Plexin D1 negatively regulates zebrafish lymphatic development.

Lymphangiogenesis is a dynamic process that involves the directed migration of lymphatic endothelial cells (LECs) to form lymphatic vessels. The molecular mechanisms that underpin lymphatic vessel patterning are not fully elucidated and, to date, no global regulator of lymphatic vessel guidance is known. In this study, we identify the transmembrane cell signalling receptor Plexin D1 (Plxnd1) as a negative regulator of both lymphatic vessel guidance and lymphangiogenesis in zebrafish. plxnd1 is expressed in developing lymphatics and is required for the guidance of both the trunk and facial lymphatic networks. Loss of plxnd1 is associated with misguided intersegmental lymphatic vessel growth and aberrant facial lymphatic branches. Lymphatic guidance in the trunk is mediated, at least in part, by the Plxnd1 ligands, Semaphorin 3AA and Semaphorin 3C. Finally, we show that Plxnd1 normally antagonises Vegfr/Erk signalling to ensure the correct number of facial LECs and that loss of plxnd1 results in facial lymphatic hyperplasia. As a global negative regulator of lymphatic vessel development, the Sema/Plxnd1 signalling pathway is a potential therapeutic target for treating diseases associated with dysregulated lymphatic growth.

Zebrafish facial lymphatics develop through sequential addition of venous and non-venous progenitors.

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.

Hydrogen-Borrowing Alkylation of 1,2-Amino Alcohols in the Synthesis of Enantioenriched γ-Aminobutyric Acids.

For the first time we have been able to employ enantiopure 1,2-amino alcohols derived from abundant amino acids in C-C bond-forming hydrogen-borrowing alkylation reactions. These reactions are facilitated by the use of the aryl ketone Ph*COMe. Racemisation of the amine stereocentre during alkylation can be prevented by the use of sub-stoichiometric base and protection of the nitrogen with a sterically hindered triphenylmethane (trityl) or benzyl group. The Ph* and trityl groups are readily cleaved in one pot to give γ-aminobutyric acid (GABA) products as their HCl salts without further purification. Both steps may be performed in sequence without isolation of the hydrogen-borrowing intermediate, removing the need for column chromatography.

Quantitative characterization of the X-ray beam at the Australian Synchrotron Imaging and Medical Beamline (IMBL).

A critical early phase for any synchrotron beamline involves detailed testing, characterization and commissioning; this is especially true of a beamline as ambitious and complex as the Imaging & Medical Beamline (IMBL) at the Australian Synchrotron. IMBL staff and expert users have been performing precise experiments aimed at quantitative characterization of the primary polychromatic and monochromatic X-ray beams, with particular emphasis placed on the wiggler insertion devices (IDs), the primary-slit system and any in vacuo and ex vacuo filters. The findings from these studies will be described herein. These results will benefit IMBL and other users in the future, especially those for whom detailed knowledge of the X-ray beam spectrum (or `quality') and flux density is important. This information is critical for radiotherapy and radiobiology users, who ultimately need to know (to better than 5%) what X-ray dose or dose rate is being delivered to their samples. Various correction factors associated with ionization-chamber (IC) dosimetry have been accounted for, e.g. ion recombination, electron-loss effects. A new and innovative approach has been developed in this regard, which can provide confirmation of key parameter values such as the magnetic field in the wiggler and the effective thickness of key filters. IMBL commenced operation in December 2008 with an Advanced Photon Source (APS) wiggler as the (interim) ID. A superconducting multi-pole wiggler was installed and operational in January 2013. Results are obtained for both of these IDs and useful comparisons are made. A comprehensive model of the IMBL has been developed, embodied in a new computer program named spec.exe, which has been validated against a variety of experimental measurements. Having demonstrated the reliability and robustness of the model, it is then possible to use it in a practical and predictive manner. It is hoped that spec.exe will prove to be a useful resource for synchrotron science in general, and for hard X-ray beamlines, whether they are based on bending magnets or insertion devices, in particular. In due course, it is planned to make spec.exe freely available to other synchrotron scientists.

Rasip1 regulates vertebrate vascular endothelial junction stability through Epac1-Rap1 signaling.

Establishment and stabilization of endothelial tubes with patent lumens is vital during vertebrate development. Ras-interacting protein 1 (RASIP1) has been described as an essential regulator of de novo lumenogenesis through modulation of endothelial cell (EC) adhesion to the extracellular matrix (ECM). Here, we show that in mouse and zebrafish embryos, Rasip1-deficient vessels transition from an angioblast cord to a hollow tube, permit circulation of primitive erythrocytes, but ultimately collapse, leading to hemorrhage and embryonic lethality. Knockdown of RASIP1 does not alter EC-ECM adhesion, but causes cell-cell detachment and increases permeability of EC monolayers in vitro. We also found that endogenous RASIP1 in ECs binds Ras-related protein 1 (RAP1), but not Ras homolog gene family member A or cell division control protein 42 homolog. Using an exchange protein directly activated by cyclic adenosine monophosphate 1 (EPAC1)-RAP1-dependent model of nascent junction formation, we demonstrate that a fraction of the RASIP1 protein pool localizes to cell-cell contacts. Loss of RASIP1 phenocopies loss of RAP1 or EPAC1 in ECs by altering junctional actin organization, localization of the actin-bundling protein nonmuscle myosin heavy chain IIB, and junction remodeling. Our data show that RASIP1 regulates the integrity of newly formed blood vessels as an effector of EPAC1-RAP1 signaling.

Microinjection of ß-glucan Into Larval Zebrafish (Danio rerio) for the Assessment of a Trained-Like Immunity Phenotype.

The innate immune system can remember previous inflammatory insults, enabling long-term heightened responsiveness to secondary immune challenges in a process termed "trained immunity." Trained innate immune cells undergo metabolic and epigenetic remodelling and, upon a secondary challenge, provide enhanced protection with therapeutic potential. Trained immunity has largely been studied in innate immune cells in vitro or following ex vivo re-stimulation where the primary insult is typically injected into a mouse, adult zebrafish, or human. While highly informative, there is an opportunity to investigate trained immunity entirely in vivo within an unperturbed, intact whole organism. The exclusively innate immune response of larval zebrafish offers an attractive system to model trained immunity. Larval zebrafish have a functional innate immune system by 2 days post fertilisation (dpf) and are amenable to high-resolution, high-throughput analysis. This, combined with their optical transparency, conserved antibacterial responses, and availability of transgenic reporter lines, makes them an attractive alternative model to study trained immunity in vivo. We have devised a protocol where ß-glucan (one of the most widely used experimental triggers of trained immunity) is systemically delivered into larval zebrafish using microinjection to stimulate a trained-like phenotype. Following stimulation, larvae are assessed for changes in gene expression, which indicate the stimulatory effect of ß-glucan. This protocol describes a robust delivery method of one of the gold standard stimulators of trained immunity into a model organism that is highly amenable to several non-invasive downstream analyses. Key features • This protocol outlines the delivery of one of the most common experimental stimulators of trained immunity into larval zebrafish. • The protocol enables the assessment of a trained-like phenotype in vivo. • This protocol can be applied to transgenic or mutant zebrafish lines to investigate cells or genes of interest in response to ß-glucan stimulation.

Infection-experienced HSPCs protect against infections by generating neutrophils with enhanced mitochondrial bactericidal activity.

Hematopoietic stem and progenitor cells (HSPCs) respond to infection by proliferating and generating in-demand neutrophils through a process called emergency granulopoiesis (EG). Recently, infection-induced changes in HSPCs have also been shown to underpin the longevity of trained immunity, where they generate innate immune cells with enhanced responses to subsequent microbial threats. Using larval zebrafish to live image neutrophils and HSPCs, we show that infection-experienced HSPCs generate neutrophils with enhanced bactericidal functions. Transcriptomic analysis of EG neutrophils uncovered a previously unknown function for mitochondrial reactive oxygen species in elevating neutrophil bactericidal activity. We also reveal that driving expression of zebrafish C/EBPß within infection-naïve HSPCs is sufficient to generate neutrophils with similarly enhanced bactericidal capacity. Our work suggests that this demand-adapted source of neutrophils contributes to trained immunity by providing enhanced protection toward subsequent infections. Manipulating demand-driven granulopoiesis may provide a therapeutic strategy to boost neutrophil function and treat infectious disease.

Analysis and interpretation of the first monochromatic X-ray tomography data collected at the Australian Synchrotron Imaging and Medical beamline.

The first monochromatic X-ray tomography experiments conducted at the Imaging and Medical beamline of the Australian Synchrotron are reported. The sample was a phantom comprising nylon line, Al wire and finer Cu wire twisted together. Data sets were collected at four different X-ray energies. In order to quantitatively account for the experimental values obtained for the Hounsfield (or CT) number, it was necessary to consider various issues including the point-spread function for the X-ray imaging system and harmonic contamination of the X-ray beam. The analysis and interpretation of the data includes detailed considerations of the resolution and efficiency of the CCD detector, calculations of the X-ray spectrum prior to monochromatization, allowance for the response of the double-crystal Si monochromator used (via X-ray dynamical theory), as well as a thorough assessment of the role of X-ray phase-contrast effects. Computer simulations relating to the tomography experiments also provide valuable insights into these important issues. It was found that a significant discrepancy between theory and experiment for the Cu wire could be largely resolved in terms of the effect of the point-spread function. The findings of this study are important in respect of any attempts to extract quantitative information from X-ray tomography data, across a wide range of disciplines, including materials and life sciences.

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.

Towards a new model of trained immunity: Exposure to bacteria and ß-glucan protects larval zebrafish against subsequent infections.

Once thought to be a feature exclusive to lymphocyte-driven adaptive immunity, immune memory has also been shown to operate as part of the innate immune system following infection to provide an elevated host response to subsequent pathogenic challenge. This evolutionarily conserved process, termed 'trained immunity', enables cells of the innate immune system to 'remember' previous pathogen encounters and mount stronger responses to the same, or different, pathogens after returning to a non-activated state. Here we show that challenging larval zebrafish, that exclusively rely on innate immunity, with live or heat-killed Salmonella typhimurium provides protection to subsequent infection with either Salmonella typhimurium or Streptococcus iniae, that lasts for at least 12 days. We also show that larvae injected with ß-glucan, the well-known trigger of trained immunity, demonstrate enhanced survival to similar live bacterial infections, a phenotype supported by increased cxcl8 expression and neutrophil recruitment to the infection site. These results support the conservation of a trained immunity-like phenotype in larval zebrafish and provide a foundation to exploit the experimental attributes of larval zebrafish to further understand this form of immunological memory.

A New Transgenic Line for Rapid and Complete Neutrophil Ablation.

Zebrafish lines expressing nitroreductase (NTR) in specific cell compartments, which sensitizes those cells to metronidazole (MTZ)-mediated ablation, have proven extremely useful for studying tissue regeneration and investigating cell function. In contrast to many cells, neutrophils are comparatively resistant to the NTR/MTZ targeted ablation strategy. Recently, a rationally engineered variant of NTR (NTR 2.0) has been described that exhibits greatly improved MTZ-mediated ablation efficacy in zebrafish. We show that a transgenic line with neutrophil-restricted expression of NTR 2.0 demonstrates complete neutrophil ablation, with an MTZ dose 100-fold less than current treatment regimens, and with treatment durations as short as 5 h.

Uricase-Deficient Larval Zebrafish with Elevated Urate Levels Demonstrate Suppressed Acute Inflammatory Response to Monosodium Urate Crystals and Prolonged Crystal Persistence.

Gout is caused by elevated serum urate leading to the deposition of monosodium urate (MSU) crystals that can trigger episodes of acute inflammation. Humans are sensitive to developing gout because they lack a functional urate-metabolizing enzyme called uricase/urate oxidase (encoded by the UOX gene). A hallmark of long-standing disease is tophaceous gout, characterized by the formation of tissue-damaging granuloma-like structures ('tophi') composed of densely packed MSU crystals and immune cells. Little is known about how tophi form, largely due to the lack of suitable animal models in which the host response to MSU crystals can be studied in vivo long-term. We have previously described a larval zebrafish model of acute gouty inflammation where the host response to microinjected MSU crystals can be live imaged within an intact animal. Although useful for modeling acute inflammation, crystals are rapidly cleared following a robust innate immune response, precluding analysis at later stages. Here we describe a zebrafish uox null mutant that possesses elevated urate levels at larval stages. Uricase-deficient 'hyperuricemic' larvae exhibit a suppressed acute inflammatory response to MSU crystals and prolonged in vivo crystal persistence. Imaging of crystals at later stages reveals that they form granuloma-like structures dominated by macrophages. We believe that uox-/- larvae will provide a useful tool to explore the transition from acute gouty inflammation to tophus formation, one of the remaining mysteries of gout pathogenesis.

Extension of hydrogen borrowing alkylation reactions for the total synthesis of (-)-γ-lycorane.

The total synthesis of (-)-γ-lycorane (10 steps) and synthesis of (±)-γ-lycorane (8 steps) was completed from cyclohexenone. A new two step hydrogen borrowing alkylation of an aziridinyl alcohol, coupled with a Ph* (Me5C6) deprotection/cyclisation procedure was developed for de novo formation of the fused 6,5 heterocyclic ring. This work is one of the first examples of hydrogen borrowing C-C bond formation being used as a key step in a total synthesis project.

Quantifying lung aeration in neonatal lambs at birth using lung ultrasound.

Background: Lung ultrasound (LUS) is a safe and non-invasive tool that can potentially assess regional lung aeration in newborn infants and reduce the need for X-ray imaging. LUS produces images with characteristic artifacts caused by the presence of air in the lung, but it is unknown if LUS can accurately detect changes in lung air volumes after birth. This study compared LUS images with lung volume measurements from high-resolution computed tomography (CT) scans to determine if LUS can accurately provide relative measures of lung aeration. Methods: Deceased near-term newborn lambs (139 days gestation, term ∼148 days) were intubated and the chest imaged using LUS (bilaterally) and phase contrast x-ray CT scans at increasing static airway pressures (0-50 cmH2O). CT scans were analyzed to calculate regional air volumes and correlated with measures from LUS images. These measures included (i) LUS grade; (ii) brightness (mean and coefficient of variation); and (iii) area under the Fourier power spectra within defined frequency ranges. Results: All LUS image analysis techniques correlated strongly with air volumes measured by CT (p < 0.01). When imaging statistics were combined in a multivariate linear regression model, LUS predicted the proportion of air in the underlying lung with moderate accuracy (95% prediction interval ± 22.15%, r 2 = 0.71). Conclusion: LUS can provide relative measures of lung aeration after birth in neonatal lambs. Future studies are needed to determine if LUS can also provide a simple means to assess air volumes and individualize aeration strategies for critically ill newborns in real time.

Zebrafish heat shock protein a4 genes in the intestinal epithelium are up-regulated during inflammation.

A number of heat shock proteins (HSPs), including Hsp70 and Hsp110, function as molecular chaperones within intestinal epithelial cells that line the mammalian digestive system. HSPs confer cellular protection against environmental stress induced by chemical toxins or pathogens. There is interest in how members of this protein family might influence the progression of inflammatory bowel disease. Using the zebrafish model system, we report the expression of the duplicated hspa4 genes within the intestinal epithelium. The hspa4 genes belong to the Hsp110 family. We show that under inflammatory stress conditions within the gut, expression of these genes is up-regulated in a similar manner to that previously observed for mammalian Hsp70. Because of the amenability of the zebrafish to whole-animal screening protocols, the hspa4 genes could be used as effective read-outs for genetic, chemical and environmental factors that might influence intestinal inflammation.

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.

First experiments on the Australian Synchrotron Imaging and Medical beamline, including investigations of the effective source size in respect of X-ray imaging.

The Imaging and Medical beamline at the Australian Synchrotron achieved ;first light' in December 2008. Here, the first experiments performed on the beamline are reported, which involved both X-ray imaging and tomography studies for a range of samples. The use of a plastic-edge phantom for quantitative measurements of contrast and resolution proved to be very instructive and helped to confirm certain parameter values such as the effective horizontal source size, detector resolution and average X-ray energy for the polychromatic beam.

The Diverse Roles of Phagocytes During Bacterial and Fungal Infections and Sterile Inflammation: Lessons From Zebrafish.

The immediate and natural reaction to both infectious challenges and sterile insults (wounds, tissue trauma or crystal deposition) is an acute inflammatory response. This inflammatory response is mediated by activation of the innate immune system largely comprising professional phagocytes (neutrophils and macrophages). Zebrafish (danio rerio) larvae possess many advantages as a model organism, including their genetic tractability and highly conserved innate immune system. Exploiting these attributes and the live imaging potential of optically transparent zebrafish larvae has greatly contributed to our understanding of how neutrophils and macrophages orchestrate the initiation and resolution phases of inflammatory responses. Numerous bacterial and fungal infection models have been successfully established using zebrafish as an animal model and studies investigating neutrophil and macrophage behavior to sterile insults have also provided unique insights. In this review we highlight how examining the larval zebrafish response to specific bacterial and fungal pathogens has uncovered cellular and molecular mechanisms behind a variety of phagocyte responses, from those that protect the host to those that are detrimental. We also describe how modeling sterile inflammation in larval zebrafish has provided an opportunity to dissect signaling pathways that control the recruitment, and fate, of phagocytes at inflammatory sites. Finally, we briefly discuss some current limitations, and opportunities to improve, the zebrafish model system for studying phagocyte biology.