Mechanical Vessel Injury in Zebrafish Embryos

Zebrafish (Danio rerio) embryos have proven to be a powerful model for studying a variety of developmental and disease processes. External development and optical transparency make these embryos especially amenable to microscopy, and numerous transgenic lines that label specific cell types with fluorescent proteins are available, making the zebrafish embryo an ideal system for visualizing the interaction of vascular, hematopoietic, and other cell types during injury and repair in vivo. Forward and reverse genetics in zebrafish are well developed, and pharmacological manipulation is possible. We describe a mechanical vascular injury model using micromanipulation techniques that exploits several of these features to study responses to vascular injury including hemostasis and blood vessel repair. Using a combination of video and timelapse microscopy, we demonstrate that this method of vascular injury results in measurable and reproducible responses during hemostasis and wound repair. This method provides a system for studying vascular injury and repair in detail in a whole animal model.     

Blood Vessel Adaptation with Fluctuations in Capillary Flow Distribution

Throughout the life of animals and human beings, blood vessel systems are continuously adapting their structures – the diameter of vessel lumina, the thickness of vessel walls, and the number of micro-vessels – to meet the changing metabolic demand of the tissue. The competition between an ever decreasing tendency of luminal diameters and an increasing stimulus from the wall shear stress plays a key role in the adaptation of luminal diameters. However, it has been shown in previous studies that the adaptation dynamics based only on these two effects is unstable. In this work, we propose a minimal adaptation model of vessel luminal diameters, in which we take into account the effects of metabolic flow regulation in addition to wall shear stresses and the decreasing tendency of luminal diameters. In particular, we study the role, in the adaptation process, of fluctuations in capillary flow distribution which is an important means of metabolic flow regulation. The fluctuation in the flow of a capillary group is idealized as a switch between two states, i.e., an open-state and a close-state. Using this model, we show that the adaptation of blood vessel system driven by wall shear stress can be efficiently stabilized when the open time ratio responds sensitively to capillary flows. As micro-vessel rarefaction is observed in our simulations with a uniformly decreased open time ratio of capillary flows, our results point to a possible origin of micro-vessel rarefaction, which is believed to induce hypertension.     

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell''s destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.Download video file.^{(57M, mov)}     

Imaging Transient Blood Vessel Fusion Events in Zebrafish by Correlative Volume Electron Microscopy

The study of biological processes has become increasingly reliant on obtaining high-resolution spatial and temporal data through imaging techniques. As researchers demand molecular resolution of cellular events in the context of whole organisms, correlation of non-invasive live-organism imaging with electron microscopy in complex three-dimensional samples becomes critical. The developing blood vessels of vertebrates form a highly complex network which cannot be imaged at high resolution using traditional methods. Here we show that the point of fusion between growing blood vessels of transgenic zebrafish, identified in live confocal microscopy, can subsequently be traced through the structure of the organism using Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) and Serial Block Face/Scanning Electron Microscopy (SBF/SEM). The resulting data give unprecedented microanatomical detail of the zebrafish and, for the first time, allow visualization of the ultrastructure of a time-limited biological event within the context of a whole organism.     

Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens

Zebrafish (Danio rerio) embryos are increasingly used as a model for studying the function of the vertebrate innate immune system in host-pathogen interactions ¹. The major cell types of the innate immune system, macrophages and neutrophils, develop during the first days of embryogenesis prior to the maturation of lymphocytes that are required for adaptive immune responses. The ease of obtaining large numbers of embryos, their accessibility due to external development, the optical transparency of embryonic and larval stages, a wide range of genetic tools, extensive mutant resources and collections of transgenic reporter lines, all add to the versatility of the zebrafish model. Salmonella enterica serovar Typhimurium (S. typhimurium) and Mycobacterium marinum can reside intracellularly in macrophages and are frequently used to study host-pathogen interactions in zebrafish embryos. The infection processes of these two bacterial pathogens are interesting to compare because S. typhimurium infection is acute and lethal within one day, whereas M. marinum infection is chronic and can be imaged up to the larval stage ^{2, 3}. The site of micro-injection of bacteria into the embryo (Figure 1) determines whether the infection will rapidly become systemic or will initially remain localized. A rapid systemic infection can be established by micro-injecting bacteria directly into the blood circulation via the caudal vein at the posterior blood island or via the Duct of Cuvier, a wide circulation channel on the yolk sac connecting the heart to the trunk vasculature. At 1 dpf, when embryos at this stage have phagocytically active macrophages but neutrophils have not yet matured, injecting into the blood island is preferred. For injections at 2-3 dpf, when embryos also have developed functional (myeloperoxidase-producing) neutrophils, the Duct of Cuvier is preferred as the injection site. To study directed migration of myeloid cells towards local infections, bacteria can be injected into the tail muscle, otic vesicle, or hindbrain ventricle ^4-6. In addition, the notochord, a structure that appears to be normally inaccessible to myeloid cells, is highly susceptible to local infection ⁷. A useful alternative for high-throughput applications is the injection of bacteria into the yolk of embryos within the first hours after fertilization ⁸. Combining fluorescent bacteria and transgenic zebrafish lines with fluorescent macrophages or neutrophils creates ideal circumstances for multi-color imaging of host-pathogen interactions. This video article will describe detailed protocols for intravenous and local infection of zebrafish embryos with S. typhimurium or M. marinum bacteria and for subsequent fluorescence imaging of the interaction with cells of the innate immune system.     

Cellular Changes in Blood Indicate Severe Respiratory Disease during Influenza Infections in Mice

Influenza A infection is a serious threat to human and animal health. Many of the biological mechanisms of the host-pathogen-interactions are still not well understood and reliable biomarkers indicating the course of the disease are missing. The mouse is a valuable model system enabling us to study the local inflammatory host response and the influence on blood parameters under controlled circumstances. Here, we compared the lung and peripheral changes after PR8 (H1N1) influenza A virus infection in C57BL/6J and DBA/2J mice using virus variants of different pathogenicity resulting in non-lethal and lethal disease. We monitored hematological and immunological parameters revealing that the granulocyte to lymphocyte ratio in the blood represents an early indicator of severe disease progression already two days after influenza A infection in mice. These findings might be relevant to optimize early diagnostic options of severe influenza disease and to monitor successful therapeutic treatment in humans.     

Analysis of Oxidative Stress in Zebrafish Embryos

High levels of reactive oxygen species (ROS) may cause a change of cellular redox state towards oxidative stress condition. This situation causes oxidation of molecules (lipid, DNA, protein) and leads to cell death. Oxidative stress also impacts the progression of several pathological conditions such as diabetes, retinopathies, neurodegeneration, and cancer. Thus, it is important to define tools to investigate oxidative stress conditions not only at the level of single cells but also in the context of whole organisms. Here, we consider the zebrafish embryo as a useful in vivo system to perform such studies and present a protocol to measure in vivo oxidative stress. Taking advantage of fluorescent ROS probes and zebrafish transgenic fluorescent lines, we develop two different methods to measure oxidative stress in vivo: i) a “whole embryo ROS-detection method” for qualitative measurement of oxidative stress and ii) a “single-cell ROS detection method” for quantitative measurements of oxidative stress. Herein, we demonstrate the efficacy of these procedures by increasing oxidative stress in tissues by oxidant agents and physiological or genetic methods. This protocol is amenable for forward genetic screens and it will help address cause-effect relationships of ROS in animal models of oxidative stress-related pathologies such as neurological disorders and cancer.     

斑马鱼胚胎中受Nodal信号调控基因的鉴定

Nodal信号在脊椎动物胚胎发育的中内胚层诱导、左右不对称性的建立、神经外胚层沿前后轴线的分化等方面起着重要的作用.为鉴定受Nodal信号调控的基因,特别是那些转录因子基因,通过将来自squint过量表达、缺失Nodal信号的MZoep突变体或野生型30%外包期胚胎的RNA与Affymetrix斑马鱼寡核苷酸芯片杂交.发现与野生型样本相比,在squint过量表达的样本中,265个转录本的表达显著增强(log2ratio>1),111个转录本的表达显著减弱(log2ratio<-1);在MZoep样本中,表达显著增强的(log2ratio>1)转录本有1495个,表达显著减弱(log2ratio<-1)的有550个.squint过量表达使26个转录因子基因的表达增强,11个转录因子基因的表达减弱;另一方面,MZoep突变体中表达增强的转录因子基因为69个,表达减弱的转录因子基因为30个.这些结果为进一步研究Nodal信号的转导机理和生物学功能提供了有益的数据.     

Optic Nerve Head Blood Flow Autoregulation during Changes in Arterial Blood Pressure in Healthy Young Subjects

Aim

In the present study the response of optic nerve head blood flow to an increase in ocular perfusion pressure during isometric exercise was studied. Based on our previous studies we hypothesized that subjects with an abnormal blood flow response, defined as a decrease in blood flow of more than 10% during or after isometric exercise, could be identified.

Methods

A total of 40 healthy subjects were included in this study. Three periods of isometric exercise were scheduled, each consisting of 2 minutes of handgripping. Optic nerve head blood flow was measured continuously before, during and after handgripping using laser Doppler flowmetry. Blood pressure was measured non-invasively in one-minute intervals. Intraocular pressure was measured at the beginning and the end of the measurements and ocular perfusion pressure was calculated as 2/3*mean arterial pressure –intraocular pressure.

Results

Isometric exercise was associated with an increase in ocular perfusion pressure during all handgripping periods (p < 0.001). By contrast no change in optic nerve head blood flow was seen. However, in a subgroup of three subjects blood flow showed a consistent decrease of more than 10% during isometric exercise although their blood pressure values increased. In addition, three other subjects showed a consistent decline of blood flow of more than 10% during the recovery periods.

Conclusion

Our data confirm previous results indicating that optic nerve head blood flow is autoregulated during an increase in perfusion pressure. In addition, we observed a subgroup of 6 subjects (15%) that showed an abnormal response, which is in keeping with our previous data. The mechanisms underlying this abnormal response remain to be shown.     

Low Temperature Mitigates Cardia Bifida in Zebrafish Embryos

The coordinated migration of bilateral cardiomyocytes and the formation of the cardiac cone are essential for heart tube formation. We investigated gene regulatory mechanisms involved in myocardial migration, and regulation of the timing of cardiac cone formation in zebrafish embryos. Through screening of zebrafish treated with ethylnitrosourea, we isolated a mutant with a hypomorphic allele of mil (s1pr2)/edg5, called s1pr2^as10 (as10). Mutant embryos with this allele expressed less mil/edg5 mRNA and exhibited cardia bifida prior to 28 hours post-fertilization. Although the bilateral hearts of the mutants gradually fused together, the resulting formation of two atria and one tightly-packed ventricle failed to support normal blood circulation. Interestingly, cardia bifida of s1pr2^as10 embryos could be rescued and normal circulation could be restored by incubating the embryos at low temperature (22.5°C). Rescue was also observed in gata5 and bon cardia bifida morphants raised at 22.5°C. The use of DNA microarrays, digital gene expression analyses, loss-of-function, as well as mRNA and protein rescue experiments, revealed that low temperature mitigates cardia bifida by regulating the expression of genes encoding components of the extracellular matrix (fibronectin 1, tenascin-c, tenascin-w). Furthermore, the addition of N-acetyl cysteine (NAC), a reactive oxygen species (ROS) scavenger, significantly decreased the effect of low temperature on mitigating cardia bifida in s1pr2^as10 embryos. Our study reveals that temperature coordinates the development of the heart tube and somitogenesis, and that extracellular matrix genes (fibronectin 1, tenascin-c and tenascin-w) are involved.     

斑马鱼及其胚胎在毒理学研究中的应用

斑马鱼作为一种新的毒理模式动物,具有其它模式动物无法比拟的优势。就急性毒理表型分析、转录组水平分析、蛋白质组学分析这3种评价化合物对斑马鱼胚胎影响的最常用方法,对斑马鱼及其胚胎在毒理学研究中的应用作一综述。通过这些方法,探究外源毒性物质的毒理学机制、筛选可用于早期预警的生物效应分子,为其合理使用提供科学依据和技术支持。     

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.     

Xenotransplantation of Human Adipose-Derived Stem Cells in Zebrafish Embryos

Zebrafish is a widely used animal model with well-characterized background in developmental biology. The fate of human adipose-derived stem cells (ADSCs) after their xenotransplantation into the developing embryos of zebrafish is unknown. Therefore, human ADSCs were firstly isolated, and then transduced with lentiviral vector system carrying a green fluorescent protein (GFP) reporter gene, and followed by detection of their cell viability and the expression of cell surface antigens. These GFP-expressing human ADSCs were transplanted into the zebrafish embryos at 3.3–4.3 hour post-fertilization (hpf). Green fluorescent signal, the proliferation and differentiation of human ADSCs in recipient embryos were respectively examined using fluorescent microscopy and immunohistochemical staining. The results indicated that human ADSCs did not change their cell viability and the expression levels of cell surface antigens after GFP transduction. Microscopic examination demonstrated that green fluorescent signals of GFP expressed in the transplanted cells were observed in the embryos and larva fish at post-transplantation. The positive staining of Ki-67 revealed the survival and proliferation of human ADSCs in fish larvae after transplantation. The expression of CD105 was observable in the xenotransplanted ADSCs, but CD31 expression was undetectable. Therefore, our results indicate that human ADSCs xenotransplanted in the zebrafish embryos not only can survive and proliferate at across-species circumstance, but also seem to maintain their undifferentiation status in a short term. This xenograft model of zebrafish embryos may provide a promising and useful technical platform for the investigation of biology and physiology of stem cells in vivo.     

Thrombin Generation in Zebrafish Blood

To better understand hypercoagulability as an underlying cause for thrombosis, the leading cause of death in the Western world, new assays to study ex vivo coagulation are essential. The zebrafish is generally accepted as a good model for human hemostasis and thrombosis, as the hemostatic system proved to be similar to that in man. Their small size however, has been a hurdle for more widespread use in hemostasis related research. In this study we developed a method that enables the measurement of thrombin generation in a single drop of non-anticoagulated zebrafish blood. Pre-treatment of the fish with inhibitors of FXa and thrombin, resulted in a dose dependent diminishing of thrombin generation, demonstrating the validity of the assay. In order to establish the relationship between whole blood thrombin generation and fibrin formation, we visualized the resulting fibrin network by scanning electron microscopy. Taken together, in this study we developed a fast and reliable method to measure thrombin generation in whole blood collected from a single zebrafish. Given the similarities between coagulation pathways of zebrafish and mammals, zebrafish may be an ideal animal model to determine the effect of novel therapeutics on thrombin generation. Additionally, because of the ease with which gene functions can be silenced, zebrafish may serve as a model organism for mechanistical research in thrombosis and hemostasis.     

斑马鱼胚胎和肿瘤细胞评价细胞毒药物活性比较研究*

目的:比较斑马鱼胚胎和肿瘤细胞作为药物筛选模型的优缺点.方法:采用MTT法检测顺铂、紫杉醇、阿霉素、5-氟尿嘧啶四种药物对HL-60和Hela细胞的增殖影响;同时,观察药物对斑马鱼胚胎发育的影响.结果:阿霉素、顺铂及紫杉醇作用于HL-60及Hela细胞的IC50均显著高于作用于斑马鱼胚胎的LD50;而5-FU作用于肿瘤细胞和斑马鱼胚胎的结果与其它药物相反;四种抗肿瘤药物对斑马鱼胚胎的生长发育均有致畸作用.结论:斑马鱼胚胎作为细胞毒类药物筛选模型,对于抗微管类药物较为敏感,但对于抗代谢药敏感性较肿瘤细胞差.     

斑马鱼整体原位杂交的技术改良

斑马鱼整体原位杂交技术广泛应用于基因表达谱、基因间相互关系和突变体筛选等方面,是研究斑马鱼发育相关基因功能的重要技术。本文从杂交探针的制备、浓度的选择和洗脱以及胚胎的脱色、蛋白酶K消化、底物显色等方面进行了摸索、改进及简化,获得了背景低、着色清晰、特异性高的实验结果,预示了简单实用、成本低廉的斑马鱼整胚原位杂交技术平台的成功建立。     

玉米胚发育过程中脱水耐性的变化

对离体玉米胚脱水耐性的变化以及不同脱水速率对其脱水耐性的影响进行了研究。授粉后16d的玉米胚能耐轻微脱水,含水量从1.45降低到0.28gH2Og-1DW时胚的萌发率为100%,但含水量低于0.1gH2Og-1DW时胚死亡。胚的脱水耐性随着发育逐渐加强,表现为电解质渗漏速率逐渐降低,萌发率和幼苗干重逐渐增加。授粉后20d胚内超氧化物歧化酶(SOD)和抗坏血酸过氧化物酶(APX)活性较高,过氧化氢酶(CAT)活性较低;授粉后24d,这些酶的活性与授粉后20d的正好相反。脂质过氧化产物丙二醛(MDA)在种子发育过程中呈下降趋势。不同脱水速率明显地影响胚的脱水耐性:在慢速脱水到含水量0.1~0.18gH2Og-1DW时,胚的萌发率和幼苗干重比快速脱水高,电解质渗漏速率比快速脱水低;在快速脱水条件下胚中的SOD、APX活性和MDA含量也比慢速脱水高;CAT活性的变化不明显。