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 ...

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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.

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

cell-tracking-using-photoconvertible-proteins-during-zebrafish

Research

JoVE Journal

Biology

11.3K vistas.  Max Delbrück Center for Molecular Medicine. 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.