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...

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKikGR,+a+monomeric+photoswitchable+fluorescent+protein.">mKikGR, a monomeric photoswitchable fluorescent protein.</a> PLoS One. 3, e3944 (2008).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nature Methods. 6, 131-133 (2009).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. 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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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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 border="0" cellpadding="0" cellspacing="0" width="821">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:303px;">PCR Purification Kit</td>
			<td style="width:233px;">Qiagen</td>
			<td align="right" style="width:119px;">28104</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">mMESSAGE mMACHINE SP6 Transcription kit</td>
			<td>Ambion</td>
			<td>AM1340</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RNeasy MiniElute Cleanup kit</td>
			<td>Qiagen</td>
			<td align="right">74204</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plastic Pasteur</td>
			<td>alpha laboratories</td>
			<td>LW4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 height="21">
			<td height="21" style="height:21px;">FemtoJet Microinjector</td>
			<td>Eppendorf</td>
			<td>5247 000.013</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Forceps (5 Inox)</td>
			<td>NeoLab</td>
			<td>2-1633</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Lab-Tek II Chambered #1.5 German Coverglass System&#160;</td>
			<td>Nunc</td>
			<td align="right">155382</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon A1R+</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nikon PLAN Apo &#955; 20x air objective&#160;</td>
			<td>Nikon GmbH Germany</td>
			<td>No Number</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">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 ...

The overall goal of the PSmOrange Photo Conversion Technology is to track cells in the living animal during embryonic development and disease. This method can answer key questions in developmental biology such as uncovering the origin of cells which develop into tissues and organs. The main advantage of this technique is that it can be applied in GFP transgenic backgrounds.This method can also be applied in different fields of investigation such as regeneration, tumor progression and invasion. After generating and embedding PSmOrange and GFP expressing embryos according to the text protocol, place the embryo under the confocal microscope and use the 20x air objective and 488 and 561 nanometer lasers to scan the specimen to identify an area to photoconvert. To detect the PSmOrange protein before photoconversion, use the 561 nanometer laser at 0.74 milliwatts of power measured at the focus plane above the objective and adjust the laser power as necessary.Adjust the zoom factors to highlight the area of interest and reduce image acquisition time and photo toxicity. Using the 488 and 561 nanometer lasers in sequential mode and a Z step between 1.0 and 2.0 micrometers, acquire a Z stack covering the structures of interest. Adjust the line average and scan frequency to optimize the image quality.Then scan the sample and select the region of interest, or ROI tool, to highlight the area to photoconvert before fixing the ROI as the stimulation area. Select the photoactivation bleaching module, check the box to activate the 488 nanometer laser, then set the 488 nanometer laser to 80%and the scan speed to 0.5 seconds per frame. Next, open the photoconversion utility and click on Add'to specify the photoconversion protocol.In the acquisition stimulation menu, set Phase One to acquisition and in the loops menu, indicate the number of images to be acquired before photoconversion. Set Phase Two to stimulation and enter the number of stimulation events to be performed. Adjust Phase Three as described for Phase One.Optionally, insert a Waiting Phase after Phase Two by entering the time to wait before the last round of image acquisition. Once the parameters are set, apply the stimulation setting and run the photoconversion. Miscroscopic settings are crucial for PSmOrange photoconversion.To ensure a successful photoconversion, the embryo is imaged directly after the experiment to detect the photoconverted PSmOrange protein. If the conversion was not successful, the procedure is repeated. Using the 488, 561 and 640 nanometer lasers, and setting the 640 nanometer laser to high power, up to 4.5 milliwatts, acquire a final z stack with the settings just described.To dismount the embryo, remove the chamber from the microscope and use forceps to remove the embryo from the agarose. Transfer the embryo into a new sterilized plastic petri dish, or sterilized 6-well plate, containing 0.2 millimolar PTU in E3.Incubate the embryo at 28 degrees Celsius until the desired stage of development. To analyze the fate of photoconverted PSmOrange protein expressing cells, re-embed the embryo according to the text protocol.Under the confocal miscroscope, scan the specimen to identify photoconverted cells in the GFP transgenic structure of interest. Use the 488, 561, and 640 nanometer lasers to acquire a z stack with a fixed stack of 1 to 2 micrometers in sequential mode covering the structures. Using Image J Fiji software, identify cells which co-express GFP and the photoconverted protein.Duplicate the original stack, use the Gaussian Blur plugin and the image calculator to subtract the smooth stacks from the original. Apply specific thresholds to the green and far red channels in order to highlight the photoconverted cells. Then use the Analyze Particles tool to detect the threshold areas in the green channel with a suitable setting.Repeat this step for the far red channel. Open the image calculator plugin to detect the co-localizing cells. Display in yellow the overlapping ROIs in the green or the far red channel using the Analyze Particles tool.Using NIS-Elements software, carry out 3D date evaluation and display the stack in 3D using the Show Volume View option. Then 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. Shown here is an example of the PSmOrange photocoversion system in zebra fish transgenic FOXD3 floating head GFP embryos.At 26 hours post-fertilization, two cell clusters in the anterior part of the pineal were photoconverted and immediately imaged. The protein was detected with a 640 nanometer far red laser but not with a 561 nanometer laser. In these cells, GFP expression was initially reduced due to photobleaching.At 52 HPF, GFP and H2B PSmOrange were detected in the photoconverted H2B PSmOrange protein expressing cells. A few of these cells formed the parapineal on the left side of the brain. This result is consistent with previous reports on the parapineal cell origin in the anterior pineal and shows the applicability of the PSmOrange technique in the living GFP transgenic vertebrate embryo.Following this procedure, the cell tracking analysis can be performed in genetically manipululated embryos in order to answer additional questions regarding the molecular hierarchies underlying the cell's development.

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

Research

JoVE Journal

Developmental Biology

8.5K visualizações.  Heidelberg University. The overall goal of the PSmOrange Photo Conversion Technology is to track cells in the living animal during embryonic development and disease. This method can answer key questions in developmental biology such as uncovering the origin of cells which develop into tissues and organs. The main advantage of this technique is that it can be applied in GFP transgenic backgrounds.This method can also be applied in different fields of investigation such as regeneration, tumor progression and i...