Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Live imaging of apoptotic cells in zebrafish.

ABSTRACT: Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. The calcium-dependent protein Annexin V (A5) binds PS with high affinity, and biochemically purified, fluorescently labeled A5 probes have been widely used to detect apoptosis in vitro. Here we show that secreted A5 fused to yellow fluorescent protein specifically labels apoptotic cells in living zebrafish. We use this fluorescent probe to characterize patterns of apoptosis in living zebrafish larvae and to visualize neuronal cell death at single-cell resolution in vivo.

SUBMITTER: van Ham TJ

PROVIDER: S-EPMC2974414 | biostudies-literature | 2010 Nov

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Live imaging of apoptotic cells in zebrafish.

van Ham Tjakko J TJ Mapes James J Kokel David D Peterson Randall T RT

FASEB journal : official publication of the Federation of American Societies for Experimental Biology 20100702 11

Many debilitating diseases, including neurodegenerative diseases, involve apoptosis. Several methods have been developed for visualizing apoptotic cells in vitro or in fixed tissues, but few tools are available for visualizing apoptotic cells in live animals. Here we describe a genetically encoded fluorescent reporter protein that labels apoptotic cells in live zebrafish embryos. During apoptosis, the phospholipid phosphatidylserine (PS) is exposed on the outer leaflet of the plasma membrane. Th ...[more]

PMID: 20601526

Similar Datasets

Imaging of nanoparticle dynamics in live and apoptotic cells using temporally-modulated polarization.

Project description:Gold nanoparticles are widely exploited in phototherapy. Owing to their biocompatibility and their strong visible-light surface plasmonic resonance, these particles also serve as contrast agents for cell image enhancement and super-resolved imaging. Yet, their optical signal is still insufficiently strong for many important real-life applications. Also, the differentiation between adjacent nanoparticles is usually limited by the optical resolution and the orientations of non-spherical particles are unknown. These limitations hamper the progress in cell research by direct optical microscopy and narrow the range of phototherapy applications. Here we demonstrate exploiting the optical anisotropy of non-spherical nanoparticles to achieve super-resolution in live cell imaging and to resolve the intracellular nanoparticle orientations. In particular, by modulating the light polarization and taking advantage of the polarization-dependence of gold nanorod optical properties, we realize the 'lock-in amplification', widely-used in electronic engineering, to achieve image enhancement in live cells and in cells that undergo apoptotic changes.

| S-EPMC6367359 | biostudies-literature

Live Imaging of Intracranial Lymphatics in the Zebrafish.

Project description:RationaleThe recent discovery of meningeal lymphatics in mammals is reshaping our understanding of fluid homeostasis and cellular waste management in the brain, but visualization and experimental analysis of these vessels is challenging in mammals. Although the optical clarity and experimental advantages of zebrafish have made this an essential model organism for studying lymphatic development, the existence of meningeal lymphatics has not yet been reported in this species.ObjectiveExamine the intracranial space of larval, juvenile, and adult zebrafish to determine whether and where intracranial lymphatic vessels are present.Methods and resultsUsing high-resolution optical imaging of the meninges in living animals, we show that zebrafish possess a meningeal lymphatic network comparable to that found in mammals. We confirm that this network is separate from the blood vascular network and that it drains interstitial fluid from the brain. We document the developmental origins and growth of these vessels into a distinct network separated from the external lymphatics. Finally, we show that these vessels contain immune cells and perform live imaging of immune cell trafficking and transmigration in meningeal lymphatics.ConclusionsThis discovery establishes the zebrafish as a important new model for experimental analysis of meningeal lymphatic development and opens up new avenues for probing meningeal lymphatic function in health and disease.

| S-EPMC7790877 | biostudies-literature

Live imaging of primary ocular vasculature formation in zebrafish.

Project description:Ocular vasculature consists of the central retinal and ciliary vascular systems, which are essential to maintaining visual function. Many researchers have attempted to determine their origins and development; however, the detailed, stepwise process of ocular vasculature formation has not been established. In zebrafish, two angioblast clusters, the rostral and midbrain organizing centers, form almost all of the cranial vasculature, including the ocular vasculature, and these are from where the cerebral arterial and venous angioblast clusters, respectively, differentiate. In this study, we first determined the anatomical architecture of the primary ocular vasculature and then followed its path from the two cerebral angioblast clusters using a time-lapse analysis of living Tg(flk1:EGFP)k7 zebrafish embryos, in which the endothelial cells specifically expressed enhanced green fluorescent protein. We succeeded in capturing images of the primary ocular vasculature formation and were able to determine the origin of each ocular vessel. In zebrafish, the hyaloid and ciliary arterial systems first organized independently, and then anastomosed via the inner optic circle on the surface of the lens by the lateral transfer of the optic vein. Finally, the choroidal vascular plexus formed around the eyeball to complete the primary ocular vasculature formation. To our knowledge, this study is the first to report successful capture of circular integration of the optic artery and vein, lateral transfer of the optic vein to integrate the hyaloidal and superficial ocular vasculatures, and formation of the choroidal vascular plexus. Furthermore, this new morphological information enables us to assess the entire process of the primary ocular vasculature formation, which will be useful for its precise understanding.

| S-EPMC5405983 | biostudies-literature

Live imaging of adult zebrafish cardiomyocyte proliferation ex vivo.

Project description:Zebrafish are excellent at regenerating their heart by reinitiating proliferation in pre-existing cardiomyocytes. Studying how zebrafish achieve this holds great potential in developing new strategies to boost mammalian heart regeneration. Nevertheless, the lack of appropriate live-imaging tools for the adult zebrafish heart has limited detailed studies into the dynamics underlying cardiomyocyte proliferation. Here, we address this by developing a system in which cardiac slices of the injured zebrafish heart are cultured ex vivo for several days while retaining key regenerative characteristics, including cardiomyocyte proliferation. In addition, we show that the cardiac slice culture system is compatible with live timelapse imaging and allows manipulation of regenerating cardiomyocytes with drugs that normally would have toxic effects that prevent their use. Finally, we use the cardiac slices to demonstrate that adult cardiomyocytes with fully assembled sarcomeres can partially disassemble their sarcomeres in a calpain- and proteasome-dependent manner to progress through nuclear division and cytokinesis. In conclusion, we have developed a cardiac slice culture system, which allows imaging of native cardiomyocyte dynamics in real time to discover cellular mechanisms during heart regeneration.

| S-EPMC8489017 | biostudies-literature

Optimized protocol for live imaging of overnutrition-elicited interactions between immune cells and β cells in zebrafish.

Project description:Here, we provide an optimized protocol to observe the interactions between infiltrating immune cells and islet β cells using live imaging. This protocol is useful for the characterization of cell-cell interactions and for the direct visualization of immune cell migration to the principal pancreatic islet during islet inflammation. We describe the preparation of zebrafish transgenic lines and detail steps for setting up the fish for live confocal imaging. For more details on the use and execution of this protocol, please refer to Yang et al. (2022).1.

| S-EPMC9860484 | biostudies-literature

A Zebrafish Live Imaging Model Reveals Differential Responses of Microglia Toward Glioblastoma Cells In Vivo.

Project description:Glioblastoma multiforme is the most common and deadliest form of brain cancer. Glioblastomas are infiltrated by a high number of microglia, which promote tumor growth and surrounding tissue invasion. However, it is unclear how microglia and glioma cells physically interact and if there are differences, depending on glioma cell type. Hence, we have developed a novel live imaging assay to study microglia-glioma interactions in vivo in the zebrafish brain. We transplanted well-established human glioblastoma cell lines, U87 and U251, into transgenic zebrafish lines with labelled macrophages/microglia. Our confocal live imaging results show distinct interactions between microglia and U87, as well as U251 glioblastoma cells that differ in number and nature. Importantly these interactions do not appear to be antitumoral as zebrafish microglia do not engulf and phagocytose the human glioblastoma cells. Finally, xenotransplants into the irf8-/- zebrafish mutant that lacks microglia, as well as pharmacological inhibition of the CSF-1 receptor (CSF-1R) on microglia, confirm a prominent role for zebrafish microglia in promoting human glioblastoma cell growth. This new model will be an important tool for drug screening and the development of future immunotherapeutics targeting microglia within glioma.

| S-EPMC5124743 | biostudies-literature

Live imaging of endogenous protein dynamics in zebrafish using chromobodies.

Project description:Chromobodies are intracellular nanoprobes that combine the specificity of antibodies with the convenience of live fluorescence imaging in a flexible, DNA-encoded reagent. Here, we present the first application of this technique to an intact living vertebrate organism. We generated zebrafish lines expressing chromobodies that trace the major cytoskeletal component actin and the cell cycle marker PCNA with spatial and temporal specificity. Using these chromobodies, we captured full localization dynamics of the endogenous antigens in different cell types and at different stages of development. For the first time, the chromobody technology enables live imaging of endogenous subcellular structures in an animal, with the remarkable advantage of avoiding target protein overexpression or tagging. In combination with improved chromobody selection systems, we anticipate a rapid adaptation of this technique to new intracellular antigens and model organisms, allowing the faithful description of cellular and molecular processes in their dynamic state.

| S-EPMC4440926 | biostudies-literature

zWEDGI: Wounding and Entrapment Device for Imaging Live Zebrafish Larvae.

Project description:Zebrafish, an established model organism in developmental biology, is also a valuable tool for imaging wound healing in space and time with cellular resolution. However, long-term imaging of wound healing poses technical challenges as wound healing occurs over multiple temporal scales. The traditional strategy of larval encapsulation in agarose successfully limits sample movement but impedes larval development and tissue regrowth and is therefore not amenable to long-term imaging of wound healing. To overcome this challenge, we engineered a functionally compartmentalized device, the zebrafish Wounding and Entrapment Device for Growth and Imaging (zWEDGI), to orient larvae for high-resolution microscopy, including confocal and second harmonic generation (SHG), while allowing unrestrained tail development and regrowth. In this device, larval viability was maintained and tail regrowth was improved over embedding in agarose. The quality of tail fiber SHG images collected from larvae in the device was similar to fixed samples but provided the benefit of time lapse data collection. Furthermore, we show that this device was amenable to long-term (>24 h) confocal microscopy of the caudal fin. Finally, the zWEDGI was designed and fabricated using readily available techniques so that it can be easily modified for diverse experimental imaging protocols.

| S-EPMC5312606 | biostudies-literature

Live-imaging of astrocyte morphogenesis and function in zebrafish neural circuits.

Project description:How astrocytes grow and integrate into neural circuits remains poorly defined. Zebrafish are well suited for such investigations, but bona fide astrocytes have not been described in this system. Here we characterize a zebrafish cell type that is remarkably similar to mammalian astrocytes that derive from radial glial cells and elaborate processes to establish their territories at early larval stages. Zebrafish astrocytes associate closely with synapses, tile with one another and express markers, including Glast and glutamine synthetase. Once integrated into circuits, they exhibit whole-cell and microdomain Ca2+ transients, which are sensitive to norepinephrine. Finally, using a cell-specific CRISPR-Cas9 approach, we demonstrate that fgfr3 and fgfr4 are required for vertebrate astrocyte morphogenesis. This work provides the first visualization of astrocyte morphogenesis from stem cell to post-mitotic astrocyte in vivo, identifies a role for Fgf receptors in vertebrate astrocytes and establishes zebrafish as a valuable new model system to study astrocyte biology in vivo.

| S-EPMC7530038 | biostudies-literature

Live imaging of the cytoskeleton in early cleavage-stage zebrafish embryos.

Project description:The large and transparent cells of cleavage-stage zebrafish embryos provide unique opportunities to study cell division and cytoskeletal dynamics in very large animal cells. Here, we summarize recent progress, from our laboratories and others, on live imaging of the microtubule and actin cytoskeletons during zebrafish embryonic cleavage. First, we present simple protocols for extending the breeding competence of zebrafish mating ensembles throughout the day, which ensures a steady supply of embryos in early cleavage, and for mounting these embryos for imaging. Second, we describe a transgenic zebrafish line [Tg(bactin2:HsENSCONSIN17-282-3xEGFP)hm1] that expresses the green fluorescent protein (GFP)-labeled microtubule-binding part of ensconsin (EMTB-3GFP). We demonstrate that the microtubule-based structures of the early cell cycles can be imaged live, with single microtubule resolution and with high contrast, in this line. Microtubules are much more easily visualized using this tagged binding protein rather than directly labeled tubulin (injected Alexa-647-labeled tubulin), presumably due to lower background from probe molecules not attached to microtubules. Third, we illustrate live imaging of the actin cytoskeleton by injection of the actin-binding fragment of utrophin fused to GFP. Fourth, we compare epifluorescence-, spinning-disc-, laser-scanning-, and two-photon-microscopic modalities for live imaging of the microtubule cytoskeleton in early embryos of our EMTB-3GFP-expressing transgenic line. Finally, we discuss future applications and extensions of our methods.

| S-EPMC6551615 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data