We would like to thank Dan Carlin for help in making caged fluorescein. In addition we would like to thank the following funding sources: Vanderbilt University Medical School Program for Developmental Biology Training Grant to JAC, and NIH HD054534to JTG.

1. Synthesis of Caged Fluorescein Dextran
<ol>
	<li>Measure out 3.5 - 4 mg of aminodextran and add into the Invitrogen-supplied tinted tube containing 1 mg of CMNB-caged fluorescein SE. In our hands this ratio gives an average loading of ~ 2.5 dye molecules per dextran.</li>
	<li>Add 500 &mu;L of 0.1 M Na2B4O7 (sodium borate) buffer to the tube.</li>
	<li>Cap and vortex for 30 seconds to dissolve the aminodextran and caged fluorescein.</li>
	<li>Let react overnight on a vortexing mixer...

<ol>
	<li>Hatada, Y., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fate+map+of+the+epiblast+of+the+early+chick+embryo.">A fate map of the epiblast of the early chick embryo.</a> Development. 120, 2879-2889 (1994).</li><li>Moody, S. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fates+of+the+blastomeres+of+the+32-cell-stage+Xenopus+embryo.">Fates of the blastomeres of the 32-cell-stage Xenopus embryo.</a> Dev Biol. 122, 300-319 (1987).</li><li>Wetts, R., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multipotent+Precursors+Can+Give+Rise+to+All+Major+Cell-Types+of+the+Frog+Retina.">Multipotent Precursors Can Give Rise to All Major Cell-Types of the Frog Retina.</a> Science. 239, 1142-1145 (1988).</li><li>Wetts, R., Fraser, S. E. <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+fluorescent+tracers+to+study+neural+cell+lineages.">Microinjection of fluorescent tracers to study neural cell lineages.</a> Development. , 1-8 (1991).</li><li>Woo, K., Fraser, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Order+and+coherence+in+the+fate+map+of+the+zebrafish+nervous+system.">Order and coherence in the fate map of the zebrafish nervous system.</a> Development. 121, 2595-2609 (1995).</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>Sato, T., Takahoko, M., Okamoto, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuC:Kaede,+a+useful+tool+to+label+neural+morphologies+in+networks+in+vivo.">HuC:Kaede, a useful tool to label neural morphologies in networks in vivo.</a> Genesis. 44, 136-142 (2006).</li><li>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=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>Haugland, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Handbook of Fluorescent Probes and Research Chemicals. , 707-711 (2002).</li><li>Kozlowski, D. J., Murakami, T., Ho, R. K., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regional+cell+movement+and+tissue+patterning+in+the+zebrafish+embryo+revealed+by+fate+mapping+with+caged+fluorescein.">Regional cell movement and tissue patterning in the zebrafish embryo revealed by fate mapping with caged fluorescein.</a> Biochem Cell Biol. 75, 551-562 (1997).</li><li>Kozlowski, D. J., Weinberg, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+(caged)+fluorescein+as+a+cell+tracer+for+fate+mapping+in+the+zebrafish+embryo.">Photoactivatable (caged) fluorescein as a cell tracer for fate mapping in the zebrafish embryo.</a> Methods Mol Biol. 135, 349-355 (2000).</li><li>Russek-Blum, N., Nabel-Rosen, H., Levkowitz, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+resolution+fate+map+of+the+zebrafish+diencephalon.">High resolution fate map of the zebrafish diencephalon.</a> Dev Dyn. 238, 1827-1835 (2009).</li><li>Summers, R. G., Piston, D. W., Harris, K. M., Morrill, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+orientation+of+first+cleavage+in+the+sea+urchin+embryo,+Lytechinus+variegatus,+does+not+specify+the+axes+of+bilateral+symmetry.">The orientation of first cleavage in the sea urchin embryo, Lytechinus variegatus, does not specify the axes of bilateral symmetry.</a> Dev Biol. 175, 177-183 (1996).</li></ol>

As described, this protocol provides a relatively quick lineage labeling method in zebrafish that is built upon the commonly used techniques of microinjection, microscopy, and immunofluorescence. We have found that laser uncaging to be the most efficient and cost effective way to uncage fluorescein in a localized fashion. This method could be used to lineage label with experimental endpoints as late as 4 dpf. However, as cells divide, the caged-fluorescein concentration per cell eventually decreases beyond detectable lev...

A correction was made to <a href="http://www.jove.com/details.php?id=2672">Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran</a>. There was an error in the authors, Ilya A. Shestopalov and James K. Chen, names. The author's names have been corrected to: Ilya A. Shestopalov and James K. Chen instead of: Ilya Shestopalov and James Chen

A correction to the article Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran was made.

Erratum: Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>CMNB-caged fluorescein SE</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>C20050</td>
<td>&nbsp;</td>
<tr>
<td>Aminodextran, 10kDa</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>D1860</td>
<td>&nbsp;</td>
<tr>
<td>Zeba desalt spin columns</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>89889</td>
<td>&nbsp;</td>
<tr>
<td>goat anti-fluorescein antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A11095</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 633 Donkey anti-goat antibody</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>A21082</td>
<td>&nbsp;</td>
<tr>
<td>35mmx10mm petri dishes</td>
<td>&nbsp;</td>
<td>Becton-Dickinson</td>
<td>351008</td>
<td>&nbsp;</td>
<tr>
<td>Upright compound microscope with 40x water immersion objective</td>
<td>&nbsp;</td>
<td>Leica</td>
<td>DM6000B</td>
<td>&nbsp;</td>
<tr>
<td>MicroPoint Manual Laser Illumination System</td>
<td>&nbsp;</td>
<td>Photonic Instruments</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Phenylthiourea (PTU)</td>
<td>&nbsp;</td>
<td>Alfa Aesar</td>
<td>L06690</td>
<td>&nbsp;</td>
<tr>
<td>Tricaine</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>E10521</td>
<td>&nbsp;</td>
<tr>
<td>Proteinase K</td>
<td>&nbsp;</td>
<td>Roche</td>
<td>03115836991</td>
<td>&nbsp;</td>
<tr>
<td>Plastic thread</td>
<td>&nbsp;</td>
<td>Any sewing supply store</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Agarose</td>
<td>&nbsp;</td>
<td>Research Products International</td>
<td>A20090</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Thermo Scientific</td>
<td>Savant SPD131DDA</td>
<td>&nbsp;</td>
<tr>
<td>Vortexing mixer</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>Vortex Genie 2</td>
<td>&nbsp;</td>
<tr>
<td>Pressure injector</td>
<td>&nbsp;</td>
<td>Applied Scientific Instrumentation</td>
<td>MPPI-3</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

lineage labeling of zebrafish cells with laser uncagable fluorescein dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

Watch this Scientific Journal Video about Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran at JoVE.com

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

lineage-labeling-zebrafish-cells-with-laser-uncagable-fluorescein

Research

JoVE Journal

Biology

13.7K visualizações.  Vanderbilt University. Este protocolo delineia uma forma de rótulo e traçar o destino de pequenos grupos de células de embriões zebrafish utilizando UV-uncaging de fluoresceína enjaulado, seguido por montar todo imunomarcação para amplificar o sinal da fluoresceína uncaged.