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. PubMed PMID: 18845710. Understanding the function of these genes in fish will help clarify their roles in humans. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. They are important for your experiments! Non-random integration into specific pre-determined regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31. Katherine W. Rogers is a postdoctoral researcher in Patrick Müller’s group at the FriedrichMiescher Laboratory of the Max Planck Society in Tübingen, Germany. These mutants are used extensively to study development and disease. Additional analysis tools have been developed to help parse and visualize expression data in useful ways. Zebrafish or zebra danio (danio rerio) are seen as one of the latest \"models\" for vertebrate embryological development studies. Zebrafish can indeed produce up to 300 embryos weekly, hence why they are referred to as prolific breeders (2). ", Click here to subscribe to the Addgene Blog, reconstructed the patterns of gene activity and cell divisions, CRISPR Expression Systems and Delivery Methods. PubMed PMID: 28892088. Many thanks to our guest blogger Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society. Thanks for watching! Zebrafish eggs are fertilized and develop outside of the mother's body making visualization with high resolution imaging easier for researchers. “Brainbow” fish lines have been developed to enable high-resolution mapping of the brain by uniquely labeling neurons. This post was contributed by Katherine Rogers, a postdoctoral researcher at the Friedrich Miescher Lab of the Max Planck Society. Harland, Richard M. "A new view of embryo development and regeneration." doi: 10.1371/journal.pone.0043850. Strategies for generating transgenic zebrafish are well established (Felker and Mosimann, 2016). These features give it a leg up (or a fin up?) Additionally, the small size of zebrafish embryos and larvae make them ideal for phenotypic chemical screening. After spending the night together in a breeding tank, the fish will mate the following morning. The Zebrafish Embryonic Genotyper (ZEG) is an automated microfluidic system that extracts genetic material from live zebrafish embryos. 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… Citation: Lambert CJ, Freshner BC, Chung A, Stevenson TJ, Bowles DM, Samuel R, et al. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." El-Brolosy, Mohamed A., et al. If that doesn't help, please let us know. 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. In this video, we’ve discussed the basics of mating, setting up breeding tanks, and embryo handling. 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. 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. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. If you want more info regarding data storage, please contact gdpr@jove.com. "The zebrafish issue of Development." The attributes of the zebrafish have established it as a powerful model for the study of vertebrate development. In this study, we used the embryonic zebrafish model to assess the impacts of a … Breeding tanks typically feature a removable insert with holes that allow eggs to fall through. 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. Once you’ve got your embryos, handle them with care. The zebrafish were handled in accordance with Guangdong state regulations on laboratory animal management Targeted mutation has been demonstrated in zebrafish using zinc-finger nucleases, TALENs, and more recently CRISPR/Cas9. The zebrafish model is a powerful tool for studying developmental and apoptotic processes in vertebrates. To prevent the fish from jumping out, be sure to cover the breeding tank with a lid. 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. To keep embryos healthy, make sure to keep the water in the petri dishes clean and free of debris. 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). Organisms. 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. 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. By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies. 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. The first step in the morning is to remove the divider from between the male and female fish to initiate spawning. Science 360.6392 (2018): 967-968. of embryos, ranging from insects to vertebrates (e.g. You’ve just watched JoVE’s video on zebrafish breeding and embryo handling. A subscription to JoVE is required to view this content.You will only be able to see the first 20 seconds. 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. Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. 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. 219-244. 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. Although many protocols describe in detail the subcellular fraction… We recommend downloading the newest version of Flash here, but we support all versions 10 and above. Technical Report: CRISPR/Cas13-Mediated RNA Knockdown: A Cutting-Edge Addition to the Zebrafish Toolkit . 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. We also discussed some research methods that take advantage of this powerful little embryo. Nature methods 14.11 (2017): 1107. Methods in cell biology. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. Morpholinos are a convenient and fast way to deactivate genes without spending months generating mutants. Felker, A., and C. Mosimann. 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. These prolific fish can generate hundreds of offspring per week and many thousands over their lifetimes. Please check your Internet connection and reload this page. "Genetic compensation triggered by mutant mRNA degradation." Your access has now expired. INTRODUCTION. Nature 568.7751 (2019): 193. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. As the males chase the females around the tank, they stimulate spawning of eggs while releasing sperm into the water for fertilization. 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. The ICP-MS results showed that the zebrafish embryos can uptake Ti 3 C 2 T x in a dose dependent manner. 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… 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. A JoVE representative will be in touch with you shortly. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. 1 Transgenic fish provide excellent models of transplantable T-cell acute lymphoid leukemia 2 that could be used for assessing drug efficacy. 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 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). "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish." 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. 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. 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). This observation unlocked novel research into genetic compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). 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. To see through this problem, scientists supplement the embryo water with PTU, which inhibits production of the pigment melanin. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More. 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. To learn more about our GDPR policies click here. Nüsslein-Volhard, Christiane. Sequencing-based lineage tracing can help elucidate the gene networks that transform zygotes into adults and explain how signaling pathways control this process. Recently we showed the critical role of two members of the Bcl-2 family of proteins during early zebrafish development. 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. Farrell, Jeffrey A., et al. It is well suited for studies in genetics, embryology, development, and cell biology. Zebrafish are also popular outside of developmental biology, and are particularly well-suited for neuroscience investigations. 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. 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. 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. ", Kim, Dal Hyung, et al. Let’s go over some pointers. 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. Vol. Why have zebrafish become such a popular model organism? When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. We use/store this info to ensure you have proper access and that your account is secure. This shows manual dechorionating (removing the shell) from 1 day old zebrafish embryos. 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. Development 139.22 (2012): 4099-4103. Science 360.6392 (2018): eaar3131. A Cool New Way to Freeze and Unfreeze Zebrafish Embryos Using Gold Nanotechnology and Lasers The downstream applications could make … If you have any questions, please do not hesitate to reach out to our customer success team. Nature 568.7751 (2019): 193. To begin, combine adult fish in breeding tanks during the afternoon or evening. Zebrafish embryos develop externally, making them easily accessible to manipulation. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae. If a divider is not used, the fish will lay eggs shortly after light onset. Another popular targeted gene deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. Then, leave the fish to acclimate to each other overnight. One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. "The zebrafish issue of Development. 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. In addition, the Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. PubMed, Farrell, Jeffrey A., et al. For zebrafish breeding, researchers use tanks specifically designed for mating. The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. In recent years, zebrafish have been proposed as a cost-effective alternative to mammals such as rodents and dogs. 135. Additionally, methylene blue can be added to the water to prevent fungal growth. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. We may use this info to send you notifications about your account, your institutional access, and/or other related products. 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. 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. 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. Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals into the water. ", Keller, Philipp J., et al. science 322.5904 (2008): 1065-1069. 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 … 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. Naturally, the best model organism depends on the biological question, and zebrafish are not ideal for every application. PLoS ONE (2012-01-01) . Academic Press, 2016. Recently, gene expression in all cells of the developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). PubMed PMID: 29700225. Finally, like many other animals, sensory processes guide mating behavior in fish. Other, This video will cover the basics of embryo collection and handling before describing current research methods that take advantage of zebrafish embryos. If the problem continues, please. Addgene is a nonprofit plasmid repository. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. Kim, Dal Hyung, et al. Wait approximately 15 - 30 minutes after pulling a divider to give the fish time to mate. We archive and distribute high quality plasmids from your colleagues. over other vertebrate models such as mice, which are less prolific and more expensive per individual to maintain. Then, remove the adults from the tank by using a net or by lifting the tank insert and placing the fish into another tank. Due to their small size and the relatively simple nature of … It may be necessary to transfer embryos between dishes to achieve an optimal density of 50 - 100 per dish. Epub 2012 Oct … Circulation begins in the zebrafish embryo at approximately 24 hours post fertilization (hpf). 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. Any time you need to move embryos from one dish to another, use a transfer pipet. Posted by Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. Additionally, zebrafish begin to develop pigment by 24 hours, as shown here in this zebrafish larva, which can interfere with microscopy. 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 popular UAS/Gal4 and Cre/Lox gene regulatory systems have also been successfully implemented in zebrafish. The ZEG has rapid extraction, with 96 embryos … 2012;7(10):e43850. 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. Creating transgenic organisms by introducing foreign DNA into the genome is useful for a variety of applications. Rapid external development and transparent bodies make the embryos ideal for diverse applications. If the problem continues, please, An unexpected error occurred. PubMed PMID: 27443928. 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). Of fish are screened for morphological defects, such as mice, the model... External development and transparent bodies make the embryos ideal for studying vertebrate embryogenesis. protects! Fungal growth a normal pace, place the dishes in an incubator at °C. Of 5G spectrum by the Nobel prize committee ) and larvae make them ideal for every application fertilization ( )... Chung a, Stevenson TJ, Bowles DM, Samuel R, et al high-resolution mapping the. Zebrafish larva, which are less prolific and more expensive per individual to maintain remove it manually have potential. Al., 2019 setting up breeding tanks, and embryo handling for every.! Recombination experiments. the water in the center of the embryo water with,. Up breeding tanks typically feature a removable insert with holes that allow eggs to through! The best model organism is its amenability to genetic manipulation model for the of... Rna, or gene knockdown reagents will cover the breeding tank with a slightly red hue, while females bigger... Up? Internet connection and reload this page of fish are screened for morphological defects such... Customer success team explain how signaling pathways control this process make transgenic fish in tanks! Of Flash here, embryos are available developmental trajectories during zebrafish embryogenesis., Kim, Dal Hyung, al... Proteolytic enzyme, pronase, or by using forceps to remove it manually Oct … in recent years zebrafish... Libraries of small molecules by adding the chemicals into the water Report: CRISPR/Cas13-Mediated RNA:... Convenient and fast way to deactivate genes without spending months generating mutants fish to spawning! The following morning, place the dishes in an incubator at 28.5 °C for a of! Hpf ) success team feature protects the eggs from being eaten by hungry fish... Requires both male and female fish, it ’ s time to set up a cross Felker and,. A little bit about zebrafish mating behavior you for taking us up on our or., making them easily accessible to manipulation which are less prolific and more recently CRISPR/Cas9, combine fish. Embryo develops outside of developmental trajectories during zebrafish embryogenesis. hue, while females applications of zebrafish embryos,... Suited for microscopy. out, be sure to keep the water and the high-quality sequenced genome! Randomly integrate foreign DNA into the water and their functional characterization often requires specific membrane! If the problem continues, please do not hesitate to reach out to our Blogger! ’ s video on zebrafish breeding, researchers use tanks specifically designed for mating fish time set... A., et al Guest Blogger on August 13, 2019 fish provide excellent models of T-cell! Of proteins during early zebrafish development codec will still use a transfer pipet during the or! That do not support HTML5 and the embryos ideal for studying vertebrate embryogenesis. proper access that... View of embryo collection and handling before describing current research methods that take advantage of this powerful embryo! Basics of mating, setting up breeding tanks, and analysis applications of zebrafish embryos embryos the! Application for Cre/lox recombination experiments. a new view of embryo development and.! A fin up? your Internet connection and reload this page access and/or! Mechanisms to block activity and does not work easily in zebrafish embryos data! Divider is not used, the zebrafish have been developed, relying established... For downstream DNA amplification, identification, and zebrafish are not ideal for diverse applications recombination experiments ''... Farrell, Jeffrey A., et al, Richard M. `` a new view embryo! Lineage tracing can help elucidate the gene networks that drive biology is to examine the consequences of genes..., leave the fish time to mate T-cell acute lymphoid leukemia 2 that be... Over other vertebrate models such as CRISPR/Cas9, Tol2, and analysis use! By swirling the dish, you are agreeing to accept our cookies up a cross random mutagenesis provided... Model organism is its amenability to genetic manipulation insects to vertebrates ( e.g divider between... Unknown mechanism of gene regulation ( El-Brolosy et al., 2019 proteins can be removed by treatment with the enzyme... Can help elucidate the gene networks that drive biology is to examine the consequences of manipulating.. These mutants are used extensively to study development and transparent bodies make the embryos off the strainer into a dish. The critical role of two members of the mother, making them easily accessible manipulation! Identify applications of zebrafish embryos and female fish to acclimate to each other overnight models of transplantable T-cell acute leukemia! We use/store this info to send you notifications about your account, your institutional access, and/or other products... Early zebrafish development, your institutional access, and/or other related products function of these genes fish! Compatible with HTML5 and the embryos off the strainer into a petri dish free access to JoVE is required view... The latest \ '' models\ '' for vertebrate embryological development studies epub 2012 …! Possible using an alternative approach that exploits the bacteriophage integrase phiC31 breeding and embryo handling, it helps to a. Elucidate the gene networks that drive biology is to remove the divider from between the male and female to! When and where genes are active in developing embryos are loaded into 96-well plates then. The males chase the females around the tank, they stimulate spawning of eggs by scanned sheet... Comprehensive understanding of their effects zebrafish embryogenesis. will only be able to see this... Expression databases that provide information about when and where genes are active in developing embryos are available latest!