Name: zebrafish brain ventricle injection
Uploaded: 2009-04-06T00:00:00
Description: Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

<p class="jove_content">The authors would like to thank Laura Anne Lowery who developed this technique in the lab.  HS is supported by NIHMH70926.  Jennifer Gutzman was supported by the CSBi/Merck postdoctoral fellowship.</p>

<p class="jove_title">Protocol</p>
<p class="jove_step">Part 1: Preparing for microinjection</p>
<ol>
	<li>Prepare for the injection by pulling capillary needles using Sutter Instruments needle puller.</li>
	<li>Fill the needle with a fluorescent dye (such as Texas-Red Dextran).</li>
	<li>Mount the needle in a micromanipulator and microinjection apparatus.</li>
	<li>Cut the needle to the appropriate size at an angle to create a beveled tip.</li>
	<li>Measure the drop size and check that it is between 1-2nl per injection in oil.</li>
</ol>
<p cl...

<ol>
	<li>Gutzman, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+the+zebrafish+midbrain-hindbrain+boundary+constriction+requires+laminin-dependent+basal+constriction.">Formation of the zebrafish midbrain-hindbrain boundary constriction requires laminin-dependent basal constriction.</a> <em style='font-style: italic'>Mech. Dev</em>. <b>125</b>, 974-983 (2008).</li><li>Lowery, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+and+Classification+of+Zebrafish+Brain+Morphology+Mutants.">Characterization and Classification of Zebrafish Brain Morphology Mutants.</a> <em style='font-style: italic'>Anat. Rec. (Hoboken)</em>. <b>292</b>, 94-106 (2009).</li><li>Lowery, L. A., Sive, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initial+formation+of+zebrafish+brain+ventricles+occurs+independently+of+circulation+and+requires+the+nagie+oko+and+snakehead/atp1a1a.1+gene+products.">Initial formation of zebrafish brain ventricles occurs independently of circulation and requires the nagie oko and snakehead/atp1a1a.1 gene products.</a> <em style='font-style: italic'>Development</em>. <b>132</b>, 2057-2067 (2005).</li><li>Kimmel, C. B. <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> <em style='font-style: italic'>Dev. Dyn</em>. <b>203</b>, 253-310 (1995).</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> <em style='font-style: italic'>The Zebrafish Book: A guide for the laboratory use of zebrafish</em>. , (1995).</li></ol>

<p class="jove_content">In this video we demonstrate how to inject fluorescent dye (Texas-Red Dextran) into developing zebrafish brain ventricles.  This method is used to visualize the brain ventricle space in contrast to the surrounding neuroepithelium and is extremely useful for determining the shape of the ventricle space as well as the shape of the surrounding brain tissue.  It allows us to better understand the process of brain ventricle formation and brain morphogenesis over time in the live embryo. This technique is an excellent tool for...

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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>Forceps</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11252-20</td>
<td>&nbsp;</td>
<tr>
<td>Capillary Tubes (needles)</td>
<td>Tools</td>
<td>Frederick Haer and Co.</td>
<td>30-30-1</td>
<td>&nbsp;</td>
<tr>
<td>Agarose-Coated dishes</td>
<td>Tools</td>
<td>Agarose (Seakem GTG; Lonza)  Dishes (Corning) </td>
<td>Agarose (50027)<br />Dishes (430166) </td>
<td>Make agarose approximately 2-3 mm deep in the dish depending on the stage of embryo you are injecting</td>
</tr>
<tr>
<td>Dissecting Microscope</td>
<td>Microscope</td>
<td>Zeiss Stereomicroscope Stemi SV 6 </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Microscope Camera with Fluorescence capabilities and with image capture software</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>1-200 &mu; pipette tips</td>
<td>Tools</td>
<td>Tip One, US Scientific</td>
<td>1111-0806</td>
<td>These are the perfect size to poke the holes in the agarose and have the embryos fit perfectly.</td>
</tr>
<tr>
<td>Photoshop</td>
<td>Image Analysis</td>
<td>Adobe</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Loop</td>
<td>Tools</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Make these yourself with a glass pipette and hair or fishing line and seal with beeswax.</td>
</tr>
<tr>
<td>Microinjector</td>
<td>Tools</td>
<td>Medical Systems Research Products Harvard Apparatus</td>
<td>PL1-100</td>
<td>&nbsp;</td>
<tr>
<td>Needle Puller</td>
<td>Tools</td>
<td>Sutter Instruments</td>
<td>&nbsp;</td>
<td>Settings (Heat 785, Pull 70, Velocity 40, Time 70).</td>
</tr>
<tr>
<td>Micromanipulator</td>
<td>Tools</td>
<td>Narishige</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent dye Texas-Red Dextran  70,000MW</td>
<td>Reagent</td>
<td>Molecular Probes</td>
<td>D1830</td>
<td>Other Molecular weights are available if desired.</td>
</tr>
<tr>
<td>Tricaine (3-aminobenzoic acid ethyl ester)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A-5040</td>
<td>&nbsp;</td>
<tr>
<td>Embryo Media</td>
<td>Reagent</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>According to Westerfield (5)</td>
</tr>		</tbody>
		</table>
		</div>

zebrafish brain ventricle injection

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved cavities within the brain that are filled with cerebrospinal fluid.  In zebrafish, after neural tube formation, the neuroepithelium undergoes a series of constrictions and folds while it fills with fluid resulting in brain ventricle formation.  In order to understand the process of ventricle formation, and the neuroepithelial shape changes that occur at the same time, we needed a way to visualize the ventricle space in comparison to the brain tissue.  However, the nature of transparent zebrafish embryos makes it difficult to differentiate the tissue from the ventricle space.  Therefore, we developed a brain ventricle injection technique where the ventricle space is filled with a fluorescent dye and imaged by brightfield and fluorescent microscopy.  The brightfield and the fluorescent images are then processed and superimposed in Photoshop.  This technique allows for visualization of the ventricle space with the fluorescent dye, in comparison to the shape of the neuroepithelium in the brightfield image.  Brain ventricle injection in zebrafish can be employed from 18 hours post fertilization through early larval stages.  We have used this technique extensively in our studies of brain ventricle formation and morphogenesis as well as in characterizing brain morphogenesis mutants (1-3).

Watch this Scientific Journal Video about Zebrafish Brain Ventricle Injection at JoVE.com

Zebrafish Brain Ventricle Injection

<p class="jove_content">After neural tube formation, the neuroepithelium constricts and folds while the tube fills with embryonic cerebrospinal fluid (eCSF) to form the embryonic brain ventricles.  We developed this ventricle injection technique to better visualize the fluid filled space in contrast to the neuroepithelial shape in a live embryo.</p>

Proper brain ventricle formation during embryonic brain development is required for normal brain function.  Brain ventricles are the highly conserved ...

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