Transgenic zebrafish larvae as a non-rodent alternative model to assess pro-inflammatory (neutrophil) responses to nanomaterials.

Hazard studies for nanomaterials (NMs) commonly assess whether they activate an inflammatory response. Such assessments often rely on rodents, but alternative models are needed to support the implementation of the 3Rs principles. Zebrafish (Danio rerio) offer a viable alternative for screening NM toxicity by investigating inflammatory responses. Here, we used non-protected life stages of transgenic zebrafish (Tg(mpx:GFP)i114) with fluorescently-labeled neutrophils to assess inflammatory responses to silver (Ag) and zinc oxide (ZnO) NMs using two approaches. Zebrafish were exposed to NMs via water following a tail fin injury, or NMs were microinjected into the otic vesicle. Zebrafish were exposed to NMs at 3 days post-fertilization (dpf) and neutrophil accumulation at the injury or injection site was quantified at 0, 4, 6, 8, 24, and 48 h post-exposure. Zebrafish larvae were also exposed to fMLF, LTB4, CXCL-8, C5a, and LPS to identify a suitable positive control for inflammation induction. Aqueous exposure to Ag and ZnO NMs stimulated an enhanced and sustained neutrophilic inflammatory response in injured zebrafish larvae, with a greater response observed for Ag NMs. Following microinjection, Ag NMs stimulated a time-dependent neutrophil accumulation in the otic vesicle which peaked at 48 h. LTB4 was identified as a positive control for studies investigating inflammatory responses in injured zebrafish following aqueous exposure, and CXCL-8 for microinjection studies that assess responses in the otic vesicle. Our findings support the use of transgenic zebrafish to rapidly screen the pro-inflammatory effects of NMs, with potential for wider application in assessing chemical safety (e.g. pharmaceuticals).

Macrophages trigger cardiomyocyte proliferation by increasing epicardial vegfaa expression during larval zebrafish heart regeneration.

Cardiac injury leads to the loss of cardiomyocytes, which are rapidly replaced by the proliferation of the surviving cells in zebrafish, but not in mammals. In both the regenerative zebrafish and non-regenerative mammals, cardiac injury induces a sustained macrophage response. Macrophages are required for cardiomyocyte proliferation during zebrafish cardiac regeneration, but the mechanisms whereby macrophages facilitate this crucial process are fundamentally unknown. Using heartbeat-synchronized live imaging, RNA sequencing, and macrophage-null genotypes in the larval zebrafish cardiac injury model, we characterize macrophage function and reveal that these cells activate the epicardium, inducing cardiomyocyte proliferation. Mechanistically, macrophages are specifically recruited to the epicardial-myocardial niche, triggering the expansion of the epicardium, which upregulates vegfaa expression to induce cardiomyocyte proliferation. Our data suggest that epicardial Vegfaa augments a developmental cardiac growth pathway via increased endocardial notch signaling. The identification of this macrophage-dependent mechanism of cardiac regeneration highlights immunomodulation as a potential strategy for enhancing mammalian cardiac repair.

Selective Cdk9 inhibition resolves neutrophilic inflammation and enhances cardiac regeneration in larval zebrafish.

Sustained neutrophilic inflammation is detrimental for cardiac repair and associated with adverse outcomes following myocardial infarction (MI). An attractive therapeutic strategy to treat MI is to reduce or remove infiltrating neutrophils to promote downstream reparative mechanisms. CDK9 inhibitor compounds enhance the resolution of neutrophilic inflammation; however, their effects on cardiac repair/regeneration are unknown. We have devised a cardiac injury model to investigate inflammatory and regenerative responses in larval zebrafish using heartbeat-synchronised light-sheet fluorescence microscopy. We used this model to test two clinically approved CDK9 inhibitors, AT7519 and flavopiridol, examining their effects on neutrophils, macrophages and cardiomyocyte regeneration. We found that AT7519 and flavopiridol resolve neutrophil infiltration by inducing reverse migration from the cardiac lesion. Although continuous exposure to AT7519 or flavopiridol caused adverse phenotypes, transient treatment accelerated neutrophil resolution while avoiding these effects. Transient treatment with AT7519, but not flavopiridol, augmented wound-associated macrophage polarisation, which enhanced macrophage-dependent cardiomyocyte number expansion and the rate of myocardial wound closure. Using cdk9-/- knockout mutants, we showed that AT7519 is a selective CDK9 inhibitor, revealing the potential of such treatments to promote cardiac repair/regeneration.

PET/CT Technology in Adult Zebrafish: A Pilot Study Toward Live Longitudinal Imaging.

Decades of research have confirmed the beneficial and advantageous use of zebrafish (Danio rerio) as a model of human disease in biomedical studies. Not only are 71% of human genes shared with the zebrafish many of these genes are linked to human diseases. Currently, numerous transgenic and mutant genetic zebrafish lines are now widely available for use in research. Furthermore, zebrafish are relatively inexpensive to maintain compared to rodents. However, a limiting factor to fully utilising adult zebrafish in research is not the fish but the technological imaging tools available. In order to increase the utilisation of adult zebrafish, which are not naturally transparent, requires new imaging approaches. Therefore, this feasibility study: (1) presents an innovative designed PET/CT adult zebrafish imaging platform, capable of maintaining normal aquatic physiology during scanning; (2) assesses the practical aspects of adult zebrafish imaging; and (3) set basic procedural guidelines for zebrafish imaging during a PET/CT acquisition. Methods: With computer aided design (CAD) software an imaging platform was developed for 3D printing. A 3D printed zebrafish model was created from a CT acquisition of a zebrafish using the CAD software. This model and subsequently euthanised zebrafish were imaged post-injection using different concentrations of the radiotracer [18F]FDG with CT contrast. Results: PET/CT imaging was successful, revealing levels as low as 0.01 MBq could be detected in the fish. In the zebrafish imaging post-injection distribution of the radiotracer was observed away from the injection site as well as tissue uptake. Potential preliminary husbandry and welfare guidelines for the fish during and after PET/CT imaging were determined. Conclusion: Using PET/CT for adult zebrafish imaging as a viable non-invasive technological tool is feasible. Adult zebrafish PET/CT imaging has the potential to be a key imaging technique offering the possibilities of enhanced biomedical understanding and new translational data sets.

Dimethyl fumarate reduces hepatocyte senescence following paracetamol exposure.

Liver disease is a major cause of premature death. Oxidative stress in the liver represents a key disease driver. Compounds, such as dimethyl fumarate (DMF), can activate the antioxidant response and are used clinically to treat disease. In this study, we tested the protective properties of DMF before or after paracetamol exposure. Following DMF administration, Nrf2 nuclear translocation was tracked at the single-cell level and target gene transactivation confirmed. Next, the protective properties of DMF were examined following paracetamol exposure. Transcriptomic and biochemical analysis revealed that DMF rescue was underpinned by reduced Nf-kB and TGF-ß signaling and cell senescence. Following on from these studies, we employed a Zebrafish model to study paracetamol exposure in vivo. We combined a genetically modified Zebrafish model, expressing green fluorescent protein exclusively in the liver, with automated microscopy. Pre-treatment with DMF, prior to paracetamol exposure, led to reduced liver damage in Zebrafish demonstrating protective properties.

Live Imaging of Heart Injury in Larval Zebrafish Reveals a Multi-Stage Model of Neutrophil and Macrophage Migration.

Neutrophils and macrophages are crucial effectors and modulators of repair and regeneration following myocardial infarction, but they cannot be easily observed in vivo in mammalian models. Hence many studies have utilized larval zebrafish injury models to examine neutrophils and macrophages in their tissue of interest. However, to date the migratory patterns and ontogeny of these recruited cells is unknown. In this study, we address this need by comparing our larval zebrafish model of cardiac injury to the archetypal tail fin injury model. Our in vivo imaging allowed comprehensive mapping of neutrophil and macrophage migration from primary hematopoietic sites, to the wound. Early following injury there is an acute phase of neutrophil recruitment that is followed by sustained macrophage recruitment. Both cell types are initially recruited locally and subsequently from distal sites, primarily the caudal hematopoietic tissue (CHT). Once liberated from the CHT, some neutrophils and macrophages enter circulation, but most use abluminal vascular endothelium to crawl through the larva. In both injury models the innate immune response resolves by reverse migration, with very little apoptosis or efferocytosis of neutrophils. Furthermore, our in vivo imaging led to the finding of a novel wound responsive mpeg1+ neutrophil subset, highlighting previously unrecognized heterogeneity in neutrophils. Our study provides a detailed analysis of the modes of immune cell migration in larval zebrafish, paving the way for future studies examining tissue injury and inflammation.

Author Correction: Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Adaptive prospective optical gating enables day-long 3D time-lapse imaging of the beating embryonic zebrafish heart.

Three-dimensional fluorescence time-lapse imaging of the beating heart is extremely challenging, due to the heart's constant motion and a need to avoid pharmacological or phototoxic damage. Although real-time triggered imaging can computationally "freeze" the heart for 3D imaging, no previous algorithm has been able to maintain phase-lock across developmental timescales. We report a new algorithm capable of maintaining day-long phase-lock, permitting routine acquisition of synchronised 3D + time video time-lapse datasets of the beating zebrafish heart. This approach has enabled us for the first time to directly observe detailed developmental and cellular processes in the beating heart, revealing the dynamics of the immune response to injury and witnessing intriguing proliferative events that challenge the established literature on cardiac trabeculation. Our approach opens up exciting new opportunities for direct time-lapse imaging studies over a 24-hour time course, to understand the cellular mechanisms underlying cardiac development, repair and regeneration.

The dyslexia susceptibility KIAA0319 gene shows a specific expression pattern during zebrafish development supporting a role beyond neuronal migration.

Dyslexia is a common neurodevelopmental disorder caused by a significant genetic component. The KIAA0319 gene is one of the most robust dyslexia susceptibility factors but its function remains poorly understood. Initial RNA-interference studies in rats suggested a role in neuronal migration whereas subsequent work with double knock-out mouse models for both Kiaa0319 and its paralogue Kiaa0319-like reported effects in the auditory system but not in neuronal migration. To further understand the role of KIAA0319 during neurodevelopment, we carried out an expression study of its zebrafish orthologue at different embryonic stages. We used different approaches including RNAscope in situ hybridization combined with light-sheet microscopy. The results show particularly high expression during the first few hours of development. Later, expression becomes localized in well-defined structures. In addition to high expression in the brain, we report for the first time expression in the eyes and the notochord. Surprisingly, kiaa0319-like, which generally shows a similar expression pattern to kiaa0319, was not expressed in the notochord suggesting a distinct role for kiaa0319 in this structure. This observation was supported by the identification of notochord enhancers enriched upstream of the KIAA0319 transcription start site, in both zebrafish and humans. This study supports a developmental role for KIAA0319 in the brain as well as in other developing structures, particularly in the notochord which, is key for establishing body patterning in vertebrates.

Adoption of in vitro systems and zebrafish embryos as alternative models for reducing rodent use in assessments of immunological and oxidative stress responses to nanomaterials.

Assessing the safety of engineered nanomaterials (NMs) is paramount to the responsible and sustainable development of nanotechnology, which provides huge societal benefits. Currently, there is no evidence that engineered NMs cause detrimental health effects in humans. However, investigation of NM toxicity using in vivo, in vitro, in chemico, and in silico models has demonstrated that some NMs stimulate oxidative stress and inflammation, which may lead to adverse health effects. Accordingly, investigation of these responses currently dominates NM safety assessments. There is a need to reduce reliance on rodent testing in nanotoxicology for ethical, financial and legislative reasons, and due to evidence that rodent models do not always predict the human response. We advocate that in vitro models and zebrafish embryos should have greater prominence in screening for NM safety, to better align nanotoxicology with the 3Rs principles. Zebrafish are accepted for use by regulatory agencies in chemical safety assessments (e.g. developmental biology) and there is growing acceptance of their use in biomedical research, providing strong foundations for their use in nanotoxicology. We suggest that investigation of the response of phagocytic cells (e.g. neutrophils, macrophages) in vitro should also form a key part of NM safety assessments, due to their prominent role in the first line of defense. The development of a tiered testing strategy for NM hazard assessment that promotes the more widespread adoption of non-rodent, alternative models and focuses on investigation of inflammation and oxidative stress could make nanotoxicology testing more ethical, relevant, and cost and time efficient.

Characterization of Triptolide-Induced Hepatotoxicity by Imaging and Transcriptomics in a Novel Zebrafish Model.

Triptolide is a vine extract used in traditional Chinese medicines and associated with hepatotoxicity. In vitro data suggest that inhibition of RNA synthesis may be the mechanism of toxicity. For studying drug-induced liver injury the zebrafish has experimental, practical and financial advantages compared with rodents. The aim of this study was to explore the mechanism of triptolide toxicity using zebrafish as the model system. The effect of triptolide exposure on zebrafish larvae was determined with regard to mortality, histology, expression of liver specific microRNA-122 and liver volume. Fluorescent microscopy was used to track toxicity in the Tg(-2.8lfabp:GFP)as3 zebrafish line. Informed by microscopy, RNA-sequencing was used to explore the mechanism of toxicity. Triptolide exposure resulted in dose-dependent mortality, a reduction in the number of copies of microRNA-122 per larva, hepatocyte vacuolation, disarray and oncotic necrosis, and a reduction in liver volume. These findings were consistent across replicate experiments. Time-lapse imaging indicated the onset of injury was 6 h after the start of exposure, at which point, RNA-sequencing revealed that 88% of genes were down-regulated. Immune response associated genes were up-regulated in the triptolide-treated larvae including nitric oxide synthase. Inhibition of nitric oxide synthase increased mortality. Triptolide induces hepatotoxicity in zebrafish larvae. This represents a new model of drug-induced liver injury that complements rodents. RNA sequencing, guided by time-lapse microscopy, revealed early down-regulation of genes consistent with previous invitro studies, and facilitated the discovery of mechanistic inflammatory pathways.

Rapid and recoverable in vivo magnetic resonance imaging of the adult zebrafish at 7T.

Increasing scientific interest in the zebrafish as a model organism across a range of biomedical and biological research areas raises the need for the development of in vivo imaging tools appropriate to this subject. Development of the embryonic and early stage forms of the subject can currently be assessed using optical based techniques due to the transparent nature of the species at these early stages. However this is not an option during the juvenile and adult stages when the subjects become opaque. Magnetic resonance imaging (MRI) techniques would allow for the longitudinal and non-invasive assessment of development and health in these later life stages. However, the small size of the zebrafish and its aquatic environment represent considerable challenges for the technique. We have developed a suitable flow cell system that incorporates a dedicated MRI imaging coil to solve these challenges. The system maintains and monitors a zebrafish during a scan and allows for it to be fully recovered. The imaging properties of this system compare well with those of other preclinical MRI coils used in rodent models. This enables the rapid acquisition of MRI data which are comparable in terms of quality and acquisition time. This would allow the many unique opportunities of the zebrafish as a model organism to be combined with the benefits of non-invasive MRI.

Cardiomyocyte proliferation in zebrafish and mammals: lessons for human disease.

Cardiomyocytes proliferate profusely during early development and for a brief period after birth in mammals. Within a month after birth, this proliferative capability is dramatically reduced in mammals unlike lower vertebrates where it persists into adult life. The zebrafish, for example, retains the ability to regenerate the apex of the heart following resection by a mechanism predominantly driven by cardiomyocyte proliferation. Differences in proliferative capacity of cardiomyocytes in adulthood between mammals and lower vertebrates are closely liked to ontogenetic or phylogenetic factors. Elucidation of these factors has the potential to provide enormous benefits if they lead to the development of therapeutic strategies that facilitate cardiomyocyte proliferation. In this review, we highlight the differences between Mammalian and Zebrafish cardiomyocytes, which could explain at least in part the different proliferative capacities in these two species. We discuss the advantages of the zebrafish as a model of cardiomyocyte proliferation, particularly at the embryonic stage. We also identify a number of key molecular pathways with potential to reveal key steps in switching cardiomyocytes from a quiescent to a proliferative phenotype.

Genetic and pharmacological inhibition of CDK9 drives neutrophil apoptosis to resolve inflammation in zebrafish in vivo.

Neutrophilic inflammation is tightly regulated and subsequently resolves to limit tissue damage and promote repair. When the timely resolution of inflammation is dysregulated, tissue damage and disease results. One key control mechanism is neutrophil apoptosis, followed by apoptotic cell clearance by phagocytes such as macrophages. Cyclin-dependent kinase (CDK) inhibitor drugs induce neutrophil apoptosis in vitro and promote resolution of inflammation in rodent models. Here we present the first in vivo evidence, using pharmacological and genetic approaches, that CDK9 is involved in the resolution of neutrophil-dependent inflammation. Using live cell imaging in zebrafish with labelled neutrophils and macrophages, we show that pharmacological inhibition, morpholino-mediated knockdown and CRISPR/cas9-mediated knockout of CDK9 enhances inflammation resolution by reducing neutrophil numbers via induction of apoptosis after tailfin injury. Importantly, knockdown of the negative regulator La-related protein 7 (LaRP7) increased neutrophilic inflammation. Our data show that CDK9 is a possible target for controlling resolution of inflammation.

Effects of Cyclin Dependent Kinase 9 inhibition on zebrafish larvae.

CDK9 is a known regulator of cellular transcription, growth and proliferation. Small molecule inhibitors are currently being developed and assessed in clinical trials as anti-cancer drugs. The zebrafish embryo provides an ideal model to explore the effects of CDK9 inhibition in-vivo. This has not been adequately explored previously at the level of a whole organism. We have compared and contrasted the effects of pharmacological and molecular inhibition of CDK9 on somatic growth, apoptosis and cellular proliferation in zebrafish larvae between 0 to 120 hours post fertilisation (hpf) using flavopiridol, a selective CDK9 antagonist, and CDK9-targeting morpholino. We demonstrate that the inhibition of CDK9 diminishes cellular proliferation and increases apoptosis. Subsequently, it affects somatic growth and development of a number of key embryonic structures including the brain, heart, eye and blood vessels. For the first time, we have localized CDK9 at a subcellular level in whole-mounted larvae. This works shows, at a high-throughput level, that CDK9 clearly plays a fundamental role in early cellular growth and proliferation.

Models for the Study of the Cross Talk Between Inflammation and Cell Cycle.

Cyclin-dependent kinases (CDKs) have been traditionally associated with the cell cycle. However, it is now known that CDK7 and CDK9 regulate transcriptional activity via phosphorylation of RNA polymerase II and subsequent synthesis of, for example, inflammatory mediators and factors that influence the apoptotic process; including apoptosis of granulocytes such as neutrophils and eosinophils. Successful resolution of inflammation and restoration of normal tissue homeostasis requires apoptosis of these inflammatory cells and subsequent clearance of apoptotic bodies by phagocytes such as macrophages. It is believed that CDK7 and CDK9 influence resolution of inflammation since they are involved in the transcription of anti-apoptotic proteins such as Mcl-1 which is especially important in granulocyte survival.This chapter describes various in vitro and in vivo models used to investigate CDKs and their inhibitors in granulocytes and particularly the role of CDKs in the apoptosis pathway. This can be performed in vitro by isolation and use of primary granulocytes and in vivo using animal models of inflammatory disease in rodents and zebrafish. Some of the methods described here to assess the role of CDKs in inflammation and apoptosis include flow cytometry and western blotting, together with imaging and quantification of apoptosis in fixed tissue, as well as in vivo models of inflammation.

CDK9 and its repressor LARP7 modulate cardiomyocyte proliferation and response to injury in the zebrafish heart.

Cyclin dependent kinase (Cdk)9 acts through the positive transcription elongation factor-b (P-TEFb) complex to activate and expand transcription through RNA polymerase II. It has also been shown to regulate cardiomyocyte hypertrophy, with recent evidence linking it to cardiomyocyte proliferation. We hypothesised that modification of CDK9 activity could both impair and enhance the cardiac response to injury by modifying cardiomyocyte proliferation. Cdk9 expression and activity were inhibited in the zebrafish (Danio rerio) embryo. We show that dephosphorylation of residue Ser2 on the C-terminal domain of RNA polymerase II is associated with impaired cardiac structure and function, and cardiomyocyte proliferation and also results in impaired functional recovery following cardiac laser injury. In contrast, de-repression of Cdk9 activity, through knockdown of La-related protein (Larp7) increases phosphorylation of Ser2 in RNA polymerase II and increases cardiomyocyte proliferation. Larp7 knockdown rescued the structural and functional phenotype associated with knockdown of Cdk9. The balance of Cdk9 and Larp7 plays a key role in cardiomyocyte proliferation and response to injury. Larp7 represents a potentially novel therapeutic target to promote cardiomyocyte proliferation and recovery from injury.

Temporal cohesion of the structural, functional and molecular characteristics of the developing zebrafish heart.

Heart formation is a complex, dynamic and highly coordinated process of molecular, morphogenetic and functional factors with each interacting and contributing to formation of the mature organ. Cardiac abnormalities in early life can be lethal in mammals but not in the zebrafish embryo which has been widely used to study the developing heart. While early cardiac development in the zebrafish has been well characterized, functional changes during development and how these relate to architectural, cellular and molecular aspects of development have not been well described previously. To address this we have carefully characterised cardiac structure, function, cardiomyocyte proliferation and cardiac-specific gene expression between 48 and 120 hpf in the zebrafish. We show that the zebrafish heart increases in volume and changes shape significantly between 48 and 72 hpf accompanied by a 40% increase in cardiomyocyte number. Between 96 and 120 hpf, while external heart expansion slows, there is rapid formation of a mature and extensive trabecular network within the ventricle chamber. While ejection fraction does not change during the course of development other determinants of contractile function increase significantly particularly between 72 and 96 hpf leading to an increase in cardinal vein blood flow. This study has revealed a number of novel aspects of cardiac developmental dynamics with striking temporal orchestration of structure and function within the first few days of development. These changes are associated with changes in expression of developmental and maturational genes. This study provides important insights into the complex temporal relationship between structure and function of the developing zebrafish heart.

Targeted laser ablation of the zebrafish larval heart induces models of heart block, valvular regurgitation, and outflow tract obstruction.

Mammalian models of cardiac disease have provided unique and important insights into human disease but have become increasingly challenging to produce. The zebrafish could provide inexpensive high-throughput models of cardiac injury and repair. We used a highly targeted laser, synchronized to fire at specific phases of the cardiac cycle, to induce regional injury to the ventricle, atrioventricular (AV) cushion, and bulbus arteriosus (BA). We assessed the impact of laser injury on hearts of zebrafish early larvae at 72 h postfertilization, to different regions, recording the effects on ejection fraction (EF), heart rate (HR), and blood flow at 2 and 24 h postinjury (hpi). Laser injury to the apex, midzone, and outflow regions of the ventricle resulted in reductions of the ventricle EF at 2 hpi with full recovery of function by 24 hpi. Laser injury to the ventricle, close to the AV cushion, was more likely to cause bradycardia and atrial-ventricular dysfunction, suggestive of an electrical conduction block. At 2 hpi, direct injury to the AV cushion resulted in marked regurgitation of blood from the ventricle to the atrium. Laser injury to the BA caused temporary outflow tract obstruction with cessation of ventricle contraction and circulation. Despite such damage, 80% of embryos showed complete recovery of the HR and function within 24 h of laser injury. Precision laser injury to key structures in the zebrafish developing heart provides a range of potentially useful models of hemodynamic overload, injury, and repair.

Zebrafish as model organisms for studying drug-induced liver injury.

Drug-induced liver injury (DILI) is a major challenge in clinical medicine and drug development. New models are needed for predicting which potential therapeutic compounds will cause DILI in humans, and new markers and mediators of DILI still need to be identified. This review highlights the strengths and weaknesses of using zebrafish as a high-throughput in vivo model for studying DILI. Although the zebrafish liver architecture is different from that of the mammalian liver, the main physiological processes remain similar. Zebrafish metabolize drugs using similar pathways to those in humans; they possess a wide range of cytochrome P450 enzymes that enable metabolic reactions including hydroxylation, conjugation, oxidation, demethylation and de-ethylation. Following exposure to a range of hepatotoxic drugs, the zebrafish liver develops histological patterns of injury comparable to those of mammalian liver, and biomarkers for liver injury can be quantified in the zebrafish circulation. The zebrafish immune system is similar to that of mammals, but the zebrafish inflammatory response to DILI is not yet defined. In order to quantify DILI in zebrafish, a wide variety of methods can be used, including visual assessment, quantification of serum enzymes and experimental serum biomarkers and scoring of histopathology. With further development, the zebrafish may be a model that complements rodents and may have value for the discovery of new disease pathways and translational biomarkers.