Phil Crosier

Vascular Lipid Accumulation, Lipoprotein Oxidation, and Macrophage Lipid Uptake in Hypercholesterolemic Zebrafish

Lipid accumulation in arteries induces vascular inflammation and atherosclerosis, the major cause of heart attack and stroke in humans. Extreme hyperlipidemia induced in mice and rabbits enables modeling many aspects of human atherosclerosis, but microscopic examination of plaques is possible only postmortem. Here we report that feeding adult zebrafish (Danio rerio) a high-cholesterol diet (HCD) resulted in hypercholesterolemia, remarkable lipoprotein oxidation, and fatty streak formation in the arteries. Feeding an HCD supplemented with a fluorescent cholesteryl ester to optically transparent fli1:EGFP zebrafish larvae in which endothelial cells express green fluorescent protein (GFP), and using confocal microscopy enabled monitoring vascular lipid accumulation and the endothelial cell layer disorganization and thickening in a live animal. The HCD feeding also increased leakage of a fluorescent dextran from the blood vessels. Administering ezetimibe significantly diminished the HCD-induced endothelial cell layer thickening and improved its barrier function. Feeding HCD to lyz:DsRed2 larvae in which macrophages and granulocytes express DsRed resulted in the accumulation of fluorescent myeloid cells in the vascular wall. Using a fluorogenic substrate for phospholipase A2 (PLA2), we observed an increased vascular PLA2 activity in live HCD-fed larvae compared to control larvae. Furthermore, by transplanting genetically modified murine cells into HCD-fed larvae, we demonstrated that toll-like receptor-4 was required for efficient in vivo lipid uptake by macrophages. These results suggest that the novel zebrafish model is suitable for studying temporal characteristics of certain inflammatory processes of early atherogenesis and the in vivo function of vascular cells.

Zebrafish granulocyte colony-stimulating factor receptor signaling promotes myelopoiesis and myeloid cell migration

Granulocyte colony-stimulating factor receptor (GCSFR) signaling participates in the production of neutrophilic granulocytes during normal hematopoietic development, with a particularly important role during emergency hematopoiesis. This study describes the characterization of the zebrafish gcsf and gcsfr genes, which showed broad conservation and similar regulation to their mammalian counterparts. Morpholino-mediated knockdown of gcsfr and overexpression of gcsf revealed the presence of an anterior population of myeloid cells during primitive hematopoiesis that was dependent on GCSF/GCSFR for development and migration. This contrasted with a posterior domain that was largely independent of this pathway. Definitive myelopoiesis was also partially dependent on a functional GCSF/GCSFR pathway. Injection of bacterial lipopolysaccharide elicited significant induction of gcsf expression and emergency production of myeloid cells, which was abrogated by gcsfr knockdown. Collectively, these data demonstrate GCSF/GCSFR to be a conserved signaling system for facilitating the production of multiple myeloid cell lineages in both homeostatic and emergency conditions, as well as for early myeloid cell migration, establishing a useful experimental platform for further dissection of this pathway.

Transgenic zebrafish reporter lines reveal conserved Toll-like receptor signaling potential in embryonic myeloid leukocytes and adult immune cell lineages

The immune response of a host to an invading pathogen is dependent on the capacity of its immune cell compartment to recognize highly conserved pathogen components using an ancient class of pattern recognition receptors known as Toll‐like receptors (TLRs). Initiation of TLR‐mediated signaling results in the induction of proinflammatory cytokines that help govern the scale and duration of any ensuing response. Specificity for TLR signaling is, in part, a result of the differential recruitment of intracellular adaptor molecules. Of these, MyD88 is required for the majority of TLR signaling. Zebrafish have been shown to possess TLRs and adaptor molecules throughout early development, including MyD88, strongly suggesting conservation of this ancient defense mechanism. However, information about which embryonic cells/tissues possess this conserved signaling potential is lacking. To help define which embryonic cells, in particular, those of the innate immune system, have the potential for MyD88‐dependent, TLR‐mediated signaling, we generated transgenic reporter lines using regulatory elements of the myd88 gene to drive the fluorescent reporters enhanced GFP and Discosoma red fluorescent protein 2 within live zebrafish. These lines possess fluorescently marked cells/tissues consistent with endogenous myd88 expression, including a subset of myeloid leukocytes. These innate immune cells were confirmed to express other TLR adaptors including Mal, trif, and Sarm. Live wound‐healing and infection assays validated the potential of these myd88‐expressing leukocytes to participate in immune responses. These lines will provide a valuable resource for further resolving the contribution of MyD88 to early vertebrate immunity.

Analysis of WASp function during the wound inflammatory response - live-imaging studies in zebrafish larvae

Wiskott-Aldrich syndrome protein (WASp) is haematopoietically restricted, and is the causative protein underlying a severe human disorder that can lead to death due to immunodeficiency and haemorrhaging. Much is known about the biochemistry of WASp and the migratory capacity of WASp-defective cells in vitro, but in vivo studies of immune-cell behaviour are more challenging. Using the translucency of zebrafish larvae, we live-imaged the effects of morpholino knockdown of WASp1 (also known as Was) on leukocyte migration in response to a wound. In embryos at 22 hours post-fertilisation, primitive macrophages were impaired in their migration towards laser wounds. Once a circulatory system had developed, at 3 days post-fertilisation, we observed significantly reduced recruitment of neutrophils and macrophages to ventral fin wounds. Cell-tracking studies indicated that fewer leukocytes leave the vessels adjacent to a wound and those that do exhibit impaired navigational capacity. Their cell morphology appears unaltered but their choice of leading-edge pseudopodia is more frequently incorrect, leading to impaired chemotaxis. We also identified two zebrafish mutants in WASp1 by TILLING, one of which was in the WIP-binding domain that is the hotspot for human lesions, and mutants exhibited the same deficiencies in wound inflammation and thrombus formation as WASp1 morphants.

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.

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.

Editorial: Maintaining the balance—fishing for drugs to treat persistent neutrophilic inflammation

Neutrophils, the most abundant white blood cell within the circulation, act as early cellular effectors of innate immunity by engulfing, killing, and digesting invading pathogens. During acute inflammation, these short-lived polymorphonuclear cells are the first to infiltrate affected tissue and after helping clear bacterial pathogens, are removed, in part, by apoptosis. This initiation of programmed neutrophil cell death marks the resolution phase of inflammation when tissue macrophages arrive to help clear the dead neutrophil debris [1, 2]. During inflammation, neutrophils provide a potent but nonspecific defense against invading pathogens, including the release of enzymes and reactive oxygen species that can result in unintended damage to host tissues. This potential to cause host tissue injury is normally attenuated by their limited lifespan, a result of an almost constitutively active apoptotic program. Programmed neutrophil cell death is an essential mechanism through which neutrophil number is balanced under normal physiological conditions and during the stressed state of inflammation. Under physiological conditions, constitutive neutrophil death ensures that homeostatic numbers of neutrophils are maintained. During inflammation, neutrophil death needs to be managed precisely to ensure preservation of immune function yet prevent/minimize their potential to damage host tissues, which may result in scarring and loss of normal organ function. Advancing understanding of how neutrophilic inflammation resolves is likely to reveal new therapeutic strategies to help manage diseases associated with inappropriate persistence of neutrophil activity during inflammation. Identifying novel drugs that possess pro-resolution properties represents an attractive strategy for the treatment of disease processes characterized by persistent neutrophil-mediated inflammation. The study presented here by Loynes and co-workers [3] highlights how the physical traits and genetic tractability of the zebrafish model system can be exploited to create a drug discovery platform to help identify novel chemical modulators of the inflammatory response. The author’s previous work helped establish a live imaging platform of neutrophilic inflammation by generating a neutrophil-specific mpx:GFP transgenic reporter line [3]. These transgenic zebrafish permit direct observation of fluorescent neutrophil behavior within an intact, transparent, whole vertebrate setting. The optical transparency of zebrafish embryos, coupled with the relative ease of generating transgenic reporter lines with fluorescently marked immune cell compartments, has helped maneuver this vertebrate to the forefront of efforts to liveimage innate immune cell behavior during inflammation. The zebrafish immune system shows remarkable conservation with that of mammals in the cell types represented and their developmental ontogeny. Zebrafish possess myeloid leukocytes, including heterophilic granulocytes/neutrophils, eosinophil/basophil-like cells, monocytes/macrophages, and a cell type resembling mast cells that help orchestrate a highly competent innate immune system [4–6]. Multiple studies have assessed the contribution of these lineages to the inflammatory response during wound healing and bacterial infection. Furthermore, the later onset of a functional adaptive immune system during zebrafish development has afforded researchers the ability to study the contribution of innate immunity exclusively to the inflammatory response. Many of these studies have given unique insights into the behavior of these cells during inflammation by viewing them within an intact, whole animal setting at single-cell resolution. Despite providing evidence of retrograde chemotaxis as a mechanism for the resolution of neutrophilic inflammation in response to zebrafish tail-fin injury, previous studies have failed to identify neutrophil apoptosis during these liveimaging experiments [7, 8]. This has brought into question whether such a mechanism operates in zebrafish. Using a similar live-imaging approach, the current study was able to observe rare events among neutrophils, as marked with the mpx:GFP transgene. The authors demonstrated cells with features typical of apoptosis, including the cessation of move-

Inflammatory cytokines provide both infection-responsive and developmental signals for blood development: Lessons from the zebrafish.

Hematopoietic stem cells (HSCs) are rare, largely dormant, long-lived cells that are capable of establishing and regenerating all mature blood cell lineages throughout the life of the host. Given their therapeutic importance, understanding factors that regulate HSC development and influence HSC proliferation and differentiation is of great interest. Exploring HSC biology through the lens of infection has altered our traditional view of the HSC. The HSC can now be considered a component of the immune response to infection. In response to inflammatory cytokine signaling, HSCs enhance their proliferative state and contribute to the production of in-demand blood cell lineages. Similar cytokine signaling pathways also participate during embryonic HSC production. With its highly conserved hematopoietic system and experimental tractability, the zebrafish model has made significant contributions to the hematopoietic field. In particular, the zebrafish system has been ideally suited to help reveal the molecular and cellular mechanisms underlying HSC development. This review highlights recent zebrafish studies that have uncovered new mechanistic insights into how inflammatory signaling pathways influence HSC behavior during infection and HSC production within the embryo.

A nursery that improves zebrafish fry survival.

Publisher Summary This chapter reviews existing methods used for raising zebrafish from the larval stage until 3–4 weeks of development and describes a new system developed to ensure consistently high rates of survival during this developmental period. The need to design a new approach stemmed from the low rates of fry survival obtained by using existing methods. The need to maximize the survival of zebrafish embryos and fry is fundamental to all research that uses zebrafish as a model system. With the increasing number of mutants and transgenics being developed by zebrafish investigators, there is a need for a system that provides consistently high survival rates of young fry. It is widely recognized that a critical period in the life of a zebrafish is in the 2 weeks following the absorption of the yolk. Two elements are essential to ensuring survival of zebrafish fry during this period: (1) an adequate supply of nutritionally high-quality feed of the appropriate particle size and (2) optimal water quality in the fry tank. The chapter also aims to develop an approach that delivers consistently high fry survival rates and minimized stress to fry during the transition phase from endogenous to exogenous feeding. In designing this system, important factors considered are minimizing physical contact with the fry and providing consistently high-quality water at a stable pH, temperature, and conductivity. There are perceived benefits of providing live feed to young fry. The best live feed to offer younger fry is rotifers, Brachionus plicatilis in particular, which are approximately 250 μm in length.

13-P017 Live imaging demand-driven myelopoiesis

ulate the transition between self-renewal and differentiation stage in different systems, and both are associated with the maintenance of the progenitor state. The general question that arises is how those signals are integrated within otic progenitors so that they result in a precise cell diversification. We have studied the interactions between Sox2 and Notch pathways in otic neurosensory progenitors. Experiments combined electroporation of c-Sox2 and hJag1 with in vitro explants, qRT-PCR and in situ hybridisation analysis. The results show that: (1) Sox2 and Ser1 are co-expressed in neurosensory otic progenitors, throughout inner ear development. (2) Forced expression of hJag1 was able to extend the domain of Sox2 expression in a nonautonomous manner, the effect being Notch-dependent. On the contrary, Sox2 did not affect Ser1 expression. (3) hJag1 activated the Notch pathway and induced typical Notch targets, including Hey1 and Hes1. (4) c-Sox2 overexpression resulted the up-regulation of Notch targets, particularly Hey1. The Sox2-induced Hey1 up-regulation was Notch-dependent, as shown by its blockade by DAPT. Taken together, the results suggest that there is a cooperation between Sox and Notch pathways in the maintenance of the commitment and self-renewal state of the proneural domain. Supported by MCINN, Spain and FCT, Portugal.