Live imaging of Runx1 expression in the dorsal aorta tracks the emergence of blood progenitors from endothelial cells.

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.

A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents.

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.

Zebrafish runx1 promoter-EGFP transgenics mark discrete sites of definitive blood progenitors.

The transcription factor Runx1 is essential for the development of definitive hematopoietic stem cells (HSCs) during vertebrate embryogenesis and is transcribed from 2 promoters, P1 and P2, generating 2 major Runx1 isoforms. We have created 2 stable runx1 promoter zebrafish-transgenic lines that provide insight into the roles of the P1 and P2 isoforms during the establishment of definitive hematopoiesis. The Tg(runx1P1:EGFP) line displays fluorescence in the posterior blood island, where definitive erythromyeloid progenitors develop. The Tg(runx1P2:EGFP) line marks definitive HSCs in the aorta-gonad-mesonephros, with enhanced green fluorescent protein-labeled cells later populating the pronephros and thymus. This suggests that a function of runx1 promoter switching is associated with the establishment of discrete definitive blood progenitor compartments. These runx1 promoter-transgenic lines are novel tools for the study of Runx1 regulation and function in normal and malignant hematopoiesis. The ability to visualize and isolate fluorescently labeled HSCs should contribute to further elucidating the complex regulation of HSC development.

Visualization of embryonic lymphangiogenesis advances the use of the zebrafish model for research in cancer and lymphatic pathologies.

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.

Dual oxidase in the intestinal epithelium of zebrafish larvae has anti-bacterial properties.

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.

Intestinal differentiation in zebrafish requires Cdx1b, a functional equivalent of mammalian Cdx2.

BACKGROUND & AIMS: The ParaHox transcription factor Cdx2 is an essential determinant of intestinal phenotype in mammals throughout development, influencing gut function, homeostasis, and epithelial barrier integrity. Cdx2 expression demarcates the zones of intestinal stem cell proliferation in the adult gut, with deregulated expression implicated in intestinal metaplasia and cancer. However, in vivo analysis of these prospective roles has been limited because inactivation of Cdx2 in mice leads to preimplantation embryonic lethality. We used the zebrafish, a valuable model for studying gut development, to generate a system to further understanding of the role of Cdx2 in normal intestinal function and in disease states. METHODS: We isolated and characterized the zebrafish cdx1b ortholog and analyzed its function by antisense morpholino gene knockdown. RESULTS: We showed that zebrafish Cdx1b replaces the role of Cdx2 in gut development. Evolutionary studies have indicated that the zebrafish cdx2 loci were lost following the genome-wide duplication event that occurred in teleosts. Zebrafish Cdx1b is expressed exclusively in the developing intestine during late embryogenesis and regulates intestinal cell proliferation and terminal differentiation. CONCLUSIONS: This work established an in vivo system to explore further the activity of Cdx2 in the gut and its impact on processes such as inflammation and cancer.

Live cell imaging of zebrafish leukocytes.

Zebrafish are ideally suited for the live imaging of early immune cell compartments. Macrophages that initially appear on the yolk surface prior to the onset of circulation are the first functional immune cells within the embryo, predating the emergence of the first granulocytic cells-the heterophilic neutrophils. Both cell types have been shown in zebrafish to contribute to a robust early innate immune system, capable of clearing systemic infections and participating in wound healing. Early imaging of these cells within zebrafish relied on differential interference contrast (DIC) optics because of their superficial locations in the embryo and the optical transparency of embryonic tissues. Recently, the creation of a number of transgenic reporter lines possessing fluorescently marked myelomonocytic compartments provides the potential to live image these cells during the inflammatory response, in real-time, within a whole animal context. Live imaging during the different stages of inflammation using this expanding library of reporter lines, coupled with the ability to model aspects of human disease in the zebrafish system, have the potential to provide significant insights into inflammation and diseases associated with its dysregulation.

The zebrafish lysozyme C promoter drives myeloid-specific expression in transgenic fish.

BACKGROUND: How different immune cell compartments contribute to a successful immune response is central to fully understanding the mechanisms behind normal processes such as tissue repair and the pathology of inflammatory diseases. However, the ability to observe and characterize such interactions, in real-time, within a living vertebrate has proved elusive. Recently, the zebrafish has been exploited to model aspects of human disease and to study specific immune cell compartments using fluorescent reporter transgenic lines. A number of blood-specific lines have provided a means to exploit the exquisite optical clarity that this vertebrate system offers and provide a level of insight into dynamic inflammatory processes previously unavailable. RESULTS: We used regulatory regions of the zebrafish lysozyme C (lysC) gene to drive enhanced green fluorescent protein (EGFP) and DsRED2 expression in a manner that completely recapitulated the endogenous expression profile of lysC. Labeled cells were shown by co-expression studies and FACS analysis to represent a subset of macrophages and likely also granulocytes. Functional assays within transgenic larvae proved that these marked cells possess hallmark traits of myelomonocytic cells, including the ability to migrate to inflammatory sources and phagocytose bacteria. CONCLUSION: These reporter lines will have utility in dissecting the genetic determinants of commitment to the myeloid lineage and in further defining how lysozyme-expressing cells participate during inflammation.

The zebrafish retinoid-related orphan receptor (ror) gene family.

The retinoid-related orphan receptors Rora, b and c are highly conserved transcription factors belonging to the steroid hormone receptor superfamily. Mammalian ROR proteins perform key regulatory roles in a number of processes during embryonic development and in the adult including neurogenesis, bone metabolism and modulation of circadian rhythms. A more recent area of interest has been their roles in the development and function of the immune system. In particular, RORA has been implicated in the regulation of inflammatory cytokine production, and RORC has been shown to be essential in the development of the T lymphocyte repertoire and of secondary lymphoid organs. We cloned the zebrafish orthologs for the Ror gene family. Assignment of orthologies was supported by analysis of the phylogenetic relationships between zebrafish and other vertebrate Ror genes based on sequence similarities, and conserved syntenies with the human Ror gene loci.

An essential role for zebrafish Fgfrl1 during gill cartilage development.

The vertebrate craniofacial skeleton develops via a complex process involving signaling cascades in all three germ layers. Fibroblast growth factor (FGF) signaling is essential for several steps in pharyngeal arch development. In zebrafish, Fgf3 and Fgf8 in the mesoderm and hindbrain have an early role to pattern the pouch endoderm, influencing craniofacial integrity. Endodermal FGF signaling is required for the differentiation and survival of postmigratory neural crest cells that form the pharyngeal skeleton. We identify a novel role for zebrafish Fgf receptor-like 1a (Fgfrl1a) that is indispensable during gill cartilage development. We show that depletion of Fgfrl1a is sufficient to abolish cartilage derivatives of the ceratobranchials. Using an Fgfrl1a-deficient model, we analyzed expression of genes critical for chondrogenesis in the different compartments of the developing pharyngeal arch. Fgfrl1a-depleted animals demonstrate typical neural crest specification and migration to populate the arch primordia as well as normal pouch segmentation. However, in the absence of Fgfrl1a, larvae fail to express the transcription factor glial cells missing 2 (gcm2), a gene necessary for cartilage and gill filament formation, in the ectodermal lining of the branchial arches. In addition, two transcription factors essential for chondrogenesis, sox9a and runx2b, fail to express within the mesenchymal condensations of the branchial arches. A duplicate zebrafish gene, fgfrl1b, has now been identified. We show that Fgfrl1b is also required for proper formation of all ventral cartilage elements and acts cooperatively with Fgfrl1a during gill cartilage formation.

A whole animal chemical screen approach to identify modifiers of intestinal neutrophilic inflammation.

By performing two high-content small molecule screens on dextran sodium sulfate- and trinitrobenzene sulfonic acid-induced zebrafish enterocolitis models of inflammatory bowel disease, we have identified novel anti-inflammatory drugs from the John Hopkins Clinical Compound Library that suppress neutrophilic inflammation. Live imaging of neutrophil distribution was used to assess the level of acute inflammation and concurrently screen for off-target drug effects. Supporting the validity of our screening strategy, most of the anti-inflammatory drug hits were known antibiotics or anti-inflammatory agents. Novel hits included cholecystokinin (CCK) and dopamine receptor agonists. Using a pharmacological approach, we show that while CCK and dopamine receptor agonists alleviate enterocolitis-associated inflammation, receptor antagonists exacerbate inflammation in zebrafish. This work highlights the utility of small molecule screening in zebrafish enterocolitis models as a tool to identify novel bioactive molecules capable of modulating acute inflammation.

Pathways in blood and vessel development revealed through zebrafish genetics.

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.

Duplicate zebrafish runx2 orthologues are expressed in developing skeletal elements.

The differentiation of cells in the vertebrate skeleton is controlled by a precise genetic program. One crucial regulatory gene in the pathway encodes the transcription factor Runx2, which in mouse is required for differentiation of all osteoblasts and the proper development of a subset of hypertrophic chondrocytes. To explore the differentiation of skeletogenic cells in the model organism zebrafish (Danio rerio), we have identified two orthologues of the mammalian gene, runx2a and runx2b. Both genes share sequence homology and gene structure with the mammalian genes, and map to regions of the zebrafish genome displaying conserved synteny with the region where the human gene is localized. While both genes are expressed in developing skeletal elements, they show evidence of partial divergence in expression pattern, possibly explaining why both orthologues have been retained through teleost evolution.

Chemically induced intestinal damage models in zebrafish larvae.

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.

Immunoresponsive gene 1 augments bactericidal activity of macrophage-lineage cells by regulating ß-oxidation-dependent mitochondrial ROS production.

Evidence suggests the bactericidal activity of mitochondria-derived reactive oxygen species (mROS) directly contributes to killing phagocytozed bacteria. Infection-responsive components that regulate this process remain incompletely understood. We describe a role for the mitochondria-localizing enzyme encoded by Immunoresponsive gene 1 (IRG1) during the utilization of fatty acids as a fuel for oxidative phosphorylation (OXPHOS) and associated mROS production. In a zebrafish infection model, infection-responsive expression of zebrafish irg1 is specific to macrophage-lineage cells and is regulated cooperatively by glucocorticoid and JAK/STAT signaling pathways. Irg1-depleted macrophage-lineage cells are impaired in their ability to utilize fatty acids as an energy substrate for OXPHOS-derived mROS production resulting in defective bactericidal activity. Additionally, the requirement for fatty acid ß-oxidation during infection-responsive mROS production and bactericidal activity toward intracellular bacteria is conserved in murine macrophages. These results reveal IRG1 as a key component of the immunometabolism axis, connecting infection, cellular metabolism, and macrophage effector function.

Retinoic acid suppresses intestinal mucus production and exacerbates experimental enterocolitis.

Exposure to retinoids for the treatment of acne has been linked to the etiology of inflammatory bowel disease (IBD). The intestinal mucus layer is an important structural barrier that is disrupted in IBD. Retinoid-induced alteration of mucus physiology has been postulated as a mechanism linking retinoid treatment to IBD; however, there is little direct evidence for this interaction. The zebrafish larva is an emerging model system for investigating the pathogenesis of IBD. Importantly, this system allows components of the innate immune system, including mucus physiology, to be studied in isolation from the adaptive immune system. This study reports the characterization of a novel zebrafish larval model of IBD-like enterocolitis induced by exposure to dextran sodium sulfate (DSS). The DSS-induced enterocolitis model was found to recapitulate several aspects of the zebrafish trinitrobenzene-sulfonic-acid (TNBS)-induced enterocolitis model, including neutrophilic inflammation that was microbiota-dependent and responsive to pharmacological intervention. Furthermore, the DSS-induced enterocolitis model was found to be a tractable model of stress-induced mucus production and was subsequently used to identify a role for retinoic acid (RA) in suppressing both physiological and pathological intestinal mucin production. Suppression of mucin production by RA increased the susceptibility of zebrafish larvae to enterocolitis when challenged with enterocolitic agents. This study illustrates a direct effect of retinoid administration on intestinal mucus physiology and, subsequently, on the progression of intestinal inflammation.

Infection-responsive expansion of the hematopoietic stem and progenitor cell compartment in zebrafish is dependent upon inducible nitric oxide.

Hematopoietic stem cells (HSCs) are rare multipotent cells that contribute to all blood lineages. During inflammatory stress, hematopoietic stem and progenitor cells (HSPCs) can be stimulated to proliferate and differentiate into the required immune cell lineages. Manipulating signaling pathways that alter HSPC capacity holds great promise in the treatment of hematological malignancies. To date, signaling pathways that influence HSPC capacity, in response to hematopoietic stress, remain largely unknown. Using a zebrafish model of demand-driven granulopoiesis to explore the HSPC response to infection, we present data supporting a model where the zebrafish ortholog of the cytokine-inducible form of nitric oxide synthase (iNOS/NOS2) Nos2a acts downstream of the transcription factor C/ebpß to control expansion of HSPCs following infection. These results provide new insights into the reactive capacity of HSPCs and how the blood system is "fine-tuned" in response to inflammatory stress.

The inflammatory bowel disease (IBD) susceptibility genes NOD1 and NOD2 have conserved anti-bacterial roles in zebrafish.

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.

Topographical distribution of antimicrobial genes in the zebrafish intestine.

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 ß-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.

Expression of zebrafish cxcl8 (interleukin-8) and its receptors during development and in response to immune stimulation.

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.