Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens
复制标题
DOI：
10.3791/3781
复制
发表时间：
2012-03-01
影响因子：
1.2
通讯作者：
Meijer, Annemarie H.
中科院分区：
文献类型：
--
作者：
Benard, Erica L.;van der Sar, Astrid M.;Meijer, Annemarie H.
斑马鱼（Danio rerio）胚胎越来越多地用作研究宿主-病原体相互作用中脊椎动物先天免疫系统功能的模型（1）。先天免疫系统的主要细胞类型，巨噬细胞和嗜中性粒细胞，在适应性免疫应答所需的淋巴细胞成熟之前的胚胎发生的第一天期间发育。获得大量胚胎的容易性，由于外部发育，胚胎和幼虫阶段的光学透明度，广泛的遗传工具，广泛的突变体资源和转基因报告系的集合，所有这些都增加了斑马鱼模型的多功能性。鼠伤寒沙门氏菌（S.鼠伤寒沙门氏菌）和海分枝杆菌可以驻留在巨噬细胞内，并经常用于研究斑马鱼胚胎中宿主-病原体相互作用。这两种细菌病原体的感染过程是有趣的比较，因为S。鼠伤寒杆菌感染是急性的，在一天之内是致命的，而M.海水感染是慢性的，可以成像到幼虫阶段（2），（3）。将细菌显微注射到胚胎中的部位（图1）决定了感染是否会迅速成为全身性的或最初保持局部性。快速全身感染可以通过经由后血岛处的尾静脉或经由居维叶管（连接心脏和躯干脉管系统的卵黄囊上的宽循环通道）将细菌直接微量注射到血液循环中来建立。在1dpf，当胚胎在这个阶段有吞噬活性的巨噬细胞，但中性粒细胞尚未成熟，注射到血岛是首选。对于2-3 dpf的注射，当胚胎也发育有功能性（产生髓过氧化物酶）的中性粒细胞时，居维叶管是首选的注射部位。为了研究骨髓细胞向局部感染的定向迁移，可以将细菌注射到尾肌、耳泡或后脑脑室中（4-6）。此外，脊索，一种似乎是正常情况下骨髓细胞无法接近的结构，对局部感染非常敏感（7）。高通量应用的一个有用的替代方案是在受精后的第一个小时内将细菌注射到胚胎的蛋黄中（8）。将荧光细菌和转基因斑马鱼系与荧光巨噬细胞或中性粒细胞相结合，为宿主-病原体相互作用的多色成像创造了理想的环境。这篇视频文章将描述用S.鼠伤寒沙门氏菌或M.海洋细菌，并随后对与先天免疫系统细胞的相互作用进行荧光成像。
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.