Live Zebrafish Embryos Research Articles

Deep-learning microscopy image reconstruction with quality control reveals second-scale rearrangements in RNA polymerase II clusters.

Fluorescence microscopy, a central tool of biological research, is subject to inherent trade-offs in experiment design. For instance, image acquisition speed can only be increased in exchange for a lowered signal quality, or for an increased rate of photo-damage to the specimen. Computational denoising can recover some loss of signal, extending the trade-off margin for high-speed imaging. Recently proposed denoising on the basis of neural networks shows exceptional performance but raises concerns of errors typical of neural networks. Here, we present a work-flow that supports an empirically optimized reduction of exposure times, as well as per-image quality control to exclude images with reconstruction errors. We implement this work-flow on the basis of the denoising tool Noise2Void and assess the molecular state and 3D shape of RNA polymerase II (Pol II) clusters in live zebrafish embryos. Image acquisition speed could be tripled, achieving 2-s time resolution and 350-nm lateral image resolution. The obtained data reveal stereotyped events of approximately 10 s duration: initially, the molecular mark for recruited Pol II increases, then the mark for active Pol II increases, and finally Pol II clusters take on a stretched and unfolded shape. An independent analysis based on fixed sample images reproduces this sequence of events, and suggests that they are related to the transient association of genes with Pol II clusters. Our work-flow consists of procedures that can be implemented on commercial fluorescence microscopes without any hardware or software modification, and should, therefore, be transferable to many other applications.

Fluorescent dendrimer-based probes for cell membrane imaging: Zebrafish epidermal labeling-based toxicity evaluation

Visualizing the plasma membrane of living mammalian cells both in vitro and in vivo is crucial for tracking their cellular activities. However, due to the complex and dynamic nature of the plasma membrane, most commercial dyes for membrane staining can only realize very limited imaging performance. Thus, precise and stable plasma membrane imaging remains technically challenging. Here, by taking advantage of the small, well-defined, and amine-rich dendrimers, we prepared poly(ethylene glycol)-cholesterol (PEG-Chol)-conjugated and cyanine dye (e.g., cyanine2, cyanine3, and cyanine5)-labeled dendrimer nanoprobes (termed DPC-Cy2, DPC-Cy3, and DPC-Cy5 NPs). It was revealed that these probes enabled universal, wash-free, long-term (at least 8 h), and multicolor (green, yellow, and red) plasma membrane labeling of a variety of live mammalian cells. Further, we confirmed that the nanoprobes (using DPC-Cy5 as a representative) could achieve high-quality, wash-free, and stable cell surface labeling of live zebrafish embryos. More importantly, we demonstrated that our probes could act as biosensors to visualize the toxicity of metal–organic frameworks (MOFs) toward the epidermal cells of zebrafish embryos, and thus they hold great potential for identifying the toxic effect of drugs/materials at the single-cell scale or in live animals. The present work highlights the advantages of utilizing dendrimers for constructing functional imaging materials, and it is also believed that the fluorescent dendrimer nanoprobes developed in this work may find wide applications like cell imaging, drug toxicity evaluation, and cellular state monitoring.

Foregut organ progenitors and their niche display distinct viscoelastic properties in vivo during early morphogenesis stages

Material properties of living matter play an important role for biological function and development. Yet, quantification of material properties of internal organs in vivo, without causing physiological damage, remains challenging. Here, we present a non-invasive approach based on modified optical tweezers for quantifying sub-cellular material properties deep inside living zebrafish embryos. Material properties of cells within the foregut region are quantified as deep as 150 µm into the biological tissue through measurements of the positions of an inert tracer. This yields an exponent, α, which characterizes the scaling behavior of the positional power spectra and the complex shear moduli. The measurements demonstrate differential mechanical properties: at the time when the developing organs undergo substantial displacements during morphogenesis, gut progenitors are more elastic (α = 0.57 ± 0.07) than the neighboring yolk (α = 0.73 ± 0.08), liver (α = 0.66 ± 0.06) and two mesodermal (α = 0.68 ± 0.06, α = 0.64 ± 0.06) progenitor cell populations. The higher elasticity of gut progenitors correlates with an increased cellular concentration of microtubules. The results infer a role of material properties during morphogenesis and the approach paves the way for quantitative material investigations in vivo of embryos, explants, or organoids.

A selective luminescent probe to monitor cellular ATP: Potential application for in vivo imaging in zebrafish embryo

Adenosine 5′-triphosphate (ATP) is the major energy currency of living cells and its efficient real-time monitoring in vivo has increased the demand for the development of fluorescent chemosensors. For the prompt detection of ATP in submolar range, we have developed an easily accessible colorimetric and fluorescence probe 4-(Di-p-tolylphosphoryl)-7-hydroxychroman-2-one (DPC). The selective nature of DPC towards ATP has been confirmed through UV–vis, fluorescence titration, NMR titration as well as computational studies. Coupled with high mitochondrial membrane potential and ATP levels, the chemosensor DPC shows intense blue fluorescence signal at intracellular compartments in murine splenocytes and A549 human lung cancer cells. Moreover, using live zebrafish (Danio rerio)embryos with high energetic demands, we have successfully achieved fluorescence imaging of ATP at distinct sites in vivo(neural tube, myotome or embryonic muscle tissue, somite boundaries and tail regions) followed by estimation of endogenous ATP level from embryo extract. Collectively, our designed probe demonstrates excellent cellular permeability, sensitivity and specificity for intracellular ATP, and may provide valuable insights in the research of ATP-regulated biological processes in living cells or organisms.

Time-Dependent Monitoring of Dopamine in the Brain of Live Embryonic Zebrafish Using Electrochemically Pretreated Carbon Fiber Microelectrodes.

Neurotransmitters are involved in functions related to signaling, stress response, and pathological disorder development, and thus, their real-time monitoring at the site of production is important for observing the changes related to these disorders. Here, we demonstrate the first time-dependent quantification of dopamine in the brains of live zebrafish embryos using electrochemically pretreated carbon fiber microelectrodes (CFMEs) utilizing differential pulse voltammetry as the measurement technique. The pretreatment of the CFMEs in 0.1 M NaOH held at a potential of +1.0 V for 600 s improves the sensitivity toward dopamine and allows for reliable measurements in low ionic strength media. We demonstrate the measurement of extracellular dopamine concentrations in the zebrafish brain during late embryogenesis. The extracellular dopamine concentration in the tectum of zebrafish varies between 200 and 400 nM. The conventional pharmacological manipulation of neurotransmitter levels in the brain demonstrates the selective detection of dopamine at the implantation site. Exposure to the dopamine transporter inhibitor nomifensine induces an increase in extracellular dopamine from 201.9 (±34.9) nM to 352.2 (±20.0) nM, while exposure to the norepinephrine transporter inhibitor desipramine does not lead to a significant modulation of the measured signal. Furthermore, we report the quantitative assessment of the catecholamine stress response of embryos to tricaine, an anesthetic frequently used in zebrafish assays. Exposure to tricaine induces a short-lived increase in brain dopamine from 198.6 (±15.7) nM to a maximum of 278.8 (±14.0) nM. Thus, in vivo electrochemistry can detect real-time changes in zebrafish neurochemical physiology resulting from drug exposure.

Engineering Advanced Environmentally Friendly Corrosion Inhibitors, Their Mechanisms, and Biological Effects in Live Zebrafish Embryos

Carbon steel is one of the most used construction materials and makes up the bulk of steel production in the world. However, this alloy is vulnerable to corrosion, which reduces its safety and costs trillions of dollars worldwide in terms of corrosion protection and infrastructure maintenance. Corrosion inhibitors are among the cheapest and most effective methods to minimize corrosion and maximize the shelf life of this ubiquitous alloy; however, they are often harmful to the environment. Currently there are no reports in terms of engineering design methodology to develop highly effective, advanced corrosion inhibitors. The possible synergistic behavior of a surface-active quaternary ammonium with a tailored carboxylate compound provides a rational molecular design toward delivering corrosion inhibitors to a metal surface through controlled speciation in solution. Here we show how entrapment and delivery of the anion inhibitor through speciation and micellar formation can lead to high inhibition efficiency, as determined by potentiodynamic polarization and electrochemical impedance spectroscopy. In particular, we demonstrate how NMR spectroscopy, pfg NMR diffusion measurements, and Cryo-TEM, taken together with molecular dynamic simulations, can reveal the micellar formation and its concentration dependence, with wormlike micelles evident at higher concentrations correlating with a dramatic increase in viscosity. This new organic corrosion inhibitor, cetrimonium 4-ethoxy cinnamate, provides significantly higher corrosion inhibition efficiency compared with the 4-hydroxy cinnamate analogue and with reduced toxic impact in the zebrafish embryo compared to the surface active CTAB often used as an antimicrobial.

In Vivo Live Imaging of Axonal Transport in Developing Zebrafish Axons.

Axonal transport is crucial for neuronal homeostasis, survival, and development. Indeed, axonal transport needs to be precisely regulated for developing axons to swiftly and accurately respond to their complex and evolving environment in space and time. A growing number of studies have started to unravel the diversity of regulatory and adaptor proteins required to orchestrate the axonal transport machinery. Despite some discrepancies between in vitro and in vivo axonal transport studies, most analyses aiming at deciphering these regulatory complexes, as well as their mode of action, were carried out in vitro in primary cultures of neurons, and mainly focused on their impact on axon specification and elongation, but rarely on axon navigation per se. Given the clear influence of the in vivo environment on axonal transport, including chemical and physical interactions with neighboring cells, it is essential to develop in vivo models to identify and characterize the molecular complexes involved in this key process. Here, we describe an experimental system to monitor axonal transport in vivo in developing axons of live zebrafish embryos with high spatial and temporal resolution. Due to its optical transparency and easy genetic manipulation, the zebrafish embryo is ideally suited to study such cellular dynamics at a single axon scale. Using this approach, we were able to unravel the key role of Fidgetin-like 1 in the regulation of bidirectional axonal transport required for motor axon targeting. Moreover, this protocol can be easily adapted to characterize a wide range of axonal transport regulators and components in physiological conditions and may additionally be used to screen new therapeutic compounds based on their ability to recue axonal transport defects in pathological conditions.

Surface-enhanced Raman scattering labeled nanoplastic models for reliable bio-nano interaction investigations

Nanoplastics (NPs) have attracted great attention as an emerging pollution. To date, their interaction with biological systems has been studied mostly by using fluorescent-labeled NPs, which suffered from serious drawbacks such as biological autofluorescence interference and false-positive results. Reliable optically labeled NP models are eagerly desired until now. Herein, a novel near-infrared (NIR) surface-enhanced Raman scattering (SERS) labeled NP model was proposed, which gained single-particle ultra-sensitivity, deep tissue detection, multiplex labeling ability, and anti-interference property. More importantly, the NP demonstrated satisfactory in vivo signal stability which completely prevented the positive-false problems. The advantages of the NPs enabled direct, dynamic in vivo behavior imaging study in living zebrafish embryo, adult zebrafish and green vegetable Brassica rapa. It was found for the first time that NPs entered blood circulation system of zebrafish larva via dermal uptake route, which only occurred in a short 48 h-window post-hatch. NPs widely distributed in roots, shoots and leaves of Brassica rapa seedlings germinating and growing in the NP-containing hydroponic culture. Different depths of one root showed varied adsorption capabilities towards NPs with fulvic acid, lipid and sodium dodecyl sulfate eco-coronas. This work provided an ideal tool for reliable bio-NP interaction study for a variety of organisms, which could promote the research of NPs.

Analysis of intracellular protein dynamics in living zebrafish embryos using light-sheet fluorescence single-molecule microscopy.

Single-molecule microscopy techniques have emerged as useful tools to image individual molecules and analyze their dynamics inside cells, but their application has mostly been restricted to cell cultures. Here, a light-sheet fluorescence microscopy setup is presented for imaging individual proteins inside living zebrafish embryos. The optical configuration makes this design accessible to many laboratories and a dedicated sample-mounting system ensures sample viability and mounting flexibility. Using this setup, we have analyzed the dynamics of individual glucocorticoid receptors, which demonstrates that this approach creates multiple possibilities for the analysis of intracellular protein dynamics in intact living organisms.

MiniSPIM-A Miniaturized Light-Sheet Microscope.

The miniSPIM is a miniaturized light-sheet microscope that enables imaging with optical sectioning on mobile camera devices such as smartphones and single-board computers. Applications of the miniSPIM include biosensing, field research, and education where maximum portability and robustness, low power consumption, and low cost are key. Here, it is shown how all of the components of a simple light-sheet microscope can be integrated within a footprint smaller than the average smartphone. Example applications include the quantification of the motion of microparticles and bacteria in fluids, the characterization of solvent polarity based on spectral shifts of the lipid probe Nile Red, and three-dimensional (3D) and time-lapse autofluorescence imaging of a live zebrafish embryo.

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Proximity Dependent Biotin Labelling in Zebrafish for Proteome and Interactome Profiling.

Identification of protein interaction networks is key for understanding intricate biological processes, but mapping such networks is challenging with conventional biochemical methods, especially for weak or transient interactions. Proximity-dependent biotin labelling (BioID) using promiscuous biotin ligases and mass spectrometry (MS)-based proteomics has emerged in the past decade as a powerful method for probing local proteomes and protein interactors. Here, we describe the application of an engineered biotin ligase, TurboID, for proteomic mapping and interactor screening in vivo in zebrafish. We generated novel transgenic zebrafish lines that express TurboID fused to a conditionally stabilised GFP-binding nanobody, dGBP, which targets TurboID to the GFP-tagged proteins of interest. The TurboID-dGBP zebrafish lines enable proximity-dependent biotin labelling in live zebrafish simply through outcrossing with existing GFP-tagged lines. Here, we outline a detailed protocol of the BLITZ method (Biotin Labelling In Tagged Zebrafish) for utilising TurboID-dGBP fish lines to map local proteomes and screen novel interactors. Graphic abstract: Schematic overview of the BLITZ method. TurboID-dGBP fish are crossed with GFP-tagged lines to obtain embryos co-expressing TurboID-dGBP (indicated by mKate2) and the GFP-POI (protein of interest). Embryos expressing only TurboID are used as a negative control. Embryos (2 to 7 dpf) are incubated overnight with a 500 μM biotin-supplemented embryo medium. This biotin incubation step allows TurboID to catalyse proximity-dependent biotinylation in live zebrafish embryos. After biotin incubation, embryos are solubilised in lysis buffer, and free biotin is removed using a PD-10 desalting column. The biotinylated proteins are captured by streptavidin affinity purification, and captured proteins are analysed by MS sequencing.

Chemical effects on dye efflux activity in live zebrafish embryos and on zebrafish Abcb4 ATPase activity.

ATP-binding cassette (ABC) transporter proteins include efflux pumps that confer multixenobiotic resistance to zebrafish embryos, a valuable toxico/pharmacological model. Here, we established an automated microscopy-based rhodamine B dye accumulation assay in which enhanced dye accumulation in live zebrafish embryos indicates inhibition of multixenobiotic efflux transporter activity. Twenty structurally divergent known substrates and/or inhibitors of human ABC transporters and environmentally relevant compounds were examined using this assay and the ATPase activity of recombinant zebrafish Abcb4 as readouts. These two assays confirmed that Abcb4 functions as an efflux transporter in zebrafish, whereas they gave discordant results for some of the tested substances. The dye accumulation assay in zebrafish embryos could be useful to screen environmental pollutants and other chemicals for efflux transporter interaction in a medium-throughput fashion.

In vivo biomolecular imaging of zebrafish embryos using confocal Raman spectroscopy

Zebrafish embryos provide a unique opportunity to visualize complex biological processes, yet conventional imaging modalities are unable to access intricate biomolecular information without compromising the integrity of the embryos. Here, we report the use of confocal Raman spectroscopic imaging for the visualization and multivariate analysis of biomolecular information extracted from unlabeled zebrafish embryos. 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 response in living zebrafish embryos. Overall, this study demonstrates the application of confocal Raman spectroscopic imaging for the comparative bimolecular analysis of fully intact and living zebrafish embryos.

Spindle Scaling Is Governed by Cell Boundary Regulation of Microtubule Nucleation

Cellular organelles such as the mitotic spindle adjust their size to the dimensions of the cell. It is widely understood that spindle scaling is governed by regulation of microtubule polymerization. Here, we use quantitative microscopy in living zebrafish embryos and Xenopus egg extracts in combination with theory to show that microtubule polymerization dynamics are insufficient to scale spindles and only contribute below a critical cell size. In contrast, microtubule nucleation governs spindle scaling for all cell sizes. We show that this hierarchical regulation arises from the partitioning of a nucleation inhibitor to the cell membrane. Our results reveal that cells differentially regulate microtubule number and length using distinct geometric cues to maintain a functional spindle architecture over a large range of cell sizes.

Fluorescent dATP for DNA Synthesis In Vivo.

Fluorescent nucleoside triphosphates are powerful probes of DNA synthesis, but their potential use in living animals has been previously underexplored. Here, we report the synthesis and characterization of 7-deaza-(1,2,3-triazole)-2'-deoxyadenosine-5'-triphosphate (dATP) derivatives of tetramethyl rhodamine ("TAMRA-dATP"), cyanine ("Cy3-dATP"), and boron-dipyrromethene ("BODIPY-dATP"). Upon microinjection into live zebrafish embryos, all three compounds were incorporated into the DNA of dividing cells; however, their impact on embryonic toxicity was highly variable, depending on the exact structure of the dye. TAMRA-EdATP exhibited superior characteristics in terms of its high brightness, low toxicity, and rapid incorporation and depletion kinetics in both a vertebrate (zebrafish) and a nematode (Caenorhabditis elegans). TAMRA-EdATP allows for unprecedented, real-time visualization of DNA replication and chromosome segregation in vivo.

An NMR-Based Biosensor to Measure Stereospecific Methionine Sulfoxide Reductase Activities in Vitro and in Vivo*.

Oxidation of protein methionines to methionine-sulfoxides (MetOx) is associated with several age-related diseases. In healthy cells, MetOx is reduced to methionine by two families of conserved methionine sulfoxide reductase enzymes, MSRA and MSRB that specifically target the S- or R-diastereoisomers of methionine-sulfoxides, respectively. To directly interrogate MSRA and MSRB functions in cellular settings, we developed an NMR-based biosensor that we call CarMetOx to simultaneously measure both enzyme activities in single reaction setups. We demonstrate the suitability of our strategy to delineate MSR functions in complex biological environments, including cell lysates and live zebrafish embryos. Thereby, we establish differences in substrate specificities between prokaryotic and eukaryotic MSRs and introduce CarMetOx as a highly sensitive tool for studying therapeutic targets of oxidative stress-related human diseases and redox regulated signaling pathways.

YAP Enhances Tumor Cell Dissemination by Promoting Intravascular Motility and Reentry into Systemic Circulation.

The oncogene YAP has been shown previously to promote tumor growth and metastasis. However, how YAP influences the behavior of tumor cells traveling within the circulatory system has not been as well explored. Given that rate-limiting steps of metastasis are known to occur while tumor cells enter, travel through, or exit circulation, we sought to study how YAP influences tumor cell behavior within the circulatory system. Intravital imaging in live zebrafish embryos revealed that YAP influenced the distribution of tumor cells within the animal following intravenous injection. Control cells became lodged in the first capillary bed encountered in the tail, whereas cells overexpressing constitutively active YAP were able to travel through this capillary plexus, reenter systemic circulation, and seed in the brain. YAP controlled transit through these capillaries by promoting active migration within the vasculature. These results were corroborated in a mouse model following intravenous injection, where active YAP increased the number of circulating tumor cells over time. Our results suggest a possible mechanism whereby tumor cells can spread to organs beyond the first capillary bed downstream from the primary tumor. These results also show that a specific gene can affect the distribution of tumor cells within an animal, thereby influencing the global pattern of metastasis in that animal. SIGNIFICANCE: These findings demonstrate that YAP endows tumor cells with the ability to move through capillaries, allowing them to return to and persist in circulation, thereby increasing their metastatic spread.See related commentary by Davidson, p. 3797.

A Fast, Simple, and Affordable Technique to Measure Oxygen Consumption in Living Zebrafish Embryos.

In all animal species, oxygen consumption is a key process that is partially impaired in a large number of pathological situations and thus provides informative details on the physiopathology of the disease. In this study, we describe a simple and affordable method to precisely measure oxygen consumption in living zebrafish larvae using a spectrofluorometer and the MitoXpress Xtra Oxygen Consumption Assay. In addition, we used zebrafish larvae treated with mitochondrial respiratory chain inhibitors, antimycin A or rotenone, to verify that our method enables precise and reliable measurements of oxygen consumption.

Translating GWAS-identified loci for cardiac rhythm and rate using an in vivo image- and CRISPR/Cas9-based approach

A meta-analysis of genome-wide association studies (GWAS) identified eight loci that are associated with heart rate variability (HRV), but candidate genes in these loci remain uncharacterized. We developed an image- and CRISPR/Cas9-based pipeline to systematically characterize candidate genes for HRV in live zebrafish embryos. Nine zebrafish orthologues of six human candidate genes were targeted simultaneously in eggs from fish that transgenically express GFP on smooth muscle cells (Tg[acta2:GFP]), to visualize the beating heart. An automated analysis of repeated 30 s recordings of beating atria in 381 live, intact zebrafish embryos at 2 and 5 days post-fertilization highlighted genes that influence HRV (hcn4 and si:dkey-65j6.2 [KIAA1755]); heart rate (rgs6 and hcn4); and the risk of sinoatrial pauses and arrests (hcn4). Exposure to 10 or 25 µM ivabradine—an open channel blocker of HCNs—for 24 h resulted in a dose-dependent higher HRV and lower heart rate at 5 days post-fertilization. Hence, our screen confirmed the role of established genes for heart rate and rhythm (RGS6 and HCN4); showed that ivabradine reduces heart rate and increases HRV in zebrafish embryos, as it does in humans; and highlighted a novel gene that plays a role in HRV (KIAA1755).