We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange mRNA In Vitro Transcription and mRNA Purification
<ol>
	<li>Linearize the H2B-PSmOrange containing pCS2+ plasmid using the NotI restriction enzyme according to manufacturer&#39;s instructions. 
	Note: Perform the next steps using appropriate protections such as gloves and lab coat to prevent mRNA contamination and degradation.</li>
	<li>Purify the linearized DNA using a PCR Purification kit or phenol-chloroform based methods according to manufacturers&#39; protocols.</li>
	<li>Use 1 &#181;g of linearized template DNA for Sp6-mRNA transcription according to manufacturer&#39;s instructions.</li>
	<li>Remove DNA by adding 1.0 U RNase free DNaseI for 10-20 min at 37 &#176;C.</li>
	<li>Clean up the mRNA using a RNA clean up kit according to manufacturer&#39;s protocol.</li>
	<li>To increase mRNA purity and concentration, precipitate the mRNA by adding 6 &#181;l sodium acetate (3.0 M, pH 5.2) and 150 &#181;l 96% ethanol. Incubate the mixed solution at -20 &#176;C for at least 30 min and up to 24 hr.</li>
	<li>Collect the mRNA by spinning the solution at 18.407 x g for 45 min at 4 &#176;C. Discard the liquid and resuspend the mRNA in 150 &#181;l 70% ethanol. Mix and centrifuge the solution for an additional 15 min at 4 &#176;C. Discard the liquid, dry the pellet for 5 min on ice and resuspend the mRNA in 20 &#181;l ultrapure RNase free water.</li>
	<li>Assess the concentration and purity of the mRNA by photometric measurement of a 1:100 mRNA dilution (1 &#181;l mRNA in 99 &#181;l ultrapure RNase free water).</li>
	<li>For short-term storage, keep the mRNA solution at -20 &#176;C. For long-term storage, keep the mRNA at -80 &#176;C.</li>
</ol>
2. H2B-PSmOrange mRNA Microinjection into Zebrafish Embryos
<ol>
	<li>Set up mating pairs the night before injection using tg(foxD3:GFP); tg(flh:GFP) double transgenic fish (or any other GFP transgenic fish). Leave the fish separated by placing a spacer between them to control the time of mating.</li>
	<li>Remove the spacer in the morning of injection and let the fish mate for 20 min.</li>
	<li>During mating time, dilute the H2B-PSmOrange mRNA to the final concentration of 130 pg/nl (in RNase free water) and transfer 6 &#181;l injection solution into a microinjection capillary using a microloader tip. Check the injection volume with a calibrated glass slide. Adjust the injection pressure to inject 2 nl mRNA solution (260 pg).</li>
	<li>Collect the eggs into a sterilized plastic petri-dish containing 1x Embryo (E3) medium.</li>
	<li>Transfer 20 to 30 embryos to an injection dish using a plastic pasteur pipette.</li>
	<li>Inject 2 nl mRNA containing solution into the cell or just below the cell into the yolk at the one-cell stage 11.</li>
	<li>Transfer injected embryos into a petri-dish containing 1x E3 medium, remove unfertilized or not normally developing embryos and grow them at 28 &#176;C. 
	Note: Light protection of the embryos is not required throughout the experiment.</li>
	<li>Raise zebrafish embryos in 1x E3 medium and add 0.2 mM PTU (1-phenyl-2 thiourea) after gastrulation to inhibit pigmentation. Change the medium twice per day to minimize the danger of bacterial contaminations.</li>
</ol>
3. Embryo Embedding
<ol>
	<li>Select the GFP (green) and PSmOrange (orange/red) co-expressing embryos using a binocular microscope equipped with a fluorescence lamp and appropriate emission filters. Transfer the positive embryos into a sterile petri-dish containing 1x E3 medium.</li>
	<li>Dechorionate embryos under a stereomicroscope using forceps 12.</li>
	<li>Transfer one embryo in a 1.5 ml tube containing 1.0 ml of pre-warmed 1.0% low melting agarose (LMA) prepared in ultrapure water by using a cut-tip pipette (P200). Note: Warm the LMA to 80 &#176;C and cool down for 3-5 min at RT before embedding the embryo.</li>
	<li>Transfer the embryo in 150 &#181;l LMA into a chambered coverglass and adjust the embryo&#39;s orientation, e.g. dorsal side down in the experiment described for the inverted confocal laser scanning microscope A1R+ (or any other suitable microscope), using a fine plastic tip. When the LMA is polymerized, fill the chamber with fish water containing 0.2 mM PTU and 0.02% ethyl 3-aminobenzoate methansulfonate (Tricaine) for anesthesia.</li>
	<li>Before imaging the sample, wait 15 min to ascertain the effect of Tricaine. The anesthesia prevents embryo movements, which can be monitored using a stereomicroscope equipped with brightfield illumination. 
	Note: The chambers can be reused twice.</li>
</ol>
4. PSmOrange Photoconversion
<ol>
	<li>Place the sample under the confocal microscope and scan the specimen to identify the area to photoconvert using 488 nm and 561 nm lasers for imaging GFP and PSmOrange respectively. 
	Note: Use the inverted confocal laser scanning microscope A1R+ on the inverted stage TiEcontrolled by the microscope imaging software and equipped with 488 nm, 561 nm and 640 nm lasers for photoconversion and imaging. However, the application is certainly not restricted to this microscope as long as the laser lines for conversion and imaging are available.
	<ol>
		<li>Put the 20X air objective into place (NA: 0.75; WD: 1.0 mm; FOV: 0.64 x 0.64 mm).</li>
		<li>Use the following microscope settings to detect the PSmOrange protein before photoconversion: 561 nm laser, 0.74 mW measured at the focus plane above the objective. 
		Note: This corresponds to 40% on this system (measuring in the focus plane is the only way to determine the effective power). Adjust the laser power according to experimental needs as the efficiency of H2B-PSmOrange injections can vary.</li>
		<li>Add zoom factors to highlight the area of interest. Higher magnifications reduce image acquisition time and therefore phototoxicity.</li>
	</ol>
	</li>
	<li>Acquire a z-stack covering the structures of interest using 488 nm and 561 nm lasers in sequential mode (line mode 1-&#62;4). Fix the z-step between 1.0 and 2.0 &#181;m. Line average and scan frequency can be used to optimize image quality.</li>
	<li>Scan the sample and select the region of interest (ROI) tool to highlight the area to photoconvert. Fix the ROI as stimulation area. Select the Photo Activation/Bleaching module, activate the 488 nm laser by checking the respective box and set the 488 nm laser to 80% (1 mW laser power measured at the objective). Set the scan speed to 0.5 sec/Frame.</li>
	<li>Open the photoconversion utility (ND Stimulation) and specify the photoconversion settings.
	<ol>
		<li>Click on the &#34;Add&#34; command to specify the photoconversion protocol.</li>
		<li>Set &#34;Phase&#34; 1 in the &#34;Acquisition/Stimulation&#34; menu to &#34;Acquisition&#34; and indicate the number of images to be acquired before photoconversion in the &#34;Loops&#34; menu.</li>
		<li>Set &#34;Phase&#34; 2 to &#34;Stimulation&#34; and enter the number of stimulation events to be performed.</li>
		<li>Adjust &#34;Phase&#34; 3 as described for &#34;Phase&#34; 1. Optionally, insert a waiting &#34;Phase&#34; after &#34;Phase&#34; 2 by entering the time to wait before the last round of image acquisition.</li>
		<li>Once all the parameters are set, apply the stimulation setting and run the photoconversion. 
		Note: Laser power, frequency of scanning and number of iterations for each round of photoconversion can vary and differ from embryo to embryo. A setting to start with is shown in Table 1.</li>
	</ol>
	</li>
	<li>Acquire a final z-stack using 488 nm, 561 nm and 640 nm lasers applying the settings as above. Set the 640 nm laser to visualize the converted PSmOrange using high laser power (up to 4.5 mW).</li>
</ol>
5. Dismount Embryo from LMA
<ol>
	<li>Remove the embryo containing chamber from the microscope. Carefully remove the embryo from the agarose using forceps.</li>
	<li>Transfer the embryo into a new sterilized plastic petri-dish or into a sterilized 6-well-plate containing 1x E3 and 0.2 mM PTU. Incubate the embryo at 28 &#176;C until the desired stage of development.</li>
</ol>
6. Analyzing the Fate of Photoconverted PSmOrange Protein Expressing Cells
<ol>
	<li>Re-embed the embryo as described under points 3.3 and 3.4.</li>
	<li>Place the sample under the confocal microscope and scan the specimen to identify photoconverted cells (640 nm) in the GFP transgenic structure of interest (488 nm).</li>
	<li>Acquire a z-stack covering the structures of interest using 488 nm, 561 nm and 640 nm lasers in sequential mode (line mode 1-&#62;4). Fix the z-step between 1.0 and 2.0 &#181;m. Line average and scan frequency can be used to optimize image quality.</li>
	<li>Use one of the following methods to identify cells, which co-express GFP and the photoconverted protein.
	<ol>
		<li>Custom made automatic Fiji ImageJ Macro3
		<ol>
			<li>Convolve the original stack using the &#34;GaussianBlur&#34; plugin ( &#8594; Process &#8594; Filters &#8594; GaussianBlur) and subtract the smooth stacks from the original using the &#34;Image Calculator&#34; plugin (&#8594; Process &#8594; Image Calculator). Use this step to visualize the structures of interest and reduce the computational time for the analysis.</li>
			<li>Apply specific thresholds to the green and far-red channels in order to highlight the photoconverted cells ( &#8594; Image &#8594; Adjust &#8594; Threshold). Use the &#34;Analyze Particles&#34; tool to detect the threshold areas with a suitable setting ( &#8594; Analyze &#8594; Analyze Particles).</li>
			<li>Open the &#34;Image Calculator&#34; plugin and display the overlapping ROIs in the green and far-red channel in yellow ( &#8594; Process &#8594; Image Calculator).</li>
		</ol>
		</li>
		<li>Microscope imaging software for 3D data evaluation
		<ol>
			<li>Display the stack in 3D using the show volume view option ( &#8594; 3D Visualization Menu &#8594; Show Volume View). Use the graphic interface to select the green, red and far-red channels, adjust the brightness and the contrast and crop the 3D stack to highlight the photoconverted area (Figure 1). 
			Note: Similar methods to analyse the data are available in most of the common software for image analysis.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nature Protocols. 2, 2024-2032 (2007).</li><li>Subach, F. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6, 153-159 (2009).</li><li>Patterson, G. 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D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), e1115 (2009).</li><li>Concha, M. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Local+tissue+interactions+across+the+dorsal+midline+of+the+forebrain+establish+CNS+laterality.">Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.</a> Neuron. 39, 423-438 (2003).</li><li>Auer, T. O., Duroure, K., Concordet, J. 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The authors have nothing to disclose.

Transgenic embryos carrying fluorescent reporters have helped fundamentally to understand embryonic development. However, there is still the essential need for promoters to facilitate the specific visualization of particular structures. In their absence, researchers rely on techniques such as the photoconversion of fluorescent proteins to find out about the origin and development of their structure of interest. This in turn is a crucial prerequisite to identify the molecular mechanisms involved in its development. Techni...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table><tbody><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Plastic Pasteur</td><td>alpha laboratories</td><td>LW4000</td><td></td></tr><tr><td>Original H2B-PSmOrange Plasmid</td><td>Addgene</td><td>31920</td><td>The plasmid described in the paper is available in the Carl lab</td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.013</td><td></td></tr><tr><td>Forceps (5 Inox)</td><td>NeoLab</td><td>2-1633</td><td></td></tr><tr><td>Lab-Tek II Chambered #1.5 German Coverglass System&amp;nbsp;</td><td>Nunc</td><td>155382</td><td></td></tr><tr><td>Nikon A1R+</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>Nikon PLAN Apo &amp;lambda; 20X air objective&amp;nbsp;</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>NIS Elements AR Software (v. 4.30.02)</td><td>Nikon GmbH Germany/Laboratory Imaging</td><td>No Number</td><td></td></tr></tbody></table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

Figure 1 illustrates an example of the PSmOrange photoconversion system. The pineal complex is a conserved structure in the vertebrate dorsal diencephalon. Like in many other vertebrates, this complex consists of the pineal organ in the center of the diencephalon and the left-sided parapineal cells. Elegant but time-consuming uncaging experiments showed that parapineal cells originate in the anterior part of the pineal organ13. In tg(foxD3:GFP); tg(flh:GFP) tra...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.5K Views.  Heidelberg University. We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Video: Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biology

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Neuroscience

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www.jove.com/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...