Epub 2012 Oct … Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs. The genetic material can then be used for downstream DNA amplification, identification, and analysis. We may use this info to send you notifications about your account, your institutional access, and/or other related products. Much of the early modern work using this embryo model began with the papers of Kimmel.Several large laboratories in the US are now developing large breeding programs to carry out \"k… It may be necessary to transfer embryos between dishes to achieve an optimal density of 50 - 100 per dish. Now that you know how to obtain and care for zebrafish embryos, let’s explore some of the ways researchers use them for scientific discovery. Your access has now expired. The Zebrafish Embryonic Genotyper (ZEG) is an automated microfluidic system that extracts genetic material from live zebrafish embryos. (RNAi, in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily in zebrafish.) For zebrafish breeding, researchers use tanks specifically designed for mating. "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. 1 Transgenic fish provide excellent models of transplantable T-cell acute lymphoid leukemia 2 that could be used for assessing drug efficacy. Disadvantages include their duplicated (i.e., redundant) genome, their long three-month generation time and labor-intensive maintenance compared to invertebrate model systems, and their resistance (so far) to facile CRISPR/Cas9-mediated insertion of foreign DNA. "Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. Zebrafish embryos develop externally, making them easily accessible to manipulation. The same technique can also be used to make transgenic fish in which specific structures are marked by the expression of fluorescent proteins. The accessibility of its externally fertilized embryos allows numerous manipulations, and the transparency of the zebrafish embryo permits direct serial visualization of biological processes in vivo (Beis and Stainier, 2006). Technical Report: CRISPR/Cas13-Mediated RNA Knockdown: A Cutting-Edge Addition to the Zebrafish Toolkit . Zebrafish one to eight-cell embryos were microinjected with 6.5 ng VaDNA together with the firefly and Renilla reporter vectors as indicated in Legend to Figure 2 alone or in combination with 4 ng of the indicated morpholinos. Over a period of 24 hours, the externally fertilized zebrafish zygote transforms into an organism with a clearly established body plan including head, eyes, and tail. Having learned how to identify male and female fish, it’s time to set up a cross. This shows manual dechorionating (removing the shell) from 1 day old zebrafish embryos. All rights reserved, Understanding Concentration and Measuring Volumes, Analysis of Apoptosis in Zebrafish Embryos by Whole-mount Immunofluorescence to, Metabolic Profile Analysis of Zebrafish Embryos, Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the. To collect early stage embryos the following morning, separate male and female fish with a divider, which stops them from breeding before you’re ready to begin an experiment. Methods in cell biology. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. Strategies for generating transgenic zebrafish are well established (Felker and Mosimann, 2016). Nature methods 14.11 (2017): 1107. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. The protocol detailed in this work describes the application of phage therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos. PubMed PMID: 28892088. A JoVE representative will be in touch with you shortly. “Brainbow” fish lines have been developed to enable high-resolution mapping of the brain by uniquely labeling neurons. To begin, combine adult fish in breeding tanks during the afternoon or evening. We recommend downloading the newest version of Flash here, but we support all versions 10 and above. Unexpectedly, phenotypes sometimes differ depending on whether a gene is deactivated via mutation or morpholino. Zebrafish or zebra danio (danio rerio) are seen as one of the latest \"models\" for vertebrate embryological development studies. "Genetic compensation triggered by mutant mRNA degradation." To keep embryos healthy, make sure to keep the water in the petri dishes clean and free of debris. A high-quality sequence assembly of the zebrafish genome was published in 2013, revealing that about 70% of human genes have a counterpart in zebrafish. Periodic mating keeps eggs healthy, but making all those eggs takes a lot of work, so individual females should only be mated once per week. The first step in the morning is to remove the divider from between the male and female fish to initiate spawning. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. Breeding tanks typically feature a removable insert with holes that allow eggs to fall through. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. 3 Transgenic zebrafish models of acute or chronic myeloid leukemia are still rare, and the models obtained so far could not be used for … Organisms. ", Keller, Philipp J., et al. After spending the night together in a breeding tank, the fish will mate the following morning. The zebrafish model is a powerful tool for studying developmental and apoptotic processes in vertebrates. The exposure of zebrafish embryos was performed according to the exposure scenario described for the fish embryo acute toxicity test with the exception of 10 instead of 5 embryos per glass vial. If you would like to continue using JoVE, please let your librarian know as they consider the most appropriate subscription options for your institution’s academic community. This observation unlocked novel research into genetic compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). By swirling the dish, you can concentrate the embryos in the center of the plate, making the pipetting easier. In this study, we used the embryonic zebrafish model to assess the impacts of a … Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More. "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish. PLoS ONE (2012-01-01) . Copyright © 2021 MyJoVE Corporation. "Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy." Additionally, zebrafish begin to develop pigment by 24 hours, as shown here in this zebrafish larva, which can interfere with microscopy. "The zebrafish issue of Development." Non-random integration into specific pre-determined regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31. The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. Keller, Philipp J., et al. Additional mutagenesis projects have since been initiated and today thousands of mutations in genes affecting diverse biological processes—not just development—are available from strain distribution centers in the US, Europe, and China. Topics: El-Brolosy, Mohamed A., et al. These mutants are used extensively to study development and disease. Morpholinos are a convenient and fast way to deactivate genes without spending months generating mutants. These embryos have the great advantage that they develop as \"see through\" embryos, that is, all internal development can be clearly observed from the outside in the living embryo. science 322.5904 (2008): 1065-1069. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. In this video, we’ve discussed the basics of mating, setting up breeding tanks, and embryo handling. Felker, A., and C. Mosimann. Finally, like many other animals, sensory processes guide mating behavior in fish. Additional analysis tools have been developed to help parse and visualize expression data in useful ways. Rapid external development and transparent bodies make the embryos ideal for diverse applications. Thanks for watching! PubMed PMID: 30944477. They are important for your experiments! Zebrafish are also popular outside of developmental biology, and are particularly well-suited for neuroscience investigations. Adult zebrafish (Danio rerio) were maintained according to standard laboratory procedures.19 Embryos and larvae were raised in embryo water (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2, 0.33 mM MgSO 4) in an incubator at 28.5°C. These prolific fish can generate hundreds of offspring per week and many thousands over their lifetimes. We also discussed some research methods that take advantage of this powerful little embryo. Recently, gene expression in all cells of the developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). We outline broad applications of this method in: (i) visualizing the biomolecular distribution of whole embryos in three dimensions, (ii) resolving anatomical features at subcellular spatial resolution, (iii) biomolecular profiling and discrimination of wild type and ΔRD1 mutant Mycobacterium marinum strains in a zebrafish embryo model of tuberculosis and (iv) in vivo temporal monitoring of the wound … Another popular targeted gene deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. The JoVE video player is compatible with HTML5 and Adobe Flash. ", Felker, A., and C. Mosimann. Then, leave the fish to acclimate to each other overnight. This microscopy-based lineage tracing defined a high-resolution map of early embryogenesis in time and space, and was also used to precisely characterize developmental defects in the one-eyed pinhead mutant from the random mutagenesis screens. Academic Press, 2016. Once you’ve got your embryos, handle them with care. Citation: Lambert CJ, Freshner BC, Chung A, Stevenson TJ, Bowles DM, Samuel R, et al. ", Nüsslein-Volhard, Christiane. Solvent Violet 47 (SV47) and Disperse Blue 14 (DB14) are two anthraquinone dyes that were previously used in different formulations for the production of violet-colored smoke. Zebrafish mutants provide a blueprint for the effects of the loss of activity of proteins and, in combination with compound derivatization, can aid the development of drugs with greater specificity in whole animals. The attributes of the zebrafish have established it as a powerful model for the study of vertebrate development. Targeted mutation has been demonstrated in zebrafish using zinc-finger nucleases, TALENs, and more recently CRISPR/Cas9. "The zebrafish issue of Development. Dead embryos will be opaque and should be removed daily. This method leads to uniform distribution of injected material throughout the embryo and can produce visible phenotypes resulting from the overexpression or … Zebrafish (Danio rerio) have been used since the 1930’s in a range of biological studies, including investigations into environmental pollutants and health, embryo growth, brain function, and disease development. over other vertebrate models such as mice, which are less prolific and more expensive per individual to maintain. Microscopy-based lineage tracing can help illuminate how signaling pathways—such as those regulated by one-eyed pinhead—contribute to the “sculpting” of tissues and organs during embryo development. The ZEG has rapid extraction, with 96 embryos … But as the field of genetics progresses and genome sequences become available, new hypotheses about specific genetic interactions arise and tools to deactivate specific genes are needed. Please check your Internet connection and reload this page. It is well suited for studies in genetics, embryology, development, and cell biology. This post was contributed by Katherine Rogers, a postdoctoral researcher at the Friedrich Miescher Lab of the Max Planck Society. Zebrafish can indeed produce up to 300 embryos weekly, hence why they are referred to as prolific breeders (2). Science 360.6392 (2018): eaar3131. INTRODUCTION. Although many protocols describe in detail the subcellular fraction… Infection of Zebrafish Embryos with Intracellular Bacterial Pathogens Transparent zebrafish embryos have proved useful model hosts to visualize and functionally study interactions between innate immune cells and intracellular bacterial pathogens, such as Salmonella… Any time you need to move embryos from one dish to another, use a transfer pipet. Science 360.6392 (2018): 967-968. "Genetic compensation triggered by mutant mRNA degradation." PubMed, Farrell, Jeffrey A., et al. Zebrafish embryos, used as the species under study in this work, were raised within the acoustic field by the authors, allowing the biological effects on such early zebrafish embryos, at each developmental stage and within the ASWF creating the acoustic levitation (AL) technology used, to be studied. ", Click here to subscribe to the Addgene Blog, reconstructed the patterns of gene activity and cell divisions, CRISPR Expression Systems and Delivery Methods. mouse, frog, fish). The chorion can be removed by treatment with the proteolytic enzyme, pronase, or by using forceps to remove it manually. Vol. If the problem continues, please, An unexpected error occurred. This can reveal previously unknown functions for genes, and lays the groundwork for understanding the gene networks that regulate processes from development to metabolism to fertility. If you want more info regarding data storage, please contact gdpr@jove.com. Posted by These features give it a leg up (or a fin up?) This study also showed that in the absence of the signaling pathway regulated by one-eyed pinhead, incorrect proportions of otherwise normal cell types are produced, rather than novel, abnormal cell types. Nüsslein-Volhard, Christiane. Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos Authors: Cafora M, Forti F, Briani F, Ghisotti D, Pistocchi A Compared to the opaque mouse embryo, where similar processes occur over days inside the mother, the zebrafish is an ideal system to observe embryogenesis live. Several different methods have been developed, relying on established reagents such as CRISPR/Cas9, Tol2, and the high-quality sequenced zebrafish genome. If the problem continues, please. Harland, Richard M. "A new view of embryo development and regeneration." Farrell, Jeffrey A., et al. Creating transgenic organisms by introducing foreign DNA into the genome is useful for a variety of applications. She studies vertebratedevelopment using optogenetic approaches. In addition, zebrafish have been successful disease models, and are used to study human illnesses such as blood disorders, diabetes, cancer, and Alzheimer’s disease. Next, pour the water and the embryos through a mesh tea strainer for collection. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. Many thanks to our guest blogger Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society. Microinjection was tested as a potentially powerful tool to introduce natural and anthropogenic pollutants directly into fish eggs to determine their toxicological impact on fish. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae PLoS One. If that doesn't help, please let us know. To illustrate some of the reasons that zebrafish have been embraced as a model organism, I’ll outline a few of the available genetic resources and highlight two recent breakthroughs in developmental biology that arose from zebrafish-based investigations. Despite these caveats, several features of zebrafish—including the available genetic resources and transparency of the embryo—have proven useful in a variety of research applications. Unable to load video. The zebrafish were handled in accordance with Guangdong state regulations on laboratory animal management Some protocols require a few additional steps for embryo maintenance. Due to their small size and the relatively simple nature of … The ICP-MS results showed that the zebrafish embryos can uptake Ti 3 C 2 T x in a dose dependent manner. The use of zebrafish embryos to assess off-target effects of drugs. This method leads to uniform distribution of injected material throughout the embryo and can produce visible phenotypes resulting from the overexpression or loss of a protein. For example, neuronal activity can be tracked in whole brains of swimming, actively learning larvae, providing unprecedented views into cognitive processes like decision making (Kim et al, 2017). Nature 568.7751 (2019): 193. With this technique, parental transfer of lipophilic contaminants may be mimicked. Circulation begins in the zebrafish embryo at approximately 24 hours post fertilization (hpf). Sequencing-based lineage tracing can help elucidate the gene networks that transform zygotes into adults and explain how signaling pathways control this process. 135. "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish." The origins, divisions, and movements of each of the thousands of cells in the developing zebrafish embryo have been tracked using microscopy and advanced computational approaches (Keller et al., 2008). Fluorescence techniques used to probe biomolecular dynamics and interactions of molecules in live zebrafish embryos are therefore highly sought-after to bridge molecular and developmental events. Random mutagenesis screens provided a major toehold into understanding how genes control biology (a fact celebrated by the Nobel prize committee). Kim, Dal Hyung, et al. We use cookies to enhance your experience on our website. doi: 10.1371/journal.pone.0043850. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. You’ve just watched JoVE’s video on zebrafish breeding and embryo handling. This feature protects the eggs from being eaten by hungry adult fish. To highlight some of the features that make zebrafish useful, I’ll outline two recent lineage tracing approaches that led to important insights in developmental biology. For example, specific tissues such as blood cells or neurons can be fluorescently labeled, allowing researchers to observe changes to these tissues under different experimental conditions. In order to generate embryos, it helps to know a little bit about zebrafish mating behavior. Please check your Internet connection and reload this page. Lineage tracing studies highlight how these advantages can translate into insights in developmental biology, and advances in this field and others are likely to continue with help from zebrafish. The acute toxicity of attached/absorbed Ti 3 C 2 T x was tested at concentrations of 25, 50, 100 and 200 μg mL −1. Easier to house and care for than rodents. When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. To prevent the fish from jumping out, be sure to cover the breeding tank with a lid. Addgene is a nonprofit plasmid repository. El-Brolosy, Mohamed A., et al. By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies. To see through this problem, scientists supplement the embryo water with PTU, which inhibits production of the pigment melanin. In recent years, zebrafish have been proposed as a cost-effective alternative to mammals such as rodents and dogs. Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. Development 139.22 (2012): 4099-4103. Nature 568.7751 (2019): 193. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." In this Application Note, we describe the microinjection procedure for targeted knock-out and knock-in using the InjectMan ® 4 micromanipulator and FemtoJet 4 micro-injector, allowing for the efficient and rapid injection of hundreds of fish (medaka and zebrafish) zygotes. Zebrafish are a small but powerful model system. Let’s go over some pointers. We use/store this info to ensure you have proper access and that your account is secure. Additionally, the small size of zebrafish embryos and larvae make them ideal for phenotypic chemical screening. Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals into the water. 219-244. Several zebrafish-specific gene expression databases that provide information about when and where genes are active in developing embryos are available. A subscription to JoVE is required to view this content.You will only be able to see the first 20 seconds. To learn more about our GDPR policies click here. In addition, the Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. PubMed Central PMCID: PMC6247916. A Cool New Way to Freeze and Unfreeze Zebrafish Embryos Using Gold Nanotechnology and Lasers The downstream applications could make … Wait approximately 15 - 30 minutes after pulling a divider to give the fish time to mate. One study profiled 38,731 cells from 694 embryos over multiple stages and reconstructed the patterns of gene activity and cell divisions that coordinate development of the body plan (Farrell et al., 2018). PubMed PMID: 29853675. ", Harland, Richard M. "A new view of embryo development and regeneration. PubMed PMID: 29700225. Bcl-2 proteins can be localized in different cellular compartments including mitochondria and endoplasmic reticulum and their functional characterization often requires specific subcellular membrane fractionation. One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Ever since the discovery of gene silencing by double-stranded RNA (dsRNA)-induced RNA interference (RNAi) in worms and plants, a number of methods have been used to interfere with functions of gene products (without genetic manipulation) across organisms including Zebrafish. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. For the first three days of life, zebrafish embryos develop inside of a chorion, which can impede manipulation of the embryo. Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. Although a large batch of embryos is preferable for statistical analysis, this also requires a lot of work for egg processing and sorting. To ensure development proceeds at a normal pace, place the dishes in an incubator at 28.5 °C. PubMed PMID: 18845710. ", Kim, Dal Hyung, et al. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. Then, remove the adults from the tank by using a net or by lifting the tank insert and placing the fish into another tank. In the 1990’s, two revolutionary large-scale random mutagenesis screens produced ~1,500 zebrafish mutants that are still used in labs across the globe (Nüsslein-Volhard et al., 2012). The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. This video will cover the basics of embryo collection and handling before describing current research methods that take advantage of zebrafish embryos. One example (which I will come back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital heart defects and cancer. Zebrafish and humans share 70% of the same genes. Live embryos expressing fluorescent proteins in specific cell types can be mounted and visualized with confocal microscopy, allowing researchers to examine cell movements in developing tissues over time. Katherine W. Rogers is a postdoctoral researcher in Patrick Müller’s group at the FriedrichMiescher Laboratory of the Max Planck Society in Tübingen, Germany. "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis." Zebrafish are also amenable to manipulation of brain activity: optogenetic approaches can be used to determine the consequences of activating or silencing specific neurons in living larvae. Finally, the transparency of the zebrafish embryos makes them well suited for microscopy. Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. Phenotype-based high content screens (PHCS) using zebrafish embryos are widely used in rapid chemical toxicity testing, which can simultaneously assess multiple apical endpoints (e.g., mortality and malformation effects). The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries.