Name: cell tracking using photoconvertible proteins during zebrafish development
Uploaded: 2012-09-28T13:00:00
Description: Watch this Scientific Journal Video about Cell Tracking Using Photoconvertible Proteins During Zebrafish Development at JoVE.com

We thank Dr. Ian C. Scott for kindly providing the zebrafish transgenic Tg(kdrl:nlsKikGR)hsc7 line at the Hospital for Sick Children, Toronto, Canada. We thank the Confocal and 2-Photon Microscopy Core Facility (Max-Delbrueck-Center for Molecular Medicine) and Dr. Zoltan Cseresnyes, for excellent technical assistance and overall imaging support. S.A.-S. is supported by a Heisenberg fellowship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

1. Obtaining Embryos for the Photoconversion Assay
Upon in-crossing of transgenic zebrafish expressing a PCFP the brightest which are homozygote embryos are chosen, and grown at 28.5 &deg;C until the desired embryonic stage15.
Note 1: This preselection will allow scanning embryos with low laser power thereby preventing phototoxicity and photobleaching.
Note 2: We suggest to use low-intensity light when visualizing embryos and to ...

<ol>
	<li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chem. Soc. Rev. 38, 2887-2921 (2009).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U. S. A. 99, 12651-12656 (2002).</li><li>Tsutsui, H., Karasawa, S., Shimizu, H., Nukina, N., Miyawaki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Semi-rational+engineering+of+a+coral+fluorescent+protein+into+an+efficient+highlighter.">Semi-rational engineering of a coral fluorescent protein into an efficient highlighter.</a> EMBO Rep. 6, 233-238 (2005).</li><li>Gurskaya, N. G., Verkhusha, V. V., Shcheglov, A. S., Staroverov, D. B., Chepurnykh, T. V., Fradkov, A. F., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Engineering+of+a+monomeric+green-to-red+photoactivatable+fluorescent+protein+induced+by+blue+light.">Engineering of a monomeric green-to-red photoactivatable fluorescent protein induced by blue light.</a> Nat. Biotechnol. 24, 461-465 (2006).</li><li>Wiedenmann, J., Ivanchenko, S., Oswald, F., Schmitt, F., Rocker, C., Salih, A., Spindler, K. D., Nienhaus, G. U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc. Natl. Acad. Sci. U.S.A. 101, 15905-15910 (2004).</li><li>Hatta, K., Tsujii, H., Omura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+a+photoconvertible+fluorescent+protein.">Cell tracking using a photoconvertible fluorescent protein.</a> Nat. Protoc. 1, 960-967 (2006).</li><li>Stark, D. A., Kulesa, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+in+vivo+comparison+of+photoactivatable+fluorescent+proteins+in+an+avian+embryo+model.">An in vivo comparison of photoactivatable fluorescent proteins in an avian embryo model.</a> Dev. Dyn. 236, 1583-1594 (2007).</li><li>Kulesa, P. M., Teddy, J. M., Stark, D. A., Smith, S. E., McLennan, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neural+crest+invasion+is+a+spatially-ordered+progression+into+the+head+with+higher+cell+proliferation+at+the+migratory+front+as+revealed+by+the+photoactivatable+protein,+KikGR.">Neural crest invasion is a spatially-ordered progression into the head with higher cell proliferation at the migratory front as revealed by the photoactivatable protein, KikGR.</a> Dev. Biol. 316, 275-287 (2008).</li><li>Nowotschin, S., Hadjantonakis, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+KikGR+a+photoconvertible+green-to-red+fluorescent+protein+for+cell+labeling+and+lineage+analysis+in+ES+cells+and+mouse+embryos.">Use of KikGR a photoconvertible green-to-red fluorescent protein for cell labeling and lineage analysis in ES cells and mouse embryos.</a> BMC Dev. Biol. 9, 49 (2009).</li><li>Griswold, S. L., Sajja, K. C., Jang, C. W., Behringer, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+and+characterization+of+iUBC-KikGR+photoconvertible+transgenic+mice+for+live+time-lapse+imaging+during+development.">Generation and characterization of iUBC-KikGR photoconvertible transgenic mice for live time-lapse imaging during development.</a> Genesis. 49, 591-598 (2011).</li><li>Baker, S. M., Buckheit, R. W., Falk, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green-to-red+photoconvertible+fluorescent+proteins:+tracking+cell+and+protein+dynamics+on+standard+wide-field+mercury+arc-based+microscopes.">Green-to-red photoconvertible fluorescent proteins: tracking cell and protein dynamics on standard wide-field mercury arc-based microscopes.</a> BMC Cell Biol. 11, 15 (2010).</li><li>Watanabe, S., Punge, A., Hollopeter, G., Willig, K. I., Hobson, R. J., Davis, M. W., Hell, S. W., Jorgensen, E. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+localization+in+electron+micrographs+using+fluorescence+nanoscopy.">Protein localization in electron micrographs using fluorescence nanoscopy.</a> Nat. Methods. 8, 80-84 (2011).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. 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>Fang, Z., Takizawa, N., Wilson, K. A., Smith, T. C., Delprato, A., Davidson, M. W., Lambright, D. G., Luna, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+membrane-associated+protein,+supervillin,+accelerates+F-actin-dependent+rapid+integrin+recycling+and+cell+motility.">The membrane-associated protein, supervillin, accelerates F-actin-dependent rapid integrin recycling and cell motility.</a> Traffic. 11, 782-799 (2010).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203, 253-310 (1995).</li><li>Isogai, S., Horiguchi, M., Weinstein, B. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+vascular+anatomy+of+the+developing+zebrafish:+an+atlas+of+embryonic+and+early+larval+development.">The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development.</a> Dev. Biol. 230, 278-301 (2001).</li><li>Lazic, S., Scott, I. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mef2cb+regulates+late+myocardial+cell+addition+from+a+second+heart+field-like+population+of+progenitors+in+zebrafish.">Mef2cb regulates late myocardial cell addition from a second heart field-like population of progenitors in zebrafish.</a> Dev. Biol. 354, 123-133 (2011).</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> Nat. Methods. 6, 131-133 (2009).</li><li>Godinho, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Live+imaging+of+zebrafish+development.">Live imaging of zebrafish development.</a> Cold Spring Harb Protoc. 2011, 770-777 (2011).</li><li>Curran, K., Lister, J. A., Kunkel, G. R., Prendergast, A., Parichy, D. M., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interplay+between+Foxd3+and+Mitf+regulates+cell+fate+plasticity+in+the+zebrafish+neural+crest.">Interplay between Foxd3 and Mitf regulates cell fate plasticity in the zebrafish neural crest.</a> Dev. Biol. 344, 107-118 (2010).</li><li>Nienhaus, G. U., Nienhaus, K., Holzle, A., Ivanchenko, S., Renzi, F., Oswald, F., Wolff, M., Schmitt, F., Rocker, C., Vallone, B., Weidemann, W., Heilker, R., Nar, H., Wiedenmann, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoconvertible+fluorescent+protein+EosFP:+biophysical+properties+and+cell+biology+applications.">Photoconvertible fluorescent protein EosFP: biophysical properties and cell biology applications.</a> Photochem. Photobiol. 82, 351-358 (2006).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li></ol>

In recent years, transgenic animal models for the expression of photoconvertible proteins such as Kaede or KikGR have been generated. These animals develop normally, indicating that these proteins have no toxic effects on embryonic development. The first report about a photoconversion assay for tracking cells in animal embryos was performed by Hatta and collaborators by injection of mRNA or DNA encoding Kaede into one-cell zebrafish embryos for ubiquitous expression, or by expression of Kaede and KikGR in neural tissue b...

<table border="1">
 <tbody>
 <tr>
 <td>Name</td>
 <td>Company</td>
 <td>Catalog #</td>
 <td>Comments</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Materials:</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Necessary equipment for raising fish and collecting eggs (see the Zebrafish Book22 for details).</td>
 </tr>
 <tr>
 <td>Stereomicroscope</td>
 <td>Leica</td>
 <td>Lecia MZ6 Stereomicroscope</td>
 <td>Equipped with laser operating at 488 nm</td>
 </tr>
 <tr>
 <td>Confocal laser scanning microscope</td>
 <td>Zeiss</td>
 <td>Zeiss LSM 710 NLO</td>
 <td>equipped with lasers operating at 488 nm, 561 nm, and 405 nm</td>
 </tr>
 <tr>
 <td>Heating block</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Pair of Dumont #5 forceps</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Cover-glass-bottomed culture dish</td>
 <td>MatTek Corp</td>
 <td>&nbsp;</td>
 <td>Ashland MA USA</td>
 </tr>
 <tr>
 <td>Microloader Pipette Tips</td>
 <td>Eppendorf</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Vortex</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>needles</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Reagents:</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>Egg water medium</td>
 </tr>
 <tr>
 <td>60 &mu;g/ml Instant Ocean Sea Salts in ddH2O</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>E3 medium </td>
 </tr>
 <tr>
 <td>5 mM NaCl</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.17 mM KCl</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.33 mM CaCl2</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.33 mM MgSO4</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>0.00001% (w/v) Methylene Blue</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>4 mg/ml Tricaine stock solution </td>
 </tr>
 <tr>
 <td>400 mg Tricaine (3-amino benzoic acidethylester)</td>
 <td>Sigma-Aldrich</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>97.9 ml ddH2O</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>2.1 ml 1M Tris (pH9)</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Adjust at pH 7, aliquot and store -20 &deg;C</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>1% LMA (Low melting temperature agarose, Lonza)</td>
 </tr>
 <tr>
 <td>1g LMA/100 ml E3 medium 1X</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 <tr>
 <td>Aliquot and store at 4 &deg;C</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 <td>&nbsp;</td>
 </tr>
 </tbody>
</table>

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.

Watch this Scientific Journal Video about Cell Tracking Using Photoconvertible Proteins During Zebrafish Development at JoVE.com

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

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.

Handy-Tracking Mit Photoconvertible Proteine ​​während der Zebrafisch-Entwicklung

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

Research

JoVE Journal

Biology

Selective Labelling of Cell-surface Proteins using CyDye DIGE Fluor Minimal Dyes

Selektive Markierung von Zelloberflächen-Proteinen unter Verwendung von CyDye DIGE Fluor Minimal Farbstoffe

Surface  proteins are central to the cell's ability to react to its environment and to interact with neighboring cells. They are known to be inducers of ...

Forschung

Biologie

Visually Mediated Odor Tracking During Flight in Drosophila

Visuell vermittelte Odor-Tracking während des Fluges in Drosophila

Flying insects use visual cues to stabilize their heading in a wind stream. Many animals additionally track odors carried in the wind. As such, visual ...

Determining Cell Number During Cell Culture using the Scepter Cell Counter

Bestimmen Zellzahl während der Zellkultur mit dem Scepter Cell Counter

Counting cells is often a necessary but tedious step for in vitro cell culture. Consistent cell concentrations ensure experimental reproducibility and ...

Closed System Cell Culture Protocol Using HYPERStack Vessels with Gas Permeable Material Technology

Closed System Cell Culture Protocol Mit HyperStack Schiffe mit gasdurchlässigen Material Technology

Large volume adherent cell culture is currently standardized on stacked plate cell growth products when microcarrier beads are not an optimal choice.   ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

Die Beurteilung Signaling Eigenschaften von ektodermale Epithel Während Craniofacial Entwicklung

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Optimized Staining and Proliferation Modeling Methods for Cell Division Monitoring using Cell Tracking Dyes

Optimiert Färbung und Proliferation Modeling-Methoden für die Zellteilung Überwachung mit Cell-Tracking Dyes

Fluorescent cell tracking dyes, in combination with flow and image cytometry, are powerful tools with which to study the interactions and fates of ...

Blastomere Explants to Test for Cell Fate Commitment During Embryonic Development

Blastomere Explantate für das Schicksal der Zelle Engagement während der embryonalen Entwicklung Testen

Fate maps, constructed from lineage tracing all of the cells of an embryo, reveal  which tissues descend from each cell of the embryo.  Although fate maps ...

Using an Automated 3D-tracking System to Record Individual and Shoals of Adult Zebrafish

Like many aquatic animals, zebrafish (Danio rerio) moves in a 3D space. It is thus preferable to use a 3D recording system to study its behavior. The ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Analyse Craniofacial Morphogenese in Zebrafisch Mit 4D konfokale Mikroskopie

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...

Handy-Tracking Mit Photoconvertible Proteine ​​während der Zebrafisch-Entwicklung

Handy-Tracking Mit Photoconvertible Proteine während der Zebrafisch-Entwicklung