Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.     

Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases

Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.     

It's time to swim! Zebrafish and the circadian clock

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.     

The zebrafish as a model system in developmental, toxicological and transgenic research

The zebrafish has long been used as a model system in fisheries biology and toxicology. More recently, it has also become the focus of a major research effort into understanding the molecular and cellular events which dictate the development of vertebrate embryos. As well, the zebrafish has proven attractive in studies examining the factors which affect the creation of transgenic fish and the expression of transgenes. The advances which have been made in these areas have firmly established this small aquarium fish as a major model system in biological and biotechnological research.     

A Novel Zebrafish Xenotransplantation Model for Study of Glioma Stem Cell Invasion

Invasion and metastasis of solid tumors are the major causes of death in cancer patients. Cancer stem cells (CSCs) constitute a small fraction of tumor cell population, but play a critical role in tumor invasion and metastasis. The xenograft of tumor cells in immunodeficient mice is one of commonly used in vivo models to study the invasion and metastasis of cancer cells. However, this model is time-consuming and labor intensive. Zebrafish (Danio rerio) and their transparent embryos are emerging as a promising xenograft tumor model system for studies of tumor invasion. In this study, we established a tumor invasion model by using zebrafish embryo xenografted with human glioblastoma cell line U87 and its derived cancer stem cells (CSCs). We found that CSCs-enriched from U87 cells spreaded via the vessels within zebrafish embryos and such cells displayed an extremely high level of invasiveness which was associated with the up-regulated MMP-9 by CSCs. The invasion of glioma CSCs (GSCs) in zebrafish embryos was markedly inhibited by an MMP-9 inhibitor. Thus, our zebrafish embryo model is considered a cost-effective approach tostudies of the mechanisms underlying the invasion of CSCs and suitable for high-throughput screening of novel anti-tumor invasion/metastasis agents.     

Modelling Delta-Notch perturbations during zebrafish somitogenesis

The discovery over the last 15 years of molecular clocks and gradients in the pre-somitic mesoderm of numerous vertebrate species has added significant weight to Cooke and Zeeman's ‘clock and wavefront’ model of somitogenesis, in which a travelling wavefront determines the spatial position of somite formation and the somitogenesis clock controls periodicity (Cooke and Zeeman, 1976). However, recent high-throughput measurements of spatiotemporal patterns of gene expression in different zebrafish mutant backgrounds allow further quantitative evaluation of the clock and wavefront hypothesis. In this study we describe how our recently proposed model, in which oscillator coupling drives the propagation of an emergent wavefront, can be used to provide mechanistic and testable explanations for the following observed phenomena in zebrafish embryos: (a) the variation in somite measurements across a number of zebrafish mutants; (b) the delayed formation of somites and the formation of ‘salt and pepper’ patterns of gene expression upon disruption of oscillator coupling; and (c) spatial correlations in the ‘salt and pepper’ patterns in Delta-Notch mutants. In light of our results, we propose a number of plausible experiments that could be used to further test the model.     

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.     

水平回转培养对斑马鱼血管发育的影响

随着空间生命科学的发展, 微重力对生命体的影响已成为科学家们日益关注的问题。多数研究表明, 微重力对生物体胚胎早期发育有着重要影响, 而血管系统作为胚胎最早行使功能的系统备受关注。目前关于微重力对血管发育影响的研究大多来自对离体培养细胞的体外回转模拟实验, 在体实验相对较少。文章利用斑马鱼作为模式动物, 在体探究水平回转培养环境下斑马鱼胚胎早期发育及水平回转培养对其血管系统发育的影响。在斑马鱼受精后24 h(24 hpf)时进行水平回转培养处理, 至36 hpf时收集胚胎, 通过体视显微镜观察斑马鱼表型变化, 通过半定量RT-PCR、qPCR及全胚原位杂交等手段对比水平回转培养环境下与对照组血管相关因子的表达情况, 并通过BrdU掺入及TUNEL法进行斑马鱼全胚细胞凋亡及增殖检测。结果表明, 在90 r/min水平回转培养环境下, 斑马鱼死亡数量没有差异, 24 hpf时破壳数量显著降低(10.3±0.41 vs. 0.0, P<0.05), 心率显著加快(223.5±2.32 vs. 185.0±3.23, P<0.05), 黑色素明显增加, 动静脉发育紊乱, 且在120 r/min转速下, 胚胎血管特异性表达因子flk1、flt4及 ephrinB2的表达均显著降低, 斑马鱼细胞凋亡明显增加, 而细胞增殖无显著差异, 提示水平回转培养对斑马鱼胚胎发育特别是早期血管发育具有重要影响。     

Zebrafish: a new model on the pharmaceutical catwalk

Zebrafish is recognized as one of the most important vertebrate model organisms; however, its value in pharmacological studies has not been extensively explored and exploited. In this review, I summarize significant findings about the effects of drugs and medicines on important physiological processes in zebrafish. Our experiments have shown that cardiovascular, anti-angiogenic and anti-cancer drugs elicit comparable responses in zebrafish embryos to those in mammalian systems. Similar observations have been reported by other laboratories, exposing zebrafish to a variety of pharmaceutical active compounds affecting a range of different processes. All the data summarized indicate that zebrafish represents a very valuable organism for different kinds of pharmacological studies, such as screenings of chemical libraries, lead validation and optimization, mode-of-action studies, analysis of gene function, predictive toxicology and teratogenicity, pharmacogenomics and toxicogenomics. Zebrafish pharmacological assays have specific advantages compared to in vitro cell culture studies and in vivo experiments using mice, complementing these assays to give valuable guides for future tests of new drugs for human therapy.     

Fgf21 is essential for haematopoiesis in zebrafish

Fibroblast growth factors (Fgfs) function as key secreted signalling molecules in many developmental events. The zebrafish is a powerful model system for the investigation of embryonic vertebrate haematopoiesis. Although the effects of Fgf signalling on haematopoiesis in vitro have been reported, the functions of Fgf signalling in haematopoiesis in vivo remain to be explained. We identified Fgf21 in zebrafish embryos. Fgf21-knockdown zebrafish embryos lacked erythroid and myeloid cells but not blood vessels and lymphoid cells. The knockdown embryos had haemangioblasts and haematopoietic stem cells. However, the knockdown embryos had significantly fewer myeloid and erythroid progenitor cells. In contrast, Fgf21 had no significant effect on cell proliferation and apoptosis in the intermediate cell mass. These results indicate that Fgf21 is a newly identified factor essential for the determination of myelo-erythroid progenitor cell fate in vivo.     

人BAFF转基因斑马鱼的构建及早期免疫基因表达检测

目的建立人BAFF转基因斑马鱼模型,探讨其在自身免疫性疾病发病中的作用。方法RT-PCR法由人淋巴瘤细胞克隆了人BAFF基因全长855bp蛋白编码区域,构建表达人BAFF重组质粒Tol2-hBAFF,体外细胞转染并通过免疫印迹法验证蛋白表达。重组载体经显微注射斑马鱼受精卵后,GFP荧光跟踪并筛选阳性鱼。qPCR法检测早期免疫相关基因表达情况。结果人BAFF-GFP融合蛋白可成功表达,利用Tol2-hBAFF重组质粒显微注射斑马鱼受精卵可获得表达人BAFF的转基因斑马鱼,且表达人BAFF斑马鱼1dpf胚胎中TCRAC明显高表达,而Ikaros则表达量显著降低,表明在斑马鱼胚胎中表达人BAFF蛋白会造成早期淋巴系统中基因的过早表达。结论建立的表达人BAFF的转基因斑马鱼,可为系统性红斑狼疮等与BAFF功能亢进密切相关的自身免疫性疾病的治疗,及相关机制研究提供一种具有诸多优点的新型工具。