Name: transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos
Uploaded: 2010-07-19T17:00:00
Description: Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

<p class="jove_content">This study was supported by a National Institutes of Health (NIH) Core Grant (5P30EY008098) and the following funds to X.W.: NIH Grant R01EY016099 and Research to Prevent Blindness Career Development Award. We thank Kira Lathrop for assistance in confocal imaging.</p>

<p class="jove_title">Day 1. Preparation</p>
<p class="jove_step">1. Set up fish crosses</p>
<p class="jove_content">Set up pairwise crosses of zebrafish in the evening. To prevent random mating before the desired time, use dividers to separate the female and male fish in the mating cassettes. GFP-expressing transgenic fish are used as a source of donor blastomeres. The pt104 transgenic line (Zou <em>et al</em>., 2008), used here, expresses GFP ubiquitously under the control of the EF1 promoter. GFP expression allows visualization of donor ce...

     

<ol>
	<li>Zou, J., Lathrop, K., Sun, M., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intact+RPE+maintained+by+Nok+is+essential+for+retinal+epithelial+polarity+and+cellular+patterning+in+zebrafish.">Intact RPE maintained by Nok is essential for retinal epithelial polarity and cellular patterning in zebrafish.</a> <em style='font-style: italic'>Journal of Neuroscience</em>. <b>28</b> (50), 13684-13695 (2008).</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+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">A guide for the laboratory use of zebrafish (Danio rerio).</a> <em style='font-style: italic'>THE ZEBRAFISH BOOK</em>. , (2007).</li><li>Ho, R. K., Kane, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-autonomous+action+of+zebrafish+spt-1+mutation+in+specific+mesodermal+precursors.">Cell-autonomous action of zebrafish spt-1 mutation in specific mesodermal precursors.</a> <em style='font-style: italic'>Nature</em>. <b>348</b>, 728-730 (1990).</li><li>Catalano, A., Raymond, P., Goldman, D., Wei, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+dou+yan+Mutation+Causes+Patterning+Defects+and+Extensive+Cell+Death+in+the+Retina.">Zebrafish dou yan Mutation Causes Patterning Defects and Extensive Cell Death in the Retina.</a> <em style='font-style: italic'>Developmental Dynamics</em>. <b>236</b> (5), 1295-1236 (2007).</li></ol>

<p class="jove_content">In this video, we demonstrate a method for blastomere transplantation to analyze the retinal and brain development in zebrafish. This protocol can also be used to analyze the development of other tissues and organs by simply releasing the donor blastomeres to corresponding locations in the host embryos. The labeling with GFP molecules yields a cleaner background than labeling with fluorescent dye-conjugated Dextran (Ho and Kane 1990). This is because the debris of Dextran dyes from dead donor cells lasts much longer than...

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<td>5 &#190;" Glass pipette</td>
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			<p class="jove_content">E3 egg water:</p> <ul> <li>5 mM NaCl</li> <li>0.17 mM KCl</li> <li>0.33 mM CaCl<sub>2</sub></li> <li>0.33 mM MgSO<sub>4</sub></li> <li>0.1% methylene blue</li> </ul>		</div>
		

transplantation of gfp-expressing blastomeres for live imaging of retinal and brain development in chimeric zebrafish embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

Watch this Scientific Journal Video about Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos at JoVE.com

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

<p class="jove_content">We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis. </p>

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

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