P.E. is supported by the Deutsche Forschungsgemeinschaft (DFG) Research Training Group 1373 and the Graduate School of the Technische Universit&#228;t M&#252;nchen (TUM-GS). G.P. was supported by TUM-GS. L.T. is supported by an EMBO fellowship (EMBO ALTF 108-2013). D.P.'s work on zebrafish was supported by the DFG through the Sonderforschungsbereich "Molecular Mechanisms of Neurodegeneration" (SFB 596); the Center for Integrated Protein Sciences (Munich) and the European Community's Seventh Framework Programme (FP7/2007-2013) under Grant agreement no. 200611 (MEMOSAD). He is currently a New York Stem Cell Foundation-Druckenmiller Fellow and was supported by a fellowship from the German Academy of Sciences Leopoldina. L.G. is supported by funding from the DFG through SFB 870 "Assembly and Function of Neuronal Circuits", Project A11. 
We are grateful to Kristina Wullimann for maintaining our fish facility, Yvonne Hufnagel for technical support and Thomas Misgeld for comments on the manuscript. We are grateful to R. K&#246;ster (Technische Universit&#228;t Braunschweig) for providing the M1 Medusa vector (pSKmemmRFP:5xUAS:H2B-CFP:5xUAS:Centrin2-YFP) from which we cloned out Centrin-YFP and S.C. Suzuki and T. Yoshimatsu (University of Washington) for providing the 14xUAS:MA-cerulean cassette which we used to generate the reporter construct to make CentrinFish. We further thank S.C. Suzuki and T. Yoshimatsu (University of Washington) for the Otx2:Gal4 transgenic line, A. Sagasti for the Sensory:Gal4-VP16 construct (UCLA) and M. Nonet (Washington University in St. Louis) for the pCold Heart Tol2 vector. We acknowledge Bettina Schmid, Alexander Hruscha and Christian Haass (German Center for Neurodegenerative Diseases Munich - DZNE) for contributing to the development of MitoFish.

The authors have nothing to disclose.

Here, we demonstrate the versatility of the Gal4-UAS expression system to fluorescently tag mitochondria, centrosomes and the cellular membranes of specific cell-types in vivo in zebrafish embryos. Many fluorescent fusion proteins that label other organelles or subcellular structures can be found in the published literature and can be obtained from the respective laboratory, commercial sources or non-commercial plasmid depositories (e.g., Addgene). To design a new fluorescent fusion protein, several par...

In vivo imaging provides direct visualization of cellular behaviors in the most physiological context. The transparency of zebrafish embryos, their rapid and external development and a rich array of genetic tools that permit fluorescent labeling have all contributed to the growing use of in vivo microscopy to elucidate the dynamics of key developmental events. Imaging studies of nervous system development in zebrafish have for example greatly expanded our knowledge of the behavior of neural progenitor cells and the fate of their progeny including their subsequent migration, differentiation and circuit integration1-8.
The stage is now set to investigate the subcellular dynamics underlying these cellular behaviors. Indeed, zebrafish are already being exploited as tools for in vivo cell biology. It is now possible to visualize mitochondria9-11, centrosomes2,8,12-14, Golgi15, the microtubule4 and actin16 cytoskeleton, endosomes17 and components of the apical membrane complex1,18, among other subcellular structures in zebrafish embryos in vivo. So far, much of what is known about the function of these organelles comes from studying their behavior in cultured cells. While in vitro studies have yielded tremendous insight into cell biology, cells in culture do not fully represent the complexity of the in vivo situation and therefore do not necessarily reflect the function and dynamics of subcellular organelles in vivo. Zebrafish embryos offer a viable in vivo alternative to examining subcellular dynamics.
As vertebrates, zebrafish possess many organ systems (e.g., neural retina) that are homologous to those found in mammalian species. Additionally, zebrafish embryos are increasingly being used to model human diseases19,20, including those related to centrosomal function (e.g., microcephaly21 and Leber&#39;s congenital amaurosis22) and to mitochondrial function (e.g., Parkinson&#39;s disease23, tauopathies10,24 and Barth syndrome25). In vivo imaging at the cellular and subcellular level in these instances will permit a better understanding of the cell biology underlying these pathological states.
The overall goal of the methods described here is to provide a comprehensive guide to investigate organelles and other subcellular structures in zebrafish embryos using in vivo light microscopy. The entire work-flow involved in visualizing and tracking subcellular structures in vivo is described - from genetic labeling approaches, to generating transiently expressing and stable transgenic fish, and finally to imaging using wide-field and confocal microscopy. While each of these procedures is used by numerous zebrafish laboratories, the protocols described are optimized and streamlined for investigating the dynamics of subcellular structures. Two specific aspects of the work described here warrant mention: First, the use of the Gal4-UAS expression system in multiple configurations to genetically label organelles in specific cell-types. Second, a direct comparison of wide-field and confocal microscopy to image subcellular structures in vivo.
Current strategies to genetically label organelles and other subcellular structures in zebrafish either make use of capped mRNA1,4,8 or DNA based constructs where promoter elements directly drive the expression of fusion proteins9,14,15. In vitro transcribed capped RNA results in rapid and broad expression, that is not tissue-specific however. Additionally, expression levels diminish over time as the capped RNA is diluted or degraded. Thus the use of RNA based constructs to examine organelle dynamics at later stages in development is limited (usually up to 3 days post-fertilization).
These limitations can be overcome by using DNA constructs, where spatial and temporal control of expression is determined by specific promoter elements. When DNA based constructs are used in the context of the Gal4-UAS system significant improvements to transgene expression levels are observed26,27. In this bipartite expression system, cell-type specific promoter elements drive the expression of a transcriptional activator Gal4, while reporter genes are cloned downstream of the Gal4-binding upstream activating sequence (UAS). By combining UAS reporters with appropriate Gal4 drivers, expression can be restricted to specific cell-types, circumventing the need to clone reporter genes behind different promoters every time a specific expression pattern is desired. Furthermore, the expression of multiple UAS reporter genes can be driven by a single Gal4 activator. The Gal4-UAS system thus provides a versatile and flexible genetic approach for subcellular labeling.
Wide-field and confocal microscopes are the workhorses of most laboratories. Wide-field systems typically use an arc lamp as a light source and detect the emitted light with a sensitive camera that is placed at the end of the light path. This imaging modality is typically restricted to thin samples as out-of focus light obscures in-focus information in thicker samples. Confocal microscopes differ from wide-field systems in that they are built to favor signals that originate from the focal plane over those that originate out of focus (i.e., &#34;optical sectioning&#34;)28. To achieve optical sectioning a pinhole is placed in the emission path in a conjugate position to the point light source. Lasers are used as light sources and signals are detected with photomultiplier tubes (PMTs). Practically, a laser beam is swiped over the sample point-by-point and the fluorescence emission at each spot (pixel) is detected by the PMT.
Here we image the very same subcellular structures in living zebrafish embryos using both wide-field and confocal microscopy to provide a direct comparison of both microscopy modalities. The underlying aim of providing such comparisons is to offer guidelines for choosing the most appropriate microscopy technique for the specific question at hand.
Using the approaches described here we demonstrate Gal4-UAS based genetic labeling of mitochondria and centrosomes. These organelles are imaged in different cell-types of the nervous system and in muscle cells using wide-field and confocal microscopy to demonstrate the suitability of each imaging modality. The methods described here can easily be adapted for investigating other organelles and subcellular structures in the living zebrafish embryo.

<table><tbody><tr><td>Agarose (2-hydroxyethylagarose)</td><td>Sigma-Aldrich</td><td>A4018-10G</td><td>Low-gelling temperature Type VII</td></tr><tr><td>Block heater</td><td>Eppendorf</td><td></td><td>Thermomixer compact</td></tr><tr><td>Ca(NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt; Calcium nitrate hydrate, 99.996%&amp;nbsp;</td><td>Aldrich</td><td>202967-50g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>CCD camera</td><td>Qimaging</td><td>Retiga Exi Fast 1394</td><td></td></tr><tr><td>Ceramic Coated Dumont #5 Forceps</td><td>Dumont - Fine Science Tools</td><td>11252-50</td><td>#5 Forceps</td></tr><tr><td>Confocal laser-scanning microscope</td><td>Olympus</td><td>FV1000 Fluoview</td><td></td></tr><tr><td>Culture dish heater</td><td>Warner Instrument Corporation</td><td>DH-35</td><td>Heating ring</td></tr><tr><td>Ethyl 3-aminobenzoate methanesulfate salt</td><td>PharmaQ</td><td>Tricaine PharmaQ-25g</td><td>Tricaine (anesthetic)</td></tr><tr><td>Fluorescence dissecting microscope</td><td>Leica</td><td>M205 FA</td><td></td></tr><tr><td>GeneClean kit</td><td>MP Biomedicals</td><td>111001200</td><td></td></tr><tr><td>Glass Bottom Culture Dishes</td><td>MatTek Corporation</td><td>P35G-0-14-C</td><td>35 mm petri dish, 14 mm microwell, No. 0 coverglass</td></tr><tr><td>Glass needles</td><td>World Precision Instruments Inc.&amp;nbsp;</td><td>TW100F-4</td><td>For microinjections</td></tr><tr><td>HEPES</td><td>Sigma</td><td>H3375-250g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>High vacuum grease</td><td>Dow Corning&amp;nbsp;</td><td>DCC000001242 150g</td><td>Silicon dioxide grease</td></tr><tr><td>KCl 99%</td><td>Sigma-Aldrich</td><td>S7643-5kg</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O &amp;nbsp;&amp;nbsp;Magnesium sulfate heptahydrate 98+% A.C.S reagent</td><td>Sigma-Aldrich</td><td>230291-500g</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>Microinjector&amp;nbsp;</td><td>Eppendorf</td><td></td><td>FemtoJet</td></tr><tr><td>Microloader tips</td><td>Eppendorf</td><td>930001007</td><td>0.5-20 ul</td></tr><tr><td>Micromanipulator</td><td>Maerzhaeuser Wetzlar</td><td>MM33 Rechts/00-42-101-0000/M3301R</td><td></td></tr><tr><td>Micropipette holder</td><td>Intracel</td><td>P/N 50-00XX-130-1</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription Kit</td><td>Ambion</td><td>AM1340</td><td>To transcribe PCS-Transposase</td></tr><tr><td>NaCl BioXtra &gt;99.5%</td><td>Sigma-Aldrich</td><td>P9541-1kg</td><td>To prepare 30x Danieau&#039;s</td></tr><tr><td>Nanophotometer</td><td></td><td></td><td>To measure DNA/RNA concentration</td></tr><tr><td>Needle puller</td><td>Sutter Instrument</td><td>P-1000</td><td>Flaming/Brown</td></tr><tr><td>NIR Apo 40x/0.80W&amp;nbsp;</td><td>Nikon</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>N-Phenylthiourea Grade I, approx. 98%</td><td>Sigma</td><td>P7629-10G</td><td>PTU (prevents pigmentation)</td></tr><tr><td>Petri dishes</td><td>Sarstedt AG&amp;nbsp;</td><td>821472</td><td>92 x 16mm&amp;nbsp;</td></tr><tr><td>Plastic molds&amp;nbsp;</td><td>Adaptive Science Tools</td><td>TU-1</td><td>For microinjections</td></tr><tr><td>Plexiglas cover-with a hole</td><td>Custom-made</td><td></td><td>The hole in the Plexiglas cover should be 3 mm larger than the diameter of the water-dipping-cone objective</td></tr><tr><td>Tea-strainer (Plastic)</td><td></td><td></td><td>To collect zebrafish eggs</td></tr><tr><td>Temperature controller</td><td>Warner Instrument Corporation</td><td>TC-344B</td><td>Dual Automatic Temperature Controller</td></tr><tr><td>Transfer pipettes</td><td>Sarstedt AG&amp;nbsp;</td><td>86.1171</td><td>3.5mL plastic transfer pipettes</td></tr><tr><td>UMPlanFI 100x/1.00W</td><td>Olympus</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>UMPlanFLN 20x/0.50W&amp;nbsp;</td><td>Olympus</td><td></td><td>Water-dipping-cone objective</td></tr><tr><td>Widefield microscope</td><td>Olympus</td><td>BX51WI</td><td></td></tr><tr><td>PTU (50x Stock)</td><td></td><td></td><td>Dissolve 76 mg PTU in 50 ml distilled water&lt;br/&gt; Stir vigorously at room temperature&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; Use at 1x working solution</td></tr><tr><td>Tricaine (20x Stock)</td><td></td><td></td><td>Dissolve 200 mg Tricaine in 48 ml distilled water&lt;br/&gt; Add 2 ml 1M Tris base (pH9)&lt;br/&gt; Adjust to pH 7&amp;nbsp;&lt;br/&gt; Store at -20 &lt;sup&gt;o&lt;/sup&gt;C in 1 ml aliquots&lt;br/&gt; Use at 1x working solution</td></tr><tr><td>Danieau&#039;s Solution (30x Stock)</td><td></td><td></td><td>1,740 mM&amp;nbsp; NaCl&amp;nbsp;&lt;br/&gt; 21 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; KCl&amp;nbsp;&lt;br/&gt; 12 mM&amp;nbsp; &amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;MgSO&lt;sub&gt;4&lt;/sub&gt;.7H&lt;sub&gt;2&lt;/sub&gt;O&amp;nbsp;&lt;br/&gt; 18 mM&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp;&amp;nbsp; Ca (NO&lt;sub&gt;3&lt;/sub&gt;)&lt;sub&gt;2&lt;/sub&gt;&lt;br/&gt; 150 mM&amp;nbsp;&amp;nbsp;&amp;nbsp; HEPES buffer&amp;nbsp;&lt;br/&gt; Distilled water upto 1 L&amp;nbsp;&lt;br/&gt; Store at 4 &lt;sup&gt;o&lt;/sup&gt;C&amp;nbsp;&lt;br/&gt; Use at 0.3x working solution</td></tr></tbody></table>

imaging subcellular structures in the living zebrafish embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Here the use of wide-field and confocal microscopy to image mitochondria and centrosomes in living zebrafish embryos is directly compared and contrasted. Depending on the location of the cells in which organelle dynamics are to be examined and the inherent frequency of the specific subcellular events, generally either wide-field or confocal microscopy is the better choice. We imaged organelles in RB neurons located on the surface of the embryo and in retinal cells located deeper. The supe...

Watch this Scientific Journal Video about Imaging Subcellular Structures in the Living Zebrafish Embryo at JoVE.com

Imaging Subcellular Structures in the Living Zebrafish Embryo

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

imaging-subcellular-structures-in-the-living-zebrafish-embryo

Research

JoVE Journal

Developmental Biology

11.5K Views.  Technische Universität München. Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy. 

Video: Imaging Subcellular Structures in the Living Zebrafish Embryo

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neuroscience

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Observing Mitotic Division and Dynamics in a Live Zebrafish Embryo

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin condensation, microtubule-kinetochore attachment, chromosome segregation, and cytokinesis in a small time frame. Errors in the delicate process can result in human disease, including birth defects and cancer. Traditional approaches investigating human mitotic disease states often rely on cell culture systems, which lack the natural physiology and developmental/tissue-specific context advantageous when studying human disease. This protocol overcomes many obstacles by providing a way to visualize, with high resolution, chromosome dynamics in a vertebrate system, the zebrafish. This protocol will detail an approach that can be used to obtain dynamic images of dividing cells, which include: in vitro transcription, zebrafish breeding/collecting, embryo embedding, and time-lapse imaging. Optimization and modifications of this protocol are also explored. Using H2A.F/Z-EGFP (labels chromatin) and mCherry-CAAX (labels cell membrane) mRNA-injected embryos, mitosis in AB wild-type, auroraBhi1045, and esco2hi2865 mutant zebrafish is visualized. High resolution live imaging in zebrafish allows one to observe multiple mitoses to statistically quantify mitotic defects and timing of mitotic progression. In addition, observation of qualitative aspects that define improper mitotic processes (i.e., congression defects, missegregation of chromosomes, etc.) and improper chromosomal outcomes (i.e., aneuploidy, polyploidy, micronuclei, etc.) are observed. This assay can be applied to the observation of tissue differentiation/development and is amenable to the use of mutant zebrafish and pharmacological agents. Visualization of how defects in mitosis lead to cancer and developmental disorders will greatly enhance understanding of the pathogenesis of disease.

Mitosis is critical to every living organism and defects often lead to cancer and developmental disorders. Using this imaging protocol and zebrafish as a model system, researchers can visualize mitosis in a live vertebrate organism and the multitude of defects that arise when mitotic processes are defective.

Mitosis is critical for organismal growth and differentiation. The process is highly dynamic and requires ordered events to accomplish proper chromatin ...

A Versatile Mounting Method for Long Term Imaging of Zebrafish Development

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

Here, we present a versatile mounting method that allows for the long-term time-lapse imaging of the posterior body development of live zebrafish embryos without perturbing normal development.

Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Biology

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Refined Multiview Light Sheet Microscopy Protocol for Zebrafish Embryos

Light Sheet Microscopy Imaging and Mounting Strategies for Early Zebrafish Embryos

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In particular, a multiview system, which allows sample rotation, enables imaging of entire embryos from different angles. However, in most imaging sessions with a multiview system, sample mounting is a troublesome process as samples are usually prepared in a polymer tube. To aid in this process, this protocol describes basic mounting strategies for imaging early zebrafish development between the 70% epiboly and early somite stages. Specifically, the study provides statistics on the various positions the embryos default to at the 70% epiboly and bud stages within the chorion. Furthermore, it discusses the optimum number of angles and the interval between angles required for imaging whole zebrafish embryos at the early stages of development so that cellular-scale information can be extracted by fusing the different views. Finally, since the embryo covers the entire field of view of the camera, which is required to obtain a cellular-scale resolution, this protocol details the process of using bead information from above or below the embryo for the registration of the different views.

Author Spotlight: Strategies for Mounting Zebrafish Embryos for High-Resolution Multiview Light-Sheet Microscopy &#8212; Techniques for Imaging and Image Reconstruction

A sample preparation strategy for imaging early zebrafish embryos within an intact chorion using a light-sheet microscope is described. It analyzes the different orientations that embryos acquire within the chorion at the 70% epiboly and bud stages and details imaging strategies for obtaining cellular-scale resolution throughout the embryo using the light-sheet system.

Light sheet microscopy has become the methodology of choice for live imaging of zebrafish embryos over long time scales with minimal phototoxicity. In ...

Imaging and 3D Reconstruction of Cerebrovascular Structures in Embryonic Zebrafish

Zebrafish are a powerful tool to study developmental biology and pathology in vivo. The small size and relative transparency of zebrafish embryos make them particularly useful for the visual examination of processes such as heart and vascular development. In several recent studies transgenic zebrafish that express EGFP in vascular endothelial cells were used to image and analyze complex vascular networks in the brain and retina, using confocal microscopy. Descriptions are provided to prepare, treat and image zebrafish embryos that express enhanced green fluorescent protein (EGFP), and then generate comprehensive 3D renderings of the cerebrovascular system. Protocols include the treatment of embryos, confocal imaging, and fixation protocols that preserve EGFP fluorescence. Further, useful tips on obtaining high-quality images of cerebrovascular structures, such as removal the eye without damaging nearby neural tissue are provided. Potential pitfalls with confocal imaging are discussed, along with the steps necessary to generate 3D reconstructions from confocal image stacks using freely available open source software.

Imaging of cerebrovascular development in larval zebrafish is described. Techniques to facilitate 3D imaging and modify cerebrovascular development using chemical treatments are also provided.

Zebrafish are a powerful tool to study developmental biology and pathology in vivo.  The small size and relative transparency of zebrafish embryos make ...

4-Dimensional Imaging of Zebrafish Optic Cup Morphogenesis

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of visual impairment, yet mechanisms of eye morphogenesis are still poorly understood. The basic organization of the embryonic eye is conserved throughout vertebrates, thus live imaging of zebrafish embryos has become a powerful approach to directly observe eye development at real time under normal and pathological conditions. Dynamic cell processes including movements, morphologies, interactions, division, and death can be visualized in the embryo. We have developed methods for uniform labeling of subcellular structures and timelapse confocal microscopy of early eye development in zebrafish. This protocol outlines the method of generating capped mRNA for injection into the 1-cell zebrafish embryo, mounting embryos at optic vesicle stage (~12 hours post fertilization, hpf), and performing multi-dimensional timelapse imaging of optic cup morphogenesis on a laser scanning confocal microscope, such that multiple datasets are acquired sequentially in the same imaging session. Such an approach yields data that can be used for a variety of purposes, including cell tracking, volume measurements, three-dimensional (3D) rendering, and visualization. Our approaches allow us to pinpoint the cellular and molecular mechanisms driving optic cup development, in both wild type and genetic mutant conditions. These methods can be employed directly by other groups or adapted to visualize many additional aspects of zebrafish eye development.

This protocol describes an approach for in toto labeling and multidimensional imaging of zebrafish early eye development. We describe labeling, embedding, and four dimensional (4D) imaging using laser scanning confocal microscopy, and considerations for optimizing acquisition of datasets for dissecting mechanisms of optic cup morphogenesis.

Visual system function requires the establishment of precise tissue and organ structures. In the vertebrate eye, structural defects are a common cause of ...