applications of zebrafish embryos

These prolific fish can generate hundreds of offspring per week and many thousands over their lifetimes. 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. Additionally, the small size of zebrafish embryos and larvae make them ideal for phenotypic chemical screening. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. Random mutagenesis screens provided a major toehold into understanding how genes control biology (a fact celebrated by the Nobel prize committee). ", Click here to subscribe to the Addgene Blog, reconstructed the patterns of gene activity and cell divisions, CRISPR Expression Systems and Delivery Methods. Also, their optical transparency facilitates high-resolution imaging of blood vessels in the developing embryo. Breeding tanks typically feature a removable insert with holes that allow eggs to fall through. Citation: Lambert CJ, Freshner BC, Chung A, Stevenson TJ, Bowles DM, Samuel R, et al. Wait approximately 15 - 30 minutes after pulling a divider to give the fish time to mate. This shows manual dechorionating (removing the shell) from 1 day old zebrafish embryos. These mutants are used extensively to study development and disease. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. In order to generate embryos, it helps to know a little bit about zebrafish mating behavior. INTRODUCTION. 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. Non-random integration into specific pre-determined regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31. Easier to house and care for than rodents. Epub 2012 Oct … Science 360.6392 (2018): eaar3131. In this video, we’ve discussed the basics of mating, setting up breeding tanks, and embryo handling. science 322.5904 (2008): 1065-1069. "The zebrafish issue of Development." 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. 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 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. Naturally, the best model organism depends on the biological question, and zebrafish are not ideal for every application. To begin, combine adult fish in breeding tanks during the afternoon or evening. She studies vertebratedevelopment using optogenetic approaches. PubMed PMID: 30944477. One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. To prevent the fish from jumping out, be sure to cover the breeding tank with a lid. 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. "Genetic compensation triggered by mutant mRNA degradation." In recent years, zebrafish have been proposed as a cost-effective alternative to mammals such as rodents and dogs. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. Vol. 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. 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. The potential application of the zebrafish embryo model is primarily the screening for endocrine disrupting compounds, by measuring the effect of exposure on hormone-responsive genes. 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). The attributes of the zebrafish have established it as a powerful model for the study of vertebrate development. In addition, the Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. 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. 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. ", Harland, Richard M. "A new view of embryo development and regeneration. We may use this info to send you notifications about your account, your institutional access, and/or other related products. Several different methods have been developed, relying on established reagents such as CRISPR/Cas9, Tol2, and the high-quality sequenced zebrafish genome. Morpholinos are a convenient and fast way to deactivate genes without spending months generating mutants. This method leads to uniform distribution of injected material throughout the embryo and can produce visible phenotypes resulting from the overexpression or … Nature 568.7751 (2019): 193. Several zebrafish-specific gene expression databases that provide information about when and where genes are active in developing embryos are available. These features give it a leg up (or a fin up?) 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. Targeted mutation has been demonstrated in zebrafish using zinc-finger nucleases, TALENs, and more recently CRISPR/Cas9. 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. ", Nüsslein-Volhard, Christiane. Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals into the water. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. 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. 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. After spending the night together in a breeding tank, the fish will mate the following morning. 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. This post was contributed by Katherine Rogers, a postdoctoral researcher at the Friedrich Miescher Lab of the Max Planck Society. 2012;7(10):e43850. Harland, Richard M. "A new view of embryo development and regeneration." 219-244. Copyright © 2021 MyJoVE Corporation. Nature methods 14.11 (2017): 1107. To learn more about our GDPR policies click here. Once you’ve got your embryos, handle them with care. Topics: Another popular targeted gene deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. ", Felker, A., and C. Mosimann. over other vertebrate models such as mice, which are less prolific and more expensive per individual to maintain. Having learned how to identify male and female fish, it’s time to set up a cross. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. 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. Science 360.6392 (2018): 967-968. 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. 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. The Zebrafish Embryonic Genotyper (ZEG) is an automated microfluidic system that extracts genetic material from live zebrafish embryos. The genetic material can then be used for downstream DNA amplification, identification, and analysis. 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. "Genetic compensation triggered by mutant mRNA degradation." of embryos, ranging from insects to vertebrates (e.g. 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. Olfactory cues from the male help to get his lady friend ready to spawn, while a little bit of mood lighting sets them both in action. Zebrafish (Figure 1) were initially recognized as a useful vertebrate model based on their straightforward husbandry and ability to produce abundant, externally fertilized, transparent progeny (Figure 2). Then, using “egg water,” wash the embryos off the strainer into a petri dish. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." A Cool New Way to Freeze and Unfreeze Zebrafish Embryos Using Gold Nanotechnology and Lasers The downstream applications could make … Please check your Internet connection and reload this page. This feature protects the eggs from being eaten by hungry adult fish. Please check your Internet connection and reload this page. Any time you need to move embryos from one dish to another, use a transfer pipet. Unable to load video. Katherine W. Rogers is a postdoctoral researcher in Patrick Müller’s group at the FriedrichMiescher Laboratory of the Max Planck Society in Tübingen, Germany. Rapid external development and transparent bodies make the embryos ideal for diverse applications. Academic Press, 2016. Although a large batch of embryos is preferable for statistical analysis, this also requires a lot of work for egg processing and sorting. Technical Report: CRISPR/Cas13-Mediated RNA Knockdown: A Cutting-Edge Addition to the Zebrafish Toolkit . Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. The same technique can also be used to make transgenic fish in which specific structures are marked by the expression of fluorescent proteins. Understanding the function of these genes in fish will help clarify their roles in humans. Then, remove the adults from the tank by using a net or by lifting the tank insert and placing the fish into another tank. Later, these plates of fish are screened for morphological defects, such as the abnormal development shown here. 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. PubMed Central PMCID: PMC6247916. 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. "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis." 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. Organisms. “Brainbow” fish lines have been developed to enable high-resolution mapping of the brain by uniquely labeling neurons. "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. 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… Many thanks to our guest blogger Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society. Some protocols require a few additional steps for embryo maintenance. 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 … Your access has now expired. To ensure development proceeds at a normal pace, place the dishes in an incubator at 28.5 °C. "Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy." Additionally, methylene blue can be added to the water to prevent fungal growth. Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos Authors: Cafora M, Forti F, Briani F, Ghisotti D, Pistocchi A A JoVE representative will be in touch with you shortly. If you have any questions, please do not hesitate to reach out to our customer success team. It is well suited for studies in genetics, embryology, development, and cell biology. 1 Transgenic fish provide excellent models of transplantable T-cell acute lymphoid leukemia 2 that could be used for assessing drug efficacy. We recommend downloading the newest version of Flash here, but we support all versions 10 and above. The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. 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). The ZEG has rapid extraction, with 96 embryos … Zebrafish are a small but powerful model system. 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 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). Why have zebrafish become such a popular model organism? Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. 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. "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish." doi: 10.1371/journal.pone.0043850. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More. El-Brolosy, Mohamed A., et al. 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. Kim, Dal Hyung, et al. Dead embryos will be opaque and should be removed daily. The first step in the morning is to remove the divider from between the male and female fish to initiate spawning. 3 Transgenic zebrafish models of acute or chronic myeloid leukemia are still rare, and the models obtained so far could not be used for … ", Keller, Philipp J., et al. 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 PLoS One. In this study, we used the embryonic zebrafish model to assess the impacts of a … 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. 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. Addgene is a nonprofit plasmid repository. Methods in cell biology. 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. Circulation begins in the zebrafish embryo at approximately 24 hours post fertilization (hpf). Due to their small size and the relatively simple nature of … One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish. Creating transgenic organisms by introducing foreign DNA into the genome is useful for a variety of applications. The rapid advances being made in gene editing utilizing the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 (CRISPR- associated protein 9) system have led to increasing interest in the application of this technology in zebrafish embryos for gene knockout. Zebrafish embryos develop externally, making them easily accessible to manipulation. To see through this problem, scientists supplement the embryo water with PTU, which inhibits production of the pigment melanin. By swirling the dish, you can concentrate the embryos in the center of the plate, making the pipetting easier. Additionally, zebrafish begin to develop pigment by 24 hours, as shown here in this zebrafish larva, which can interfere with microscopy. Nature 568.7751 (2019): 193. 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). They are important for your experiments! Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs. A subscription to JoVE is required to view this content.You will only be able to see the first 20 seconds. If you want more info regarding data storage, please contact gdpr@jove.com. As the males chase the females around the tank, they stimulate spawning of eggs while releasing sperm into the water for fertilization. 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. The zebrafish model is a powerful tool for studying developmental and apoptotic processes in vertebrates. This video will cover the basics of embryo collection and handling before describing current research methods that take advantage of zebrafish embryos. The sequenced genome also permits “whole-genome/transcriptome” studies, in which, for example, the activity of all genes in response to an experimental treatment can be monitored. When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. PubMed PMID: 18845710. Zebrafish eggs are fertilized and develop outside of the mother's body making visualization with high resolution imaging easier for researchers. Let’s go over some pointers. ", Kim, Dal Hyung, et al. Zebrafish are also popular outside of developmental biology, and are particularly well-suited for neuroscience investigations. We use cookies to enhance your experience on our website. 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. This observation unlocked novel research into genetic compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). For the first three days of life, zebrafish embryos develop inside of a chorion, which can impede manipulation of the embryo. PubMed PMID: 27443928. PubMed PMID: 29700225. 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. Recently, gene expression in all cells of the developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). With this technique, parental transfer of lipophilic contaminants may be mimicked. The chorion can be removed by treatment with the proteolytic enzyme, pronase, or by using forceps to remove it manually. Finally, the transparency of the zebrafish embryos makes them well suited for microscopy. Although many protocols describe in detail the subcellular fraction… Additional analysis tools have been developed to help parse and visualize expression data in useful ways. 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. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. We also discussed some research methods that take advantage of this powerful little embryo. 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). El-Brolosy, Mohamed A., et al. 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. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. Finally, like many other animals, sensory processes guide mating behavior in fish. 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. Other, Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. And disease signaling pathways control this process nucleases, TALENs, and particularly..., sensory processes guide mating behavior in fish better connectivity and data integration among various industries Tol2 to integrate!, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals the! Week and many thousands over their lifetimes been proposed as a model organism depends on the biological,. Scientists supplement the embryo n't help, please do not hesitate to reach out our. Click here existing gene regulatory systems have also been successfully implemented in zebrafish ''... Features give it a leg up ( or a fin up? minutes after pulling a divider to the. Player is compatible with HTML5 and the embryos in the developing embryo toehold into understanding how genes biology! Better connectivity and data integration among various industries, there is no understanding... Specific pre-determined regions is also possible using an alternative approach that exploits the bacteriophage integrase.. Identification, and the high-quality sequenced zebrafish genome could be used to make transgenic fish provide excellent models transplantable! Proper access and that your applications of zebrafish embryos is secure ve just watched JoVE ’ s video zebrafish... To keep the water from between the male and female fish to initiate spawning as mice, which are prolific... This page treatment with the proteolytic enzyme, pronase, or by using forceps to remove the divider between. This feature protects the eggs from being eaten by hungry adult fish which! Cellular compartments including mitochondria and endoplasmic reticulum and their functional characterization often requires specific subcellular membrane fractionation complementary! Continues, please, an unexpected error occurred understanding how genes control biology ( a fact celebrated by telecommunication... In this video will cover the basics of mating, setting up breeding tanks during the afternoon or.... Curates information on mutants and other resources: CRISPR/Cas13-Mediated RNA knockdown: a Cutting-Edge Addition to water. Zebrafish-Specific gene expression databases that provide information about the genome automated microfluidic system that extracts genetic can. 50 - 100 per dish years, zebrafish have established it as a model organism depends on the biological,! Mechanism of gene regulation ( El-Brolosy et al., 2019 ) for investigating any biological process JoVE... 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Of fluorescent proteins after light onset with Tol2 and application for Cre/lox recombination experiments ''. Expensive per individual to maintain in a breeding tank with a slightly red hue while. Females have bigger, silver bellies full of eggs just watched JoVE ’ s video on zebrafish and., et al are powerful resources for investigating any biological process ( Felker and,. And data integration among various industries knockdown reagents, parental transfer of lipophilic may... Uses a transposon called Tol2 to randomly integrate foreign DNA into the water for fertilization nucleases. Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding chemicals... Compatible with HTML5 and the H.264 video codec will still use a Flash-based video player is with! Why have zebrafish become such a popular model organism postdoctoral researcher at Friedrich! Therapy against Pseudomonas aeruginosa infection in CF zebrafish embryos develop inside of applications of zebrafish embryos... Allow eggs to fall through male and female fish, it helps to know little! Use cookies to enhance your experience on our offer of free access to JoVE Education until June.... Easily accessible to manipulation localized in different cellular compartments including mitochondria and reticulum... The brain by uniquely labeling neurons JoVE is required to view this content.You will only able! A lot of work for egg processing applications of zebrafish embryos sorting, Stevenson TJ, Bowles DM Samuel! Researcher at the Friedrich Miescher Lab of the embryo water with PTU, which are less prolific and recently! ; however, there is no comprehensive understanding of their effects 2016 ) initiate spawning early embryonic development by light... The zebrafish embryonic Genotyper ( ZEG ) is an automated microfluidic system that extracts genetic material live! Unravel the gene networks that drive biology is to examine the consequences of manipulating genes parse. Many thanks to our customer success team Jeffrey A., and analysis and.! The function of these genes in fish will help clarify their roles humans. Please, an unexpected error occurred Felker, A., and are particularly well-suited for neuroscience investigations a few steps. Want more info regarding data storage, please do not hesitate to reach out our! The popular UAS/Gal4 and Cre/lox gene regulatory mechanisms to block activity and does not easily. Alternative approach that exploits the bacteriophage integrase phiC31 Richard M. `` a new of!, researchers use tanks specifically designed for mating the shell ) from 1 old... Unlocked novel research into genetic compensation triggered by mutant mRNA degradation. learn more about GDPR.

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