Name: Large Scale Zebrafish-Based In vivo Small Molecule Screen (Video) | JoVE Journal
Uploaded: 2010-12-30T17:00:00.000+00:00
Duration: 7 min 3 s
Description: Learn a large-scale zebrafish-based in vivo small molecule screening method for drug discovery. This technique identifies compounds that modulate embryonic development and physiology efficiently.

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5 minutes

1) Zebrafish Egg Collection
<ol>
	<li>On the afternoon prior to the day of the chemical screen, set up 10 to 20 zebrafish breeding tanks. Fill each tank with water from the aquaculture system. Using a fish net, transfer one adult male and one to two adult females to inner container in each breeding tank. Separate the male and female fish from each other with a divider. Label the cages and put a lid over them.</li>
	<li>On the morning of the screen, remove the dividers from breeding tanks and allow zebrafish to mate. Over the course of next 1 hour, allow fertilized eggs to fall through grid at the bottom of each inner container.</li>
	<li>After 1 hour, return adult zebrafish back to permanent storage tanks, remove the inner container and collect the eggs by straining the water in each breeding tank through a plastic tea strainer. </li>
	<li>Invert the strainer over a Petri dish and rinse the strainer gently to flush the eggs into the Petri dish by using a wash bottle containing the E3 medium. </li>
	<li>All unfertilized eggs, which appear opaque, should be removed using a disposable plastic pipette. Each mating cross should yield approximately 200 embryos.</li>
</ol>
2) Arraying Embryos in 96-well Plates
<ol>
	<li>Transfer about 5 embryos in E3 medium into each well of a 96-well plate by using a glass Pasteur pipette.</li>
	<li> Once embryos are arrayed onto the 96-well plate, remove as much of the E3 medium as possible out of the wells using a 12-channel (30 - 300 &mu;L) pipette, taking care not to puncture the embryo. Using the 12-channel pipette, deliver 250 &mu;L of E3 medium containing 0.5&mu;g/ml kanamycin to each well as quickly as possible so as not to allow embryos to dry up.</li>
	<li> Put the 96-well plates into 28.5&deg;C incubator until they reach the desired stage when the compounds to be added.</li>
</ol>
3) Transfer of Small Molecule Library
While compound transfer can be automated with robotic transfer methods, we will describe the manual transfer method.
<ol>
	<li>Small molecule libraries are typically supplied in a 96-well format, with each compound stored in DMSO as a 10 mM stock. About 60 minutes before the embryos reach the stage when the compounds are to be added, thaw a desired number of 96-well plates containing aliquots of small molecules (source plate). Take note of the serial or other identification number of the source plates. To minimize condensation on the plates, thawing can occur in a desiccation chamber containing Drierite (W.A. HAMMOND DRIERITE CO, Xenia, OH).</li>
	<li>Briefly spin down the plates in a tabletop centrifuge equipped with multi-well plate adaptor.</li>
	<li>Remove the aluminum sealing tape from source plate. Using a 12-channel pipette, dilute the compounds in the source plate to the concentration of 0.5 mM (for example, if starting with 250 nL aliquots of 10mM stock, add 4.75 &mu;L of DMSO to each well). </li>
	<li>When the embryos in the 96-well plate (recipient plate) reach the desired stage, use a 12-channel (2-20 &mu;L) pipette to transfer 2.5&mu;L of compounds (0.5mM) from the source plates into the recipient plates containing the embryos.</li>
	<li>Record the identification number of the source plates on the recipient embryo plates. Cover the recipient plates now containing the embryos and compounds with lids, gently mix the plates by gently swirling, and place them in a 28.5&deg;C incubator.</li>
	<li>Cover each source plate containing unused small molecules (0.5 mM) with an aluminum sealing tape and place in a -80&deg;C freezer for long-term storage.</li>
</ol>
4) Screening for Effects of Small Molecules by Visual Inspection of Phenotypes
<ol>
	<li>Prior to performing the screen, formulate a specific criterion for what would constitute a "hit".</li>
	<li>At desired times in development, remove the 96-well plates containing compound-treated embryos from incubator and examine each well under a stereomicroscope. For better visualization of subtle changes, such as changes in circulatory pattern, a phase-contrast inverted microscope can be used. Fluorescent microscopy can be used to examine perturbation of expression of GFP or DsRed proteins under a tissue-specific promoter.</li>
	<li>Quickly scan the 96-well plates for any well in which at least 3 out of 5 embryos exhibit the prescribed "hit" phenotype. Record the identity of the plate and the well location of each potential hit. </li>
	<li>Reconfirm a potential hit by retesting the effects of the compound at several doses (1 uM, 5 uM, 10 &mu;M and 50 &mu;M). For each dose, 10 embryos are tested in 0.5 mL of E3 media in a 48-well plate format. The timing of compound addition for retesting should be identical to that of the original screening. A hit is confirmed when the elicited phenotype is reproduced on retesting of the compound</li>
	<li>Identify the hit compound from the database of small molecules in the chemical library.</li>
</ol>

<ol><li>Bayliss, P. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+modulation+of+receptor+signaling+inhibits+regenerative+angiogenesis+in+adult+zebrafish%5BTitle%5D)+AND+%22Nature+chemical+biology%22%5BJournal%5D)">Chemical modulation of receptor signaling inhibits regenerative angiogenesis in adult zebrafish.</a> Nature chemical biology. 2, 265-273 (2006).</li>
<li>Burns, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+assay+for+small+molecules+that+modulate+zebrafish+embryonic+heart+rate%5BTitle%5D)+AND+%22Nature+chemical+biology%22%5BJournal%5D)">High-throughput assay for small molecules that modulate zebrafish embryonic heart rate.</a> Nature chemical biology. 1, 263-264 (2005).</li>
<li>Hao, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+structure-activity+relationship+study+of+dorsomorphin+analogues+identifies+selective+VEGF+and+BMP+inhibitors%5BTitle%5D)+AND+%22ACS+chemical+biology%22%5BJournal%5D)">In vivo structure-activity relationship study of dorsomorphin analogues identifies selective VEGF and BMP inhibitors.</a> ACS chemical biology. 5, 245-253 (2010).</li>
<li>Hong, C. C., Peterson, Q. P., Hong, J. Y., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Artery%2Fvein+specification+is+governed+by+opposing+phosphatidylinositol-3+kinase+and+MAP+kinase%2FERK+signaling%5BTitle%5D)+AND+%22Curr+Biol%22%5BJournal%5D)">Artery/vein specification is governed by opposing phosphatidylinositol-3 kinase and MAP kinase/ERK signaling.</a> Curr Biol. 16, 1366-1372 (2006).</li>
<li>Milan, D. J., Peterson, T. A., Ruskin, J. N., Peterson, R. T., MacRae, C. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Drugs+that+induce+repolarization+abnormalities+cause+bradycardia+in+zebrafish%5BTitle%5D)+AND+%22Circulation%22%5BJournal%5D)">Drugs that induce repolarization abnormalities cause bradycardia in zebrafish.</a> Circulation. 107, 1355-1358 (2003).</li>
<li>Peterson, R. T., Link, B. A., Dowling, J. E., Schreiber, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecule+developmental+screens+reveal+the+logic+and+timing+of+vertebrate+development%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">Small molecule developmental screens reveal the logic and timing of vertebrate development.</a> Proceedings of the National Academy of Sciences of the United States of America. 97, 12965-12969 (2000).</li>
<li>Yu, P. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dorsomorphin+inhibits+BMP+signals+required+for+embryogenesis+and+iron+metabolism%5BTitle%5D)+AND+%22Nature+chemical+biology%22%5BJournal%5D)">Dorsomorphin inhibits BMP signals required for embryogenesis and iron metabolism.</a> Nature chemical biology. 4, 33-41 (2008).</li>
<li>Zon, L. I., Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((In+vivo+drug+discovery+in+the+zebrafish%5BTitle%5D)+AND+%22Nature+reviews%22%5BJournal%5D)">In vivo drug discovery in the zebrafish.</a> Nature reviews. 4, 35-44 (2005).</li>
<li>Stern, H. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Small+molecules+that+delay+S+phase+suppress+a+zebrafish+bmyb+mutant%5BTitle%5D)+AND+%22Nature+chemical+biology%22%5BJournal%5D)">Small molecules that delay S phase suppress a zebrafish bmyb mutant.</a> Nature chemical biology. 1, 366-370 (2005).</li>
<li>Peterson, R. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Chemical+suppression+of+a+genetic+mutation+in+a+zebrafish+model+of+aortic+coarctation%5BTitle%5D)+AND+%22Nature+biotechnology%22%5BJournal%5D)">Chemical suppression of a genetic mutation in a zebrafish model of aortic coarctation.</a> Nature biotechnology. 22, 595-599 (2004).</li>
</ol>

No conflicts of interest declared.

When planning a zebrafish-based chemical screen, particular attention must be paid to the robustness of the phenotype under consideration and the background rate of such phenotype. This is particularly important for screens for chemical suppressors of an induced phenotype. For example, for a phenotype caused by heat-shock induction of a transgene, the condition that induces the phenotype reproducibly must be precisely mapped out prior to initiating the screen to avoid unacceptably high false positive rates. With care...

<table><tbody><tr><td>Material Name</td><td>Type</td><td>Company</td><td>Catalogue Number</td></tr><tr><td>Minimum of 20 pairs of adult zebrafish of desired genotype.</td><td></td><td></td><td></td></tr><tr><td>Fish nets, Breeding tanks, with removable inner container and dividers (Aquatic Habitats).</td><td></td><td></td><td></td></tr><tr><td>Petri dishes (10 cm).</td><td></td><td></td><td></td></tr><tr><td>Plastic tea strainer.</td><td></td><td></td><td></td></tr><tr><td>Wash bottle containg embryo water.</td><td></td><td></td><td></td></tr><tr><td>Disposable polyethylene transfer pipettes.</td><td></td><td></td><td></td></tr><tr><td>Polystyrene 96-well round-bottom assay plates (Corning COSTAR; Lowell, MA).</td><td></td><td></td><td></td></tr><tr><td>Glass Pasteur pipette (Fisher Scientific).</td><td></td><td></td><td></td></tr><tr><td>Manual pipette pump, 10 mL (Bel-Art Products, Pequannock, NJ).</td><td></td><td></td><td></td></tr><tr><td>E3 embryo medium: 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl&lt;sub&gt;2&lt;/sub&gt; , 0.33 mM MgSO&lt;sub&gt;4&lt;/sub&gt; , containing 0.003% PTU (phenylthio-carbamide, Sigma; St. Louis, MO). PTU can be prepared as a 10x solution by dissolving 0.3-g PTU in 1 L of E3 embryo media. Solutions containing PTU should be protected from light by covering with aluminum foil.</td><td></td><td></td><td></td></tr><tr><td>12-channel pipettes, 2-20 &amp;#956;L (Eppendorf).</td><td></td><td></td><td></td></tr><tr><td>12-channel pipettes, 3-300 &amp;#956;L (Eppendorf).</td><td></td><td></td><td></td></tr><tr><td>Disposable polystyrene pipette basin, 50 mL (Fisher Scientific).</td><td></td><td></td><td></td></tr><tr><td>Small molecule library of structurally diverse compounds arrayed in a 96-wll format at 10 mM stock in DMSO. Each master plate is aliquoted into 96-well polypropylene storage plates (Corning), and stored at -80&amp;#x00B0;C until use.</td><td></td><td></td><td></td></tr><tr><td>Aluminum sealing tape for 96-well plates (Nunc, Rochester, NY).</td><td></td><td></td><td></td></tr><tr><td>DMSO (Sigma, St. Louis, MO).</td><td></td><td></td><td></td></tr><tr><td>Basic incubator, 28.5&amp;#x00B0;C (Fisher Scientific).</td><td></td><td></td><td></td></tr><tr><td>Stereomicroscope with transmitted light base (Leica Microsystems, Bannockburn, IL).</td><td></td><td></td><td></td></tr></tbody></table>

large scale zebrafish-based in vivo small molecule screen

Large Scale Zebrafish-Based In vivo Small Molecule Screen

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to mammals, zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Zebrafish has emerged as a powerful in vivo platform for phenotype-based drug screens and chemical genetic analysis. Here, we demonstrate a simple, practical method for large-scale screening of small molecules using zebrafish embryos.

Given their small embryo size, rapid development, transparency, fecundity, and numerous molecular, morphological and physiological similarities to ...

The overall goal of the following experiment is to discover small molecules that specifically modulate key aspects of embryonic development and physiology. This is achieved by placing a large number of fertilized eggs into 96 while screening plates as a second step. Aliquots of a library of small molecules are added to the 96 well plates next embryos in the 96.Well plates are examined for phenotypes elicited by the small molecules. Results are obtained that show which small molecules specifically affect super fish development or physiology. Hello, my name is Charles Hong at Vanderbilt University School of Medicine.In our laboratory, we do high throughput chemical screens using difish embryos. This is a relatively easy technique that can be widely adapted to discover novel bioactive compounds. On the afternoon prior to the day of the chemical screen, set up 10 to 20 zebrafish breeding tanks.Place a divider into each tank and fill each tank with water from the aquaculture system using a fish net transfer, one adult male and one to two adult females to the inner container. In each breeding tank, label the tanks and put a lid over them on the morning of the screen. Remove the dividers from the breeding tanks and allow zebrafish to mate.Over the course of the next hour. Allow fertilized eggs to fall through the grid at the bottom of each inner container. After one hour return the adult seber fish back to the permanent storage tanks.Remove the inner container and collect the eggs by straining the water in each breeding tank through a plastic tea strainer. Invert the strainer over a Petri dish and rinse the strainer gently to flush the eggs into the Petri dish. By using a wash bottle containing E three medium, all unfertilized eggs which appear opaque should be removed.Using a disposable plastic pipette, each mating cross should yield approximately 200 embryos. Then transfer about five embryos in E three medium into each well of a 96 well plate by using a glass paster pipette. Once embryos are arrayed onto a 96 well plate.Remove as much of the E three medium as possible out of the wells using a 12 channel pipette. Taking care not to puncture the embryos. Using the 12 channel pipette, deliver 250 microliters of E three.Medium containing 0.5 Micrograms per milliliter can mycin to each well as quickly as possible so as not to allow the embryos to dry up. Put the 96 well plates into a 28.5 degree Celsius incubator until they reach the desired stage when the compounds are to be added. In this protocol, we look for compounds that perturb early embryonic patterning and will incubate the embryos for three hours or until the 1000 cell stage is achieved by greater than 90%of the embryos small molecule libraries are typically supplied.In a 96 well source plate with each compound stored in DMSO as a 10 millimolar stock, about 60 minutes before the embryos reach the desired stage thaw an appropriate number of 96 well plates containing aliquots of small molecules. Take note of the serial or other identification number of the source plates to minimize condensation on the plates. T fall in a desiccation chamber containing dryer.Right.Briefly spin down the plates in a tabletop centrifuge equipped with a multi-well plate adapter. Remove the aluminum ceiling tape from the source plate using a 12 channel pipette dilute the compounds in the source plate to the desired concentration with DMSO. In this protocol, we use a 0.5 millimolar final concentration and add 4.75 microliters of DMSO to dilute the 10 millimolar stock plate.When the embryos in the 96 well plate reach the desired stage, use a 12 channel pipette to transfer 2.5 microliters of compounds from the source plates into the recipient plates containing the embryos. Cover the plates now containing the embryos and compounds with lids, and record the identification number of the source plates on the embryo plates. Mix the plates by gently swirling and place them in a 28.5 degree Celsius incubator.Cover each source plate containing unused small molecules with aluminum ceiling, tape and place in a minus 80 degrees Celsius freezer for long-term storage. Prior to performing the visual screen, formulate a specific hit criterion. For example, in a screen for small molecule inhibitors of four brainin development, a hit may be a compound that confers the loss of the forebrain.Absence of eyes is an easy visual indicator of the loss of forebrain and requires no fluorescence to assay at desired times in development. Remove the 96 well plates containing compound treated embryos from the incubator and examine each well under a stereo microscope when screening for small molecule inhibitors of four brainin development, examine plates at 28 hours for loss of the eye as a marker of four brainin developmental inhibition. Record the identity of the plate and well location for any well in which at least three out of five embryos exhibit the prescribed hi phenotype Place the 96 well plate back in the incubator.Reconfirm the loss of eyes and forebrain at 48 hours. Even though the embryos will not have a forebrain, they will survive to at least four days. Reconfirm a potential hit by retesting the effects of the compound at several doses for each dose, 10 embryos are tested in 0.5 milliliters of E three media in a 48 well plate format.The timing of compound edition for retesting should be identical to that of the original screening. A HIIT is confirmed when the elicited phenotype is reproduced in a dose dependent manner on retesting of the compound. Finally, identify the hi compound from the database of small molecules in the chemical library.Using the zebrafish based visual chemical screen, we discovered a small molecule hedgehog signal inhibitor based on its effect on body axis. This image shows embryos exposed to hedgehog inhibitor displaying marked ventral curvature of the tail. At 24 hours, the same embryos examined at 48 hours still display the ventrally curved phenotype.This image displays the compound treated embryo compared to the hedgehog pathway mutant embryo. Once this technique is mastered, one person can screen approximately 400 compounds a week in just a couple hours. This technique has also paved the way for chemical biologists to study things such as cell development, cell signaling pathways, and embryonic development.When working with uncharacterized small molecules, it's important to have proper safety use lab coats and gloves as these uncharacterized molecules could be potentially hazardous.

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Learn a large-scale zebrafish-based in vivo small molecule screening method for drug discovery. This technique identifies compounds that modulate embryonic development and physiology efficiently.

Large Scale Zebrafish-Based In vivo Small Molecule Screen

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