Name: Microdissection of Zebrafish Embryonic Eye Tissues (Video) | JoVE Journal
Uploaded: 2010-06-27T23:34:00.000+00:00
Duration: 9 min 1 s
Description: Learn microdissection of zebrafish embryonic retina with and without retinal pigment epithelium using fine forceps and stereo microscopy. This method preserves intact tissues for RNA expression studies in developmental biology.

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5 minutes

This work is supported by a startup fund from the Department of Biological Sciences at Purdue University.

Part 1: Preparations before microdissection 
<ol>
	<li>Solutions
	<ol class="lalpha">
		<li>E3 medium4 (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2 and 0.33 mM MgSO4).</li> 
		<li>Ringer's solution1, 5 (116 mM NaCl, 2.9 mM KCl, 1.8 mM CaCl2, 5 mM HEPES, pH7.2), filter sterilized.</li> 
		<li>5N NaOH.</li>
	</ol>
	</li> 
	<li>Chemical etching of tungsten needles 
	<ol class="lalpha"> 
		<li>Secure a small beaker containing 5N NaOH on a Petri dish by clay.</li> 
		<li>Attach a paper clip on the side of the beaker, so that it comes in contact with the NaOH solution.</li> 
		<li>Connect the negative electrode from a DC power supply to the paper clip and the positive electrode to one end of a short piece (~2cm) of tungsten wire.</li> 
		<li>Wrap the other end of tungsten wire with a small clay ball and dip this end into the NaOH solution.</li> 
		<li>Increase the voltage to approximately 3V and dip the wire up and down in the NaOH solution until a good etching rate is attained. A needle in good quality can usually be etched in 10 minutes.</li> 
		<li>The tungsten wire exposed to the solution will become gradually thinner. When the clay ball drops off, the other side of the wire that is still attached to the positive electrode will have a sharp needle shape.</li> 
		<li>Rinse the sharp needle with Ringer's solution or E3 to remove salt deposits.</li> <li>Attach the needle to a wooden applicator by tape or a needle holder for easy handling.</li> 
	</ol>
	</li> 
	<li>Culture plates 
	<ol class="lalpha"> 
		<li>The Falcon 60 x 15 mm polystyrene culture plates are ready to use for retinal microdissection.</li>
		<li>For dissecting RPE-attached retina, crush two embryos completely in a culture plate containing Ringer's solution to get rid of the adhesiveness of the surface 30 minutes before dissection. Shortly before dissection, wash the plate extensively with fresh Ringer's solution.</li>
		</ol>
	</li>
	<li>Embryos collection and staging 
	<ol class="lalpha">
		<li>Adult zebrafish are maintained as described4, 5.</li>
		<li>Separate the parent fish in static breeding tanks pairwise by a divider the night before breeding.</li>
		<li>Remove the divider after the room light is turned on in the next morning. Allow the parents to cross at 10-minute intervals every half an hour. Separate the fish after each crossing.</li>
		<li>Maintain the embryos collected from each crossing in E3 medium separately at 28°C.</li>
		<li>Stage embryos at 10 to 12 hours post fertilization (hpf) and restage immediately before dissection at the specific time points, according to established criteria1, 6.</li>
	</ol>
	</li> 
</ol>
Part 2: Dissection - brain removal and eye exposure1
<ul>
	<li>The procedures described in this section are common for dissecting retina (Part 3A) and RPE-attached retina (Part 3B).</li>
	<li>All fine dissections are done by a tip of the Dumont forceps and assisted by another pair of Dumont forceps for positioning the tissue. A chemically-etched tungsten needle can be used for finer manipulations if necessary.</li>
	<li>All fine dissections are performed at ~ 5-8x magnification under an Olympus SZX16 microscope equipped with an 1X objective or equivalent.</li>
	<li>Maintain the embryos in E3 medium in a bench top incubator at 28°C next to the microscope for easy embryo access during dissection.</li>
	<li>The microscope stage is warmed to 28°C by a thermo plate to minimize the influence of temperature fluctuation on gene expression.</li>
</ul>
<ol>
	<li>Transfer an embryo at a specific developmental stage to Ringer's solution in a Falcon 60 X 15 mm culture plate prepared as described in Part 1#3.</li>
	<li>Cut off the head with part of the anterior trunk quickly from the body.</li> 
	<li>Pin the posterior end of this tissue on culture plate with the assisting forceps.</li> 
	<li>Open the head on the dorsal side starting from the anterior forehead.</li>
	<li>Remove the brain so that the medial side of the eyes is exposed and is facing upward for further manipulations.</li>
</ol>
 Part 3A: Retinal dissection1
 <ol> 
	<li>Prepare the embryo as described in Part 2.</li>
	<li>Carefully brush the exposed RPE on the medial side of the eye ball that is facing upward by the tip of the forceps until a small opening to the retina is seen.</li>
	<li>Continue the brushing and peeling action until the medial side of the retina is almost all exposed. Avoid scratching and punching the exposed retina.</li>
	<li>To remove the lateral RPE on the retina which is now facing downward, approach that and brush at an angle of approximately 45° from the culture plate surface.</li>
	<li>Remove the RPE that is attached relatively firmly to the ora serrata by pinning the detached portion of the RPE to the culture plate for dissection, and then lift the retina gently.</li>
	<li>Roll the retina on the bottom of the culture dish to clean up the residual RPE. 
	<ol class="lalpha"> 
		<li>We have noticed that this particular Falcon polystyrene culture plate has a preferential adherence of RPE for approximately 20 minutes once an embryo is crushed. This property is utilized for the removal of RPE remnants. However, the retinal cells do stick to the surface to a lesser extent, which can be inspected closely under high magnification. One has to strike a balance between the completeness of RPE removal and retinal integrity.</li>
		<li>The lens often adheres to the culture plate and detaches from the retina during the rolling process. Occasionally, it is necessary to detach the lens by an etched tungsten needle with a swirling action on the lens surface.</li>
	</ol>
	</li>
	<li>These procedures can successfully remove RPE from the retina without compromising the integrity of the tissue. This is indicated by a good overall morphology (Figure 1B and B') and histology (Figure 1B"). In particular, the preservation of the extracellular matrix in between the photoreceptor layer and the RPE is exceptionally good (Figure 1B", arrow; compare with the histology from the whole embryo (Figure 1A")).</li>
</ol>
Part 3B: RPE-attached retinal dissection3 
<ol>
	<li>Prepare the embryo as described in Part 2.</li>
	<li>Pin the head to the culture plate by the assistant forceps. Lift the eye gently from the posterior lateral side and roll to the anterior side.
	<ol class="lalpha">
		<li>The presumptive choroid and scleral tissue on the outside of the RPE layer are attached relatively tightly to the skin and can be mostly peeled off by rolling the eye carefully to the anterior side.</li>
	</ol>
	</li>
	<li>Remove the lens with a tungsten needle after the lateral side of the eye is exposed and when the eye is still attached to the skin.</li>
	<li>These procedures can successfully preserve the whole RPE layer with the retina. Presumptive choroids and scleral tissue can be largely removed (Figure 1C, C' and C").</li>
</ol>
Part 4: Tissue sample collection for RNA work
<ol>
	<li>The dissected samples can be collected in TRIzol in an RNase-free microfuge tube for downstream RNA characterization as described before1.</li>
</ol>
Representative Results
<img src="/files/ftp_upload/2028/2028fig1.jpg" alt="Sequential embryo development; microscope images; cellular differentiation analysis." data-altupdatedat="1788235205000" data-alt="Figure 1"> Figure 1. (A) Lateral and (A') dorsal view of a zebrafish larval head at 52 hpf before dissection. (A") The corresponding histological section of the larval head in (A) &amp; (A'). (B) Lateral and (B') dorsal view of a dissected retina at 54 hpf. The surface of retina was intact from both lateral and dorsal views. (B") The corresponding histological section of the dissected retina in (B) &amp; (B'). The structure of the retina and retinal lamination was intact. In particular, the extracellular matrix between the photoreceptor layer and RPE (A", arrows) was well preserved in the dissected retina (B", arrows). (C) Lateral and (C') medial view of a dissected RPE-attached retina at 52 hpf. RPE layer was intact and continuous, which was also indicated by the histological section of the dissected tissue (C"). The white area in C' is the optic nerve (arrow). For histology, tissue samples were collected and fixed in 4% paraformaldehyde. Plastic embedding and sectioning of these samples were performed as described3. Scale bar = 50 μm.

<ol><li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profiling+of+zebrafish+embryonic+retina%5BTitle%5D)+AND+%22Zebrafish%22%5BJournal%5D)">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li>
<li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Factorial+microarray+analysis+of+zebrafish+retinal+development%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li>
<li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo%5BTitle%5D)+AND+%22Invest+Ophthalmol+Vis+Sci%22%5BJournal%5D)">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li>
<li>Nusslein-Volhard, C., Dahm, R. Zebrafish : a practical approach. , 1st ed, Oxford University Press. Oxford ; New York. (2002).</li>
<li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+zebrafish+book+%3A+a+guide+for+the+laboratory+use+of+zebrafish+%28Danio+rerio%29%5BTitle%5D)+AND+%22%22%5BJournal%5D)">The zebrafish book : a guide for the laboratory use of zebrafish (Danio rerio).</a> , 4th ed, University of Oregon Press. Eugene, OR. (2000).</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/pubmed?term=((Stages+of+embryonic+development+of+the+zebrafish%5BTitle%5D)+AND+%22Dev+Dyn%22%5BJournal%5D)">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li>
<li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Zebrafish%3A+a+model+system+for+the+study+of+eye+genetics%5BTitle%5D)+AND+%22Prog+Retin+Eye+Res%22%5BJournal%5D)">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li>
</ol>

Experiments on animals were performed in accordance with the guidelines and regulations set forth by Purdue Animal Care and Use Committee.

Microdissection of zebrafish eye tissues can effectively obtain intact retinas and RPE-attached retinas. This substantially assists expression studies pertaining to a specific eye tissue (i.e. retina or RPE). In fact, we have successfully utilized these procedures to obtain RNA expression profiles of the whole retina1 and RPE3. The utility of these profiles is strongly supported by our recent identification of pathways and gene families that are perturbed in a retinal differentiation mutant2</s...

<table><tbody><tr><td>Cordless pestle motor</td><td>VWR international</td><td>47747-370</td><td></td></tr><tr><td>DC power supply</td><td>Lascar</td><td>PSU130</td><td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td></tr><tr><td>Disposable pestle &amp;amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td><td>VWR international</td><td>47747-366</td><td>These are used for tissue collection in TRIzol for expression analysis.</td></tr><tr><td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td><td>World Precision Instruments, Inc.</td><td>500341</td><td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td></tr><tr><td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td><td>Fine Science Tools</td><td>11252-00</td><td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td></tr><tr><td>Falcon polystyrene culture plates, 60 X 15 mm</td><td>BD Biosciences</td><td>351007</td><td>These plates are used as dissection plates.</td></tr><tr><td>Olympus SZX16 Stereomicroscope</td><td>Olympus Corporation</td><td>SZX16</td><td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies&lt;sup&gt;1-3&lt;/sup&gt; without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td></tr><tr><td>Sharpening stone</td><td>Fine Science Tools</td><td>29008-01</td><td>Use this to sharpen the tip of the forceps if necessary</td></tr><tr><td>Thermo plate</td><td>Tokai Hit</td><td>MATS-U55SZX2B</td><td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&amp;#x00B0;C during dissection in previous studies&lt;sup&gt;1-3&lt;/sup&gt; with good success.</td></tr><tr><td>Trizol, 100 mL</td><td>Invitrogen</td><td>15596-026</td><td></td></tr><tr><td>tungsten wire, 0.015 inch diameter</td><td>World Precision Instruments, Inc.</td><td>TGW1510</td><td></td></tr><tr><td>Wooden Applicator</td><td>Puritan</td><td>807</td><td>This is used for holding the chemically-etched tungsten needle.</td></tr></tbody></table>

microdissection of zebrafish embryonic eye tissues

Microdissection of Zebrafish Embryonic Eye Tissues

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 &mu;m, while the diameter of the lens is less 100 &mu;m. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina1, 2 and retinal pigment epithelium3.

This article describes an approach to microdissect zebrafish retinas with and without retinal pigment epithelium attached, from one to three days postfertilization embryos.

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

This video displays microdissection of retina and retina with retinal pigment, epithelium, or RPE attached from zebrafish embryos at one to three days post fertilization. All procedures can be done with fine forceps and general laboratory supplies. Under standard stereo microscopes for retinal dissection, the single layer RPE is removed and peeled off by brushing action and adherence of the RPE to the surface of the culture plate.For RPE attached retinal dissection, the adhesive properties of the culture plate are removed by embryonic crush and washing prior to the dissection. To preserve the RPE intact careful lifting of this tissue then efficiently separates the choroid and sclera from the RPE attached retina, followed by lens removal using a chemically etched tungsten needle. This dissection method can obtain intact retina tissues and has been successfully utilized to study tissue specific RNA expression, profiles of zebrafish retina and retinal pigment epithelium.Hi, I'm Li Jang from the laboratory of Professor UFE Leon in the Department of Biological Sciences at Purdue University. Today we'll show you how to dissect zebrafish embryonic eye tissues. We use this procedure in our laboratory to study the gene network regulating eye development.So let's get started.Eight. Prior to micro dissection, prepare the following solutions. E three, medium filter sterilized ringer solution and five normal sodium hydroxide.Next, chemically etch tungsten needles. Set a small beaker in a Petri dish by clay to facilitate handling of the beaker and prevent spillage of sodium hydroxide. Attach a standard paperclip to the side of the beaker.Pour five normal sodium hydroxide into the beaker until the clip contacts sodium hydroxide. Connect the negative electrode from a DC power supply to the paperclip and the positive electrode to one end of a short piece of tungsten wire. Wrap the other end of tungsten wire with a four millimeter diameter clay ball and dip this end into the sodium hydroxide solution.Increase the voltage to approximately three volts and dip the wire up and down in one second, intervals in the sodium hydroxide solution for about 10 minutes. The tungsten wire exposed to sodium hydroxide will become gradually thinner when the clay ball drops off the other side of the wire attached to the positive electrode will have a sharp needle shape. Rinse the sharp needle with ringer solution or E three to remove salt deposits and attach the needle to a wooden applicator by tape or a needle holder for easy handling To prepare embryos for dissection.Maintain adult super fish according to standard protocols the night before breeding. Separate the parent fish in static breeding tanks pairwise using a divider. Remove the divider after the room light is turned on the next morning and allow the parents to cross at 10 minute intervals every half an hour, separating the fish after each crossing.Maintain the embryos collected from each crossing in E three medium separately at 28 degrees Celsius. Stage embryos at 10 to 12 hours post fertilization and restage immediately before dissection at the specific time points. Use Falcon 60 by 15 millimeter polystyrene culture plates for dissection.A new unused plate has an intrinsic adhesive property for RPE tissue up to approximately 20 minutes and can aid in the removal of RPE for retinal dissections. If RPE attached retina is desired, this adhesive property can be overcome by crushing two embryos completely in a culture plate containing ringer solution and left for 30 minutes prior to dissection, followed by extensive washing with fresh ringer solution prior to dissection. All fine dissections are done at about five to eight x magnification on an Olympus SC X 16 microscope with a one x objective.Using dumont forceps or a chemically etched tungsten needle, the microscope stage is warmed to 28 degrees Celsius. To minimize the influence of temperature fluctuation to begin retinal dissection, transfer an embryo at a specific developmental stage to five milliliters of ringer solution in a Falcon 60 by 15 millimeter culture plate prepared as described for retinal dissection without RRPE, cut off the head and part of the anterior trunk using the sharp tip of the forceps quickly removing the zebrafish body Next pin, the posterior end of the head trunk section on a culture plate. With the assisting forceps, open the head on the dorsal side starting from the anterior forehead.Remove the brain so that the medial side of the eyes is exposed and is facing upward for further manipulations. To dissect the retina, use the top of the forceps to carefully brush the exposed RPE on the medial side of the eye facing upward until a small opening to the retina is seen. Continue the brushing and peeling action until the medial side of the retina is almost all exposed.Avoid scratching and punching the exposed retina for removal of lateral RPE on the retina of the eye facing the culture dish approach. Had an angle of approximately 45 degrees from the culture plate surface and brush the eye with the forceps. Remove the RPE that is attached relatively firmly to the aura serrata by pinning the detached portion of the RPE to the culture plate for dissection.And then lift the retina gently to clean up the residual RPE and remove the lens using the adhesive property of the culture dish for adherence. Be careful not to damage the retinal cells, which will also mildly adhere to the dish surface. If the lens does not detach with rolling, use an etched tungsten needle with a swirling action on the lens surface.To begin RPE attached retinal dissection, use a Falcon 60 by 15 millimeter culture plate prepared as described for an RPE attached retinal dissection. Transfer an embryo at a specific developmental stage to five milliliters of ringer solution. Cut off the head and part of the anterior trunk.Quickly removing the zebrafish body as done previously. Pin the posterior end of the head trunk section on a culture plate. With the assisting forceps, open the head on the dorsal side starting from the anterior forehead.Remove the brain so that the medial side of the eyes is exposed and is facing upward. For further manipulations using forceps, lift the eye gently from the posterior lateral side and roll to the anterior side. The choroid and scleral tissue on the outside of the RPE layer are attached relatively tightly to the skin and can be mostly peeled off by rolling the eye carefully to the anterior side.Remove the lens with the tungsten needle After the lateral side of the eye is exposed and when the eye is still attached to the skin for the retinal dissection. Without RPE and RPE attached retina, the dissected samples can be collected in triol in an RNA re micro tube for downstream. RNA characterization.A successful retinal dissection without RPE is shown here where the retinal integrity is maintained as indicated by the intact retina surface. The corresponding histological section also shows that the retinal architecture is very well maintained. In particular, the preservation of the extracellular matrix in between the photoreceptor layer and the RPE is exceptionally good.A successful RPE attached retinal dissection is shown by the intact and continuous RPE layer. The corresponding histological section shows that both retinal and RPE cells are in good shape. The total RNA extracted from the dissected retinas is of high quality.This is indicated by the electropherogram and gel picture obtained from the bioanalyzer analysis. The baseline is flat and the 18 s and 28 s ribosomal peaks are intact. The RNA integrity number is typically larger than 9.8.We've just shown you the micro dissection procedures for obtaining zebrafish embryonic retina with and without RPE. When doing this procedure, it's important to remember the most critical steps in retinal dissection are the brushing action by the tip of faucets and the tissue rolling. For RPE remnants removal, we found putting elbows on adjustable arm rest in your chair during dissection can substantially stabilize the hand movements.So that's it. Thanks for watching and good luck with your experiments.

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Learn microdissection of zebrafish embryonic retina with and without retinal pigment epithelium using fine forceps and stereo microscopy. This method preserves intact tissues for RNA expression studies in developmental biology.

Microdissection of Zebrafish Embryonic Eye Tissues

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