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Experimental manipulation and other lines of evidence indicate that the lens plays a prominent role in the growth and differentiation of the vertebrate eye. Here we describe a lens transplantation method for studying the role of the lens in teleost eye development. The method involves three steps: (1) preparing embryos for the operations by embedding them in agar, (2) microsurgery with tungsten needles...
Mutagenesis screens in zebrafish have uncovered several hundred mutant alleles affecting the development of the retina and established the zebrafish as one of the leading models of vertebrate eye development. In addition to forward genetic mutagenesis approaches, gene function in the zebrafish embryo is being studied using several reverse genetic techniques. Some of these rely on the overexpression...
The ability to image neural activity in populations of neurons inside an intact animal, while obtaining single-cell or subcellular spatial resolution, has led to several advances in our understanding of vertebrate locomotor control. This result, first reported in a 1995 study of motoneurons in larval zebrafish, was the beginning of a series of technical developments that exploited the transparency...
Heat shock proteins (HSPs) indicate exposure to cellular stress and adverse cellular effects, thus serving as biomarkers of these effects. The highly conserved Hsp70 proteins are expressed under proteotoxic conditions, whereas small HSPs are expressed in response to stressors acting on the cytoskeleton and cell signaling pathways. Poeciliopsis lucida hepatocellular carcinoma line 1 (PLHC-1) cells...
The Tol2 transposon system can create chromosomal insertions in the zebrafish germ lineage very efficiently. We constructed a Tol2-based gene trap vector, T2KSAG, which contains a splice accepter, the GFP gene and the polyA signal. In the pilot screen for gene trapping using T2KSAG, we identified 38 fish lines expressing GFP in specific organs and tissues. In the SAGp53A line, GFP is expressed in...
The zebrafish is an ideal organism for small molecule studies. The ability to use the whole organism allows complex in vivo phenotypes to be assayed and combines animal testing with screening. Embryos are easily treatable by waterborne exposure. The small size and abundance of embryos make zebrafish suitable for screening in a high-throughput manner in 96- or 48-well plates. Zebrafish embryos have...
The combination of accessible embryology and forward genetic techniques has made zebrafish a powerful model system for the study of vertebrate development. One limitation of genetic analysis is that the study of gene function is usually limited to the first developmental event affected by a gene. In vivo electroporation has recently matured as a method for studying gene function at different developmental...
Animal models provide an in vivo system to study gene function by transgenic and knockout approaches. Targeted knockout approaches have been very successful in mice, but are currently not feasible in zebrafish due to the inability to grow embryonic stem cells. As an alternative, a reverse genetic approach that utilizes screening by resequencing and/or TILLING (Targeting Induced Local Lesions IN Genomes)...
Recent reports make use of the zebrafish to study complex behavior such as addiction, anxiety, or learning and memory. We have established reliable tests and appropriate controls to measure these behavioral parameters in the zebrafish adult. Our assays are robust enough to permit the detection of dominant mutations affecting drug-induced reward, and therefore can be used in forward genetic screens...
The morphological effects of mutation and disease are often critical to our understanding of normal and abnormal function. The power and popularity of zebrafish as a forward and reverse genetic vertebrate model system, combined with its small size, have made it an ideal model in which to study the genetics of histologically scorable phenotypes. The presence of multiple tissue types in this organism’s...
At the turn of the 20th century, classical regenerative biology – the study of organismal/tissue/limb regeneration in animals such as crayfish, snails, and planaria – garnered much attention. However, scientific luminaries such as Thomas Hunt Morgan eventually turned to other fields after concluding that inquiries into regenerative mechanisms were largely intractable beyond observational intrigues...
Zebrafish have been extensively used in biomedical research as a model to study vertebrate development but it is only recently that it has also been adopted into varied fields such as immunology and host–pathogen interactions. Zebrafish have a rapid life cycle, small size and the adults exhibit no territorial behavior in relatively dense cages. Under standard conditions each female lays an average...
Zebrafish gain increasing popularity as animal model for the study of various aspects of modern cell biology as well as model organism for human diseases. This is owed to the fact that zebrafish represent a cost effective and versatile in vivo alternative to in vitro cell culture systems and to invertebrate- and classic rodent models as they combine many strengths of each of these systems. Zebrafish...
The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per...
Genome-wide profiling of DNA methylation and histone modifications answered many questions as to how the genes are regulated on a global scale and what their epigenetic makeup is. Yet, little is known about the function of these marks during early vertebrate embryogenesis. Here we provide detailed protocols for ChIP-seq and MethylCap-seq procedures applied to zebrafish (Danio rerio) embryonic material...
Video processing is increasingly becoming a standard procedure in zebrafish behavior investigations as it enables higher research throughput and new or better measures. This trend, fostered by the ever increasing performance-to-price ratio of the required recording and processing equipment, should be expected to continue in the foreseeable future, with video-processing based methods permeating more...
Rapidly developing imaging technologies including two-photon microscopy and genetically encoded calcium indicators have opened up new possibilities for recording neural population activity in awake, behaving animals. In the small, transparent zebrafish, it is even becoming possible to image the entire brain of a behaving animal with single-cell resolution, creating brain-wide functional maps. In this...
The zebrafish mutation project (ZMP) aims to generate a loss of function allele for every protein-coding gene, but importantly to also characterise the phenotypes of these alleles during the first five days of development. Such a large-scale screen requires a systematic approach both to identifying phenotypes, and also to linking those phenotypes to specific mutations. This phenotyping pipeline simultaneously...
The ability to identify a phenotype causing mutation is essential for successful use of mutagenesis screens in many model organisms. Mapping mutations was for a long time a bottleneck in zebrafish research, as the standard method for mapping and identification of mutations was time consuming and expensive. The development of new sequencing technologies in the last couple of years has enabled the rapid...
The zebrafish Danio rerio has emerged as a powerful vertebrate model system that lends itself particularly well to quantitative investigations with live imaging approaches, owing to its exceptionally high optical clarity in embryonic and larval stages. Recent advances in light microscopy technology enable comprehensive analyses of cellular dynamics during zebrafish embryonic development, systematic...
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