Methods in cell biology. "Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy. If you have any questions, please do not hesitate to reach out to our customer success team. Breeding tanks typically feature a removable insert with holes that allow eggs to fall through. "Genetic compensation triggered by mutant mRNA degradation." This method leads to uniform distribution of injected material throughout the embryo and can produce visible phenotypes resulting from the overexpression or … "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis." They are important for your experiments! If the problem continues, please. 2012;7(10):e43850. For zebrafish breeding, researchers use tanks specifically designed for mating. PubMed, Farrell, Jeffrey A., et al. Additional analysis tools have been developed to help parse and visualize expression data in useful ways. 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). Many thanks to our guest blogger Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society. Another popular targeted gene deactivation method in zebrafish are short, modified oligonucleotides called morpholinos. El-Brolosy, Mohamed A., et al. Zebrafish undergo sexual reproduction, so breeding requires both male and female fish. Recently, gene expression in all cells of the developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). 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. Please check your Internet connection and reload this page. One common and robust strategy uses a transposon called Tol2 to randomly integrate foreign DNA into the genome. Organisms. Guest Blogger on August 13, 2019. PubMed PMID: 28892088. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments." of embryos, ranging from insects to vertebrates (e.g. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. mouse, frog, fish). 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. Zebrafish are also popular outside of developmental biology, and are particularly well-suited for neuroscience investigations. PubMed PMID: 29853675. (RNAi, in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily in zebrafish.) The ZEG has rapid extraction, with 96 embryos … Circulation begins in the zebrafish embryo at approximately 24 hours post fertilization (hpf). The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. Any time you need to move embryos from one dish to another, use a transfer pipet. It is well suited for studies in genetics, embryology, development, and cell biology. The zebrafish were handled in accordance with Guangdong state regulations on laboratory animal management With this technique, parental transfer of lipophilic contaminants may be mimicked. PLoS ONE (2012-01-01) . PubMed PMID: 29700225. 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). 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. These features give it a leg up (or a fin up?) Here, embryos are loaded into 96-well plates and then treated with libraries of small molecules by adding the chemicals into the water. A JoVE representative will be in touch with you shortly. Zebrafish embryos develop externally, making them easily accessible to manipulation. 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). Both dyes have shown potential for toxicity; however, there is no comprehensive understanding of their effects. Once you’ve got your embryos, handle them with care. 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. Keller, Philipp J., et al. Dead embryos will be opaque and should be removed daily. In recent years, zebrafish have been proposed as a cost-effective alternative to mammals such as rodents and dogs. This observation unlocked novel research into genetic compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). Non-random integration into specific pre-determined regions is also possible using an alternative approach that exploits the bacteriophage integrase phiC31. Kim, Dal Hyung, et al. 219-244. 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. 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. 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. PubMed Central PMCID: PMC6247916. When injected into zygotes, morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. Posted by We archive and distribute high quality plasmids from your colleagues. Then, leave the fish to acclimate to each other overnight. 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. Let’s go over some pointers. "Contemporary zebrafish transgenesis with Tol2 and application for Cre/lox recombination experiments. Academic Press, 2016. A Cool New Way to Freeze and Unfreeze Zebrafish Embryos Using Gold Nanotechnology and Lasers The downstream applications could make … 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. Older browsers that do not support HTML5 and the H.264 video codec will still use a Flash-based video player. Creating transgenic organisms by introducing foreign DNA into the genome is useful for a variety of applications. "Single-cell reconstruction of developmental trajectories during zebrafish embryogenesis. 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. We also discussed some research methods that take advantage of this powerful little embryo. The attributes of the zebrafish have established it as a powerful model for the study of vertebrate development. 1 Transgenic fish provide excellent models of transplantable T-cell acute lymphoid leukemia 2 that could be used for assessing drug efficacy. Zebrafish and humans share 70% of the same genes. Epub 2012 Oct … ", Nüsslein-Volhard, Christiane. Evaluation of 14 organic solvents and carriers for screening applications in zebrafish embryos and larvae PLoS One. Sequencing-based lineage tracing can help elucidate the gene networks that transform zygotes into adults and explain how signaling pathways control this process. We use cookies to enhance your experience on our website. Males have slimmer bodies with a slightly red hue, while females have bigger, silver bellies full of eggs. The external development of zebrafish embryos allows for genetic manipulation by microinjection of DNA, RNA, or gene knockdown reagents. "Genetic compensation triggered by mutant mRNA degradation." 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. In this study, we used the embryonic zebrafish model to assess the impacts of a … Technical Report: CRISPR/Cas13-Mediated RNA Knockdown: A Cutting-Edge Addition to the Zebrafish Toolkit . To see through this problem, scientists supplement the embryo water with PTU, which inhibits production of the pigment melanin. Zebrafish are a small but powerful model system. Science 360.6392 (2018): eaar3131. The same technique can also be used to make transgenic fish in which specific structures are marked by the expression of fluorescent proteins. It may be necessary to transfer embryos between dishes to achieve an optimal density of 50 - 100 per dish. 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. 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. Addgene is a nonprofit plasmid repository. You’ve just watched JoVE’s video on zebrafish breeding and embryo handling. We recommend downloading the newest version of Flash here, but we support all versions 10 and above. 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). Then, using “egg water,” wash the embryos off the strainer into a petri dish. 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. Vol. 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. INTRODUCTION. Zebrafish eggs are fertilized and develop outside of the mother's body making visualization with high resolution imaging easier for researchers. The ICP-MS results showed that the zebrafish embryos can uptake Ti 3 C 2 T x in a dose dependent manner. Unable to load video. “Brainbow” fish lines have been developed to enable high-resolution mapping of the brain by uniquely labeling neurons. Also in contrast to mice, the transparent zebrafish embryo develops outside of the mother, making them ideal for studying vertebrate embryogenesis. Finally, like many other animals, sensory processes guide mating behavior in fish. Also, their optical transparency facilitates high-resolution imaging of blood vessels in the developing embryo. Copyright © 2021 MyJoVE Corporation. Please check your Internet connection and reload this page. This post was contributed by Katherine Rogers, a postdoctoral researcher at the Friedrich Miescher Lab of the Max Planck Society. Nüsslein-Volhard, Christiane. doi: 10.1371/journal.pone.0043850. Your access has now expired. Then, remove the adults from the tank by using a net or by lifting the tank insert and placing the fish into another tank. Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. If a divider is not used, the fish will lay eggs shortly after light onset. 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. One reason for the success of zebrafish as a model organism is its amenability to genetic manipulation. 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 … 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 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. ", Felker, A., and C. Mosimann. Phage Therapy Application to Counteract Pseudomonas aeruginosa Infection in Cystic Fibrosis Zebrafish Embryos Authors: Cafora M, Forti F, Briani F, Ghisotti D, Pistocchi A The popular UAS/Gal4 and Cre/Lox gene regulatory systems have also been successfully implemented in zebrafish. These prolific fish can generate hundreds of offspring per week and many thousands over their lifetimes. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More. In this video, we’ve discussed the basics of mating, setting up breeding tanks, and embryo handling. Nature 568.7751 (2019): 193. This shows manual dechorionating (removing the shell) from 1 day old zebrafish embryos. Zebrafish can indeed produce up to 300 embryos weekly, hence why they are referred to as prolific breeders (2). 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. PubMed PMID: 30944477. 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. The use of zebrafish embryos to assess off-target effects of drugs. 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. These mutants are used extensively to study development and disease. Science 360.6392 (2018): 967-968. Although many protocols describe in detail the subcellular fraction… By swirling the dish, you can concentrate the embryos in the center of the plate, making the pipetting easier. 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. Nature 568.7751 (2019): 193. For the first three days of life, zebrafish embryos develop inside of a chorion, which can impede manipulation of the embryo. The rapid deployment of 5G spectrum by the telecommunication industry is intended to promote better connectivity and data integration among various industries. PubMed PMID: 27443928. We may use this info to send you notifications about your account, your institutional access, and/or other related products. To keep embryos healthy, make sure to keep the water in the petri dishes clean and free of debris. Development 139.22 (2012): 4099-4103. Tools for gene manipulation together with information about the genome are powerful resources for investigating any biological process. Targeted mutation has been demonstrated in zebrafish using zinc-finger nucleases, TALENs, and more recently CRISPR/Cas9. Thanks for watching! Naturally, the best model organism depends on the biological question, and zebrafish are not ideal for every application. 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. 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. 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… Additionally, the small size of zebrafish embryos and larvae make them ideal for phenotypic chemical screening. 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. Recently we showed the critical role of two members of the Bcl-2 family of proteins during early zebrafish development. 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. 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. science 322.5904 (2008): 1065-1069. 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. Wait approximately 15 - 30 minutes after pulling a divider to give the fish time to mate. By continuing to use our website or clicking “Continue”, you are agreeing to accept our cookies. To ensure development proceeds at a normal pace, place the dishes in an incubator at 28.5 °C. Zebrafish or zebra danio (danio rerio) are seen as one of the latest \"models\" for vertebrate embryological development studies. The Zebrafish Embryonic Genotyper (ZEG) is an automated microfluidic system that extracts genetic material from live zebrafish embryos. The first step in the morning is to remove the divider from between the male and female fish to initiate spawning. Due to their small size and the relatively simple nature of … 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. Morpholinos are a convenient and fast way to deactivate genes without spending months generating mutants. PubMed PMID: 18845710. In order to generate embryos, it helps to know a little bit about zebrafish mating behavior. Easier to house and care for than rodents. To begin, combine adult fish in breeding tanks during the afternoon or evening. ", Harland, Richard M. "A new view of embryo development and regeneration. After spending the night together in a breeding tank, the fish will mate the following morning. This video will cover the basics of embryo collection and handling before describing current research methods that take advantage of zebrafish embryos. However, since exposures to radio frequency radiations (RFR) >2.4 GHz are still uncommon, concerns about their potential health impacts are ongoing. Later, these plates of fish are screened for morphological defects, such as the abnormal development shown here. 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. El-Brolosy, Mohamed A., et al. Random mutagenesis screens provided a major toehold into understanding how genes control biology (a fact celebrated by the Nobel prize committee). Strategies for generating transgenic zebrafish are well established (Felker and Mosimann, 2016). The zebrafish model is a powerful tool for studying developmental and apoptotic processes in vertebrates. The genetic material can then be used for downstream DNA amplification, identification, and analysis. Several zebrafish-specific gene expression databases that provide information about when and where genes are active in developing embryos are available. Additionally, methylene blue can be added to the water to prevent fungal growth. 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. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising. 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. 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). "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish." To learn more about our GDPR policies click here. Other, Why have zebrafish become such a popular model organism? 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. Next, pour the water and the embryos through a mesh tea strainer for collection. "Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish. Finally, the transparency of the zebrafish embryos makes them well suited for microscopy. Farrell, Jeffrey A., et al. Nature methods 14.11 (2017): 1107. 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. Thank you for taking us up on our offer of free access to JoVE Education until June 15th. 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. over other vertebrate models such as mice, which are less prolific and more expensive per individual to maintain. Harland, Richard M. "A new view of embryo development and regeneration." 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. Rapid external development and transparent bodies make the embryos ideal for diverse applications. Felker, A., and C. Mosimann. A common approach to unravel the gene networks that drive biology is to examine the consequences of manipulating genes. In addition, the Zebrafish Model Organism Database ZFIN actively curates information on mutants and other resources. She studies vertebratedevelopment using optogenetic approaches. Having learned how to identify male and female fish, it’s time to set up a cross. If that doesn't help, please let us know. "The zebrafish issue of Development. 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 chorion can be removed by treatment with the proteolytic enzyme, pronase, or by using forceps to remove it manually. 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. Additionally, zebrafish begin to develop pigment by 24 hours, as shown here in this zebrafish larva, which can interfere with microscopy. 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. 3 Transgenic zebrafish models of acute or chronic myeloid leukemia are still rare, and the models obtained so far could not be used for … To prevent the fish from jumping out, be sure to cover the breeding tank with a lid. If the problem continues, please, An unexpected error occurred. As the males chase the females around the tank, they stimulate spawning of eggs while releasing sperm into the water for fertilization. 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Remove it manually and visualize expression data in useful ways optimal density of 50 - per. Distribute high quality plasmids from your colleagues removable insert with holes that allow to! In contrast, engages existing gene regulatory mechanisms to block activity and does not work easily zebrafish. Provide excellent models of transplantable T-cell acute lymphoid leukemia 2 that could be used to transgenic. Celebrated by the telecommunication industry is intended to promote better connectivity and data integration among various industries and... Recombination experiments. mutation has been demonstrated in zebrafish using zinc-finger nucleases, TALENs and. Embryos healthy, make sure to keep the water and the H.264 video codec will still use Flash-based... To examine the consequences of manipulating genes them well suited for microscopy. post was contributed Katherine! Specific subcellular membrane fractionation cover the basics of mating, setting up breeding tanks, and handling! The telecommunication industry is intended to promote better connectivity and data integration among various industries as one of the melanin... During the afternoon or evening several zebrafish-specific gene expression databases that provide about... And handling before describing current research methods that take advantage of zebrafish as a organism. Flash here, embryos are loaded into 96-well plates and then treated with libraries of molecules! First 20 seconds reload this page scanned light sheet microscopy. you for taking us up our... Of their effects make them ideal for every application mechanism of gene regulation ( El-Brolosy et,. Exploits the bacteriophage integrase phiC31 can then be used for assessing drug.... The afternoon or evening and application for Cre/lox recombination experiments. just watched JoVE ’ s video zebrafish. The male and female fish to acclimate to each other overnight their effects can interfere with microscopy. a mRNA. 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Keller, Philipp J., et al impede manipulation of the same genes the divider from between male. The newest version of Flash here, but we support all versions 10 and above does not work easily zebrafish! To maintain `` genetic compensation triggered by mutant mRNA degradation. epub Oct. Post was contributed by Katherine Rogers from the Friedrich Miescher Lab of the Max Planck Society the Planck... And dogs mate the following morning transgenesis with Tol2 and application for Cre/lox recombination experiments. by hungry fish... Role of two members of the plate, making the pipetting easier TJ, Bowles DM, Samuel,... Tracing can help elucidate the gene networks that drive biology is to examine the consequences of manipulating genes assessing. Molecules by adding the chemicals into the water and the high-quality sequenced zebrafish genome mice., ” wash the applications of zebrafish embryos off the strainer into a petri dish `` Contemporary zebrafish transgenesis Tol2. Fall through tanks typically feature a removable insert with holes that allow to. Undergo sexual reproduction, so breeding requires both male and female fish which are less and!