findings provide evidence for a novel role for cohesin in development, and indicate potential for monoallelic loss of cohesin subunits to alter gene expression.

The precise assembly of an integrated network of blood vessels is essential for the survival of vertebrate embryos. However, the processes by which primitive endothelial cells form mature vessels capable of supplying oxygen and nutrients to developing tissues remain incompletely understood. Here, we propose a role for Radar, one of the zebrafish orthologues of gdf6, in establishing integrity of the trunk vasculature in zebrafish embryos. We show that radar expression is appropriately placed, both spatially and temporally, to perform such a role. Transcripts for radar are detected in the hypochord and the primitive gut endoderm. These tissues intimately flank developing axial vessels in the trunk and have been previously implicated in the regulation of vascular development. Morpholino-based targeted gene knock-down has generated a Radar-specific loss-of-function zebrafish model. These embryos display normal initiation of vascular patterning and commencement of circulation. However, by day 2 of development, the integrity of the axial vasculature is compromised with hemorrhages and circulation short-circuits throughout the developing trunk. We show that this aberrant vascular development is specific to a reduction of the radar gene product. These results suggest that Radar is involved in a signaling pathway required for establishing the integrity of the axial vessels during zebrafish development.

Studies in zebrafish have potential to contribute to understanding of the vertebrate hematopoietic and vasculogenic systems. Our research has examined the roles of several molecules in pathways that lead to the development of blood and vessels in zebrafish, and has provided insights into the regulation of these processes. Gdf6a/radar, a member of the bone morphogenetic protein (BMP) family, is expressed in the zebrafish hypochord and primitive gut endoderm; structures that flank the developing dorsal aorta and posterior cardinal vein. This pattern of expression positions Gdf6a/radar as a candidate regulator of vasculogenesis. Support for such a role has come from experiments where Gdf6a/radar function was depleted with antisense morpholino oligonucleotides. This resulted in vascular leakiness, suggesting that Gdf6a/radar is involved in maintenance of vascular integrity. The transcription factor Runx1 is known to play a critical role in mammalian definitive hematopoiesis. When Runx1 expression domains and function were analyzed in zebrafish, the importance of this gene in definitive hematopoiesis was confirmed. However there was also evidence for a wider role, including involvement in vascular development and neuropoiesis. This work has laid the foundation for an ethylnitrosourea (ENU) mutagenesis screen based on runx1 whole-mount in situ hybridzation, that aims to identify genes operative in the runx1 pathway. An additional member of the Runx family, Runx3, is also involved in developmental hematopoiesis, with a function distinct from that of Runx1. We hypothesize that Runx1 and Runx3 form a continuum of transcriptional control within the hematopoietic system. An added attraction of zebrafish is that models of human disease can be generated, and we have shown that this system has potential for the study of Runx1-mediated leukemogenesis.

An in situ hybridization expression screen using a signal sequence trap system has been conducted in zebrafish to isolate cDNAs that encode secreted proteins. Random clones (secreted expressed sequence tags; sESTs) were sequenced from zebrafish embryonic (18-24 hr postfertilization) and adult kidney libraries. From the two RNA sources, 627 random sEST cDNAs were identified as being homologous or identical to known genes and 166 clones encode currently unidentified genes. The sESTs represent a broad range of enzymes and other regulatory molecules. Whole-mount in situ hybridization analysis was carried out by using antisense probes generated from 244 selected sESTs, and a range of expression patterns was obtained. Genetic mapping undertaken with sEST sequences demonstrated that assignment of map position was attainable by using 5' primers. The signal sequence trap system used in this work has yielded a range of cDNAs that encode secreted proteins and, together with analysis of patterns of expression and genetic mapping, has the potential to facilitate analysis of signaling pathways central to development and physiology., (Copyright 2001 Wiley-Liss, Inc.)

The Growth/differentiation factor (Gdf) 5, 6, 7 genes form a closely related subgroup belonging to the TGF-beta superfamily. In zebrafish, there are three genes that belong to the Gdf5, 6, 7 subgroup that have been named radar, dynamo, and contact. The genes radar and dynamo both encode proteins most similar to mouse GDF6. The orthologous identity of these genes on the basis of amino acid similarities has not been clear. We have identified gdf7, a fourth zebrafish gene belonging to the Gdf5, 6, 7 subgroup. To assign correct orthologies and to investigate the evolutionary relationships of the human, mouse, and zebrafish Gdf5, 6, 7 subgroup, we have compared genetic map positions of the zebrafish and mammalian genes. We have mapped zebrafish gdf7 to linkage group (LG) 17, contact to LG9, GDF6 to human chromosome (Hsa) 8 and GDF7 to Hsa2p. The radar and dynamo genes have been localized previously to LG16 and LG19, respectively. A comparison of syntenies shared among human, mouse, and zebrafish genomes indicates that gdf7 is the ortholog of mammalian GDF7/Gdf7. LG16 shares syntenic relationships with mouse chromosome (Mmu) 4, including Gdf6. Portions of LG16 and LG19 appear to be duplicate chromosomes, thus suggesting that radar and dynamo are both orthologs of Gdf6. Finally, the mapping data is consistent with contact being the zebrafish ortholog of mammalian GDF5/Gdf5.