Name: surgical size reduction of zebrafish for the study of embryonic pattern scaling
Uploaded: 2019-05-03T14:30:00
Description: Watch this Scientific Journal Video about Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling at JoVE.com

The work was supported by the PRESTO program of the Japan Science and Technology Agency&#160;(JPMJPR11AA)&#160; and a National Institutes of Health grant (R01GM107733).

All fish-related procedures were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School.
1. Tool and Reagent Preparation
<ol>
	<li>Make a wire loop to chop embryos
	<ol>
		<li>Take 20 cm of stainless steel wire that is stiff and non-corrosive with a diameter of 40 &#956;m. Loop the wire through into glass capillary (1.0 mm outer diameter, 0.5 mm inner diameter, no filament), making a small loop at t...

<ol>
	<li>Cooke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale+of+body+pattern+adjusts+to+available+cell+number+in+amphibian+embryos.">Scale of body pattern adjusts to available cell number in amphibian embryos.</a> Nature. 290, 775-778 (1981).</li><li>Driesch, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Entwicklungsmechanische+Studien:+I.+Der+Werthe+der+beiden+ersten+Furchungszellen+in+der+Echinogdermenentwicklung.+Experimentelle+Erzeugung+von+Theil-+und+Doppelbildungen.">Entwicklungsmechanische Studien: I. Der Werthe der beiden ersten Furchungszellen in der Echinogdermenentwicklung. Experimentelle Erzeugung von Theil- und Doppelbildungen.</a> Zeitschrift fur wissenschaftliche Zoologie. , (1892).</li><li>Morgan, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Half+embryos+and+whole+embryos+from+one+of+the+first+two+blastomeres.">Half embryos and whole embryos from one of the first two blastomeres.</a> Anatomischer Anzeiger. 10, 623-638 (1895).</li><li>Ishimatsu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Size-reduced+embryos+reveal+a+gradient+scaling-based+mechanism+for+zebrafish+somite+formation.">Size-reduced embryos reveal a gradient scaling-based mechanism for zebrafish somite formation.</a> Development. 145, (2018).</li><li>Megason, S. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+toto+imaging+of+embryogenesis+with+confocal+time-lapse+microscopy.">In toto imaging of embryogenesis with confocal time-lapse microscopy.</a> Methods in Molecular Biology. 546, 317-332 (2009).</li><li>Graeden, E., 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=Live+imaging+of+the+zebrafish+embryonic+brain+by+confocal+microscopy.">Live imaging of the zebrafish embryonic brain by confocal microscopy.</a> Journal of Visualized Experiments. (26), e1217 (2009).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <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+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> Journal of Visualized Experiments. (25), e1115 (2009).</li><li>Yuan, S., Sun, Z. <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+mRNA+and+morpholino+antisense+oligonucleotides+in+zebrafish+embryos.">Microinjection of mRNA and morpholino antisense oligonucleotides in zebrafish embryos.</a> Journal of Visualized Experiments. (27), e1113 (2009).</li><li>Sorlien, E. L., Witucki, M. A., Ogas, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+Production+and+Identification+of+CRISPR/Cas9-generated+Gene+Knockouts+in+the+Model+System+Danio+rerio.">Efficient Production and Identification of CRISPR/Cas9-generated Gene Knockouts in the Model System Danio rerio.</a> Journal of Visualized Experiments. (138), e56969 (2018).</li><li>Kemp, H. A., Carmany-Rampey, A., Moens, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generating+chimeric+zebrafish+embryos+by+transplantation.">Generating chimeric zebrafish embryos by transplantation.</a> Journal of Visualized Experiments. (29), e1394 (2009).</li><li>Mizuno, T., Shinya, M., Takeda, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+and+tissue+transplantation+in+zebrafish+embryos.">Cell and tissue transplantation in zebrafish embryos.</a> Methods in Molecular Biology. 127, 15-28 (1999).</li><li>Cooke, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+somite+number+during+morphogenesis+of+a+vertebrate,+Xenopus+laevis.">Control of somite number during morphogenesis of a vertebrate, Xenopus laevis.</a> Nature. 254, 196-199 (1975).</li><li>Inomata, H., Shibata, T., Haraguchi, T., Sasai, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+of+dorsal-ventral+patterning+by+embryo+size-dependent+degradation+of+Spemann's+organizer+signals.">Scaling of dorsal-ventral patterning by embryo size-dependent degradation of Spemann's organizer signals.</a> Cell. 153, 1296-1311 (2013).</li><li>Gomez, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+segment+number+in+vertebrate+embryos.">Control of segment number in vertebrate embryos.</a> Nature. 454, 335-339 (2008).</li><li>Lauschke, V. M., Tsiairis, C. D., Francois, P., Aulehla, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scaling+of+embryonic+patterning+based+on+phase-gradient+encoding.">Scaling of embryonic patterning based on phase-gradient encoding.</a> Nature. 493, 101-105 (2013).</li><li>Schindelin, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fiji:+an+open-source+platform+for+biological-image+analysis.">Fiji: an open-source platform for biological-image analysis.</a> Nature Methods. 9, 676-682 (2012).</li><li>Almuedo-Castillo, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scale-invariant+patterning+by+size-dependent+inhibition+of+Nodal+signalling.">Scale-invariant patterning by size-dependent inhibition of Nodal signalling.</a> Nature Cell Biology. 20, 1032-1042 (2018).</li><li>Koos, D. S., Ho, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+nieuwkoid+gene+characterizes+and+mediates+a+Nieuwkoop-center-like+activity+in+the+zebrafish.">The nieuwkoid gene characterizes and mediates a Nieuwkoop-center-like activity in the zebrafish.</a> Current Biology. 8, 1199-1206 (1998).</li><li>Yamanaka, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+homeobox+gene,+dharma,+can+induce+the+organizer+in+a+non-cell-autonomous+manner.">A novel homeobox gene, dharma, can induce the organizer in a non-cell-autonomous manner.</a> Genes and Development. 12, 2345-2353 (1998).</li><li>Jesuthasan, S., Stahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+microtubules+and+specification+of+the+zebrafish+embryonic+axis.">Dynamic microtubules and specification of the zebrafish embryonic axis.</a> Current Biology. 7, 31-42 (1997).</li><li>Schier, A. F., Talbot, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish+organizer.">The zebrafish organizer.</a> Current Opinion in Genetics and Development. 8, 464-471 (1998).</li></ol>

Historically, among vertebrate animals, size reduction has been mainly performed using amphibian embryos, by bisecting the embryos along animal-vegetal axis at a blastula stage12. However, there are mainly two differences between frog and zebrafish embryos when we bisect embryos. First, at the stage when zebrafish embryos become tolerant of bisecting (blastula stage), the organizer is located in a restricted area of blastula margin18,...

Scientists have long known that embryos have a remarkable ability to form constant body proportions although embryo size can vary greatly both under natural and experimental conditions1,2,3. Despite decades of theoretical and experimental studies, this robustness to size variation, termed scaling, and its underlying mechanisms remain unknown in many tissues and organs. In order to directly capture the dynamics of the developing system, we established a reproducible and simple size reduction technique in zebrafish4, which has the great advantage in in vivo live imaging5.
Zebrafish has served as a model vertebrate animal to study multiple disciplines of biology, including developmental biology. In particular, zebrafish is ideal for in vivo live imaging6 because 1) development can proceed normally outside the mother and the egg shell, and 2) the embryos are transparent. In addition, the embryos can withstand some temperature and environmental fluctuations, which allows them to be studied in laboratory conditions. Also, in addition to conventional gene expression perturbation by morpholino and mRNA injection7,8, recent advances in CRISPR/Cas9 technology has made reverse genetics in zebrafish highly efficient9. Furthermore, many classical techniques in embryology, such as cell transplantation or tissue surgery can be applied4,10,11.
Size reduction techniques were originally developed in amphibian and other non-vertebrate animals12. For example, in Xenopus laevis, another popular vertebrate animal model, bisection along the animal-vegetal axis at blastula stage can produce size-reduced embryos12,13. However, in our hands this one-step approach results in dorsalized or ventralized embryos in zebrafish, presumably because dorsal determinants are distributed unevenly and one cannot know their localization from the morphology of embryos. Here we demonstrate an alternative two-step chopping technique for zebrafish that produces normally developing but smaller embryos. With this technique, cells are first removed from the animal pole, a region of na&#239;ve cells lacking in organizer activity. To balance the amount of yolk and cells, which is important for epiboly and subsequent morphogenesis, yolk is then removed. Here, we detail this protocol and provide two examples of size invariance in pattern formation; somite formation and ventral neural tube patterning. Combined with quantitative imaging, we utilized the size reduction technique to examine the how the sizes of somites and neural tube are affected in size reduced embryos.

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			<td height="21" style="height:21px;width:216px;">60 mm PYREX Petri dish</td>
			<td style="width:107px;">CORNING</td>
			<td style="width:119px;">&#160;3160-60</td>
			<td style="width:167px;"></td>
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			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:107px;">affymetrix</td>
			<td align="right" style="width:119px;">75817</td>
			<td>For making a mount for live imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Agarose, low gelling temperature Type VII-A</td>
			<td style="width:107px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">A0701-25G</td>
			<td></td>
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			<td height="21" style="height:21px;width:216px;">CaCl2</td>
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			<td>For 1/3 Ringer&#39;s solution</td>
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			<td height="21" style="height:21px;width:216px;">CaSO4</td>
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			<td>For egg water</td>
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			<td height="42" style="height:42px;width:216px;">Cover slip (25 mm x 25 mm, Thickness 1)</td>
			<td style="width:107px;">CORNING</td>
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			<td height="21" style="height:21px;width:216px;">Glass pipette</td>
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			<td>For microscope incubator</td>
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			<td height="21" style="height:21px;width:216px;">Instant sea salt</td>
			<td style="width:107px;">Instant Ocean</td>
			<td align="right" style="width:119px;">138510</td>
			<td>For egg water</td>
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		<tr height="42">
			<td height="42" style="height:42px;width:216px;">KCl</td>
			<td style="width:107px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">P4504</td>
			<td>For 1/3 Ringer&#39;s solution</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methyl cellulose</td>
			<td style="width:107px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">M0387-100G</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">NaCl</td>
			<td style="width:107px;">SIGMA-ALDRICH</td>
			<td style="width:119px;">S7653</td>
			<td>For 1/3 Ringer&#39;s solution</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Petri dish</td>
			<td style="width:107px;">Falcon</td>
			<td align="right" style="width:119px;">351029</td>
			<td>For making a mount for live imaging</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Phenol red</td>
			<td style="width:107px;">SIGMA Life Science</td>
			<td style="width:119px;">P0290</td>
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			<td height="42" style="height:42px;width:216px;">Pipette pump</td>
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			<td height="42" style="height:42px;width:216px;">Tricaine-S (MS222)</td>
			<td style="width:107px;">WESTERN CHEMICAL INC</td>
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			<td height="21" style="height:21px;">Ultra thin bright annealed 316L dia. 0.035 mm Stainless Steel Weaving Wires</td>
			<td style="width:107px;">Sandra</td>
			<td style="width:119px;"></td>
			<td>The wire we used was obtained ~20 years ago and we could not find exactly the same one. This product has the same material and diameter as the one we use.</td>
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</table>

surgical size reduction of zebrafish for the study of embryonic pattern scaling

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.&#160;

Yolk volume reduction is important for normal morphology 
As recently described in Almuedo-Castillo et al.17, size reduction of embryos can be achieved without reducing yolk volume. To compare with and without yolk volume reduction, we performed both two-step chopping (both blastula and yolk) and blastula-only chopping (Figure 2 and Supplemental Movie 1). Two-step chopped embryos showed seemingly normal overall morphology compare...

Watch this Scientific Journal Video about Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling at JoVE.com

Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.

In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained ...

Although scaling is one of the universal and fundamental attributes of developmental systems, the underlying mechanisms have not been understood in many organs and tissues. The size reduction technique of zebrafish presented in this video will advance your understanding of mechanisms underlying dynamic scaling processes. Advantages of working with zebrafish are that it is easy to perform live imaging, genetics, and quantitative analysis.Our technique is also very simple and easy to apply without any specialized equipment or intensive training. The health of embryos is the key for success with this technique. Use young fish as parents and be careful to minimize the damage by removing the chorions.And also play around in the size of the wound on the yolk, and how many cells are removed. As the embryos we use are very young and fragile, they need to be handled very carefully when transferring and chopping. At the same time, it needs to be fast so the procedure can be done in the right time window of development.Visual demonstration can give a good sense of how to produce smaller embryos effectively. To make a wire loop for embryo chopping, feed 20 centimeter long stiff and non-corrosive stainless steel wire through a glass capillary, making a small, one millimeter long loop at the top. Place a little droplet of clear nail polish onto the tip of the class capillary, between the capillary and the wire loop to hold it in place, making sure not to get any nail polish onto the loop portion, as it may damage the embryos.And then let it dry. Use lab tape to attach the glass capillary with the loop onto a wooden chopstick, leaving about two and a half centimeters of the glass capillary extending beyond the chopstick so that the chopstick part of the tool does not dip into the water later. In a 100 millimeter glass Petri dish with a plastic spatula, spread approximately 0.5 milliliters of 2%methyl cellulose near the center of the bottom thinly and evenly to a thickness of approximately 0.5 millimeters.Pour approximately 30 milliliters of one-third Ringer's solution to the side of the dish, and allow it to spread onto the rest of the dish and cover the methyl cellulose. Place previously de-chorionated embryos at the 256 cell to one K cell stage on 2%methyl cellulose, making sure the embryos are oriented onto their side. To chop blastula, use the wire loop to cut near the animal pole, perpendicular to the animal vegetal axis and chop off approximately 30 to 40%of blastoderm cells.Then gently tap the ends together to help the remaining cells stick back. For yolk, use the mounted wire to make a small wound near the vegetal pole by nicking the egg membrane, which will cause the yolk to ooze out for a few minutes, and then heal. When the yolk stops oozing out, use a pipette to move the embryo outside of the methyl cellulose in the same dish for recovery.Repeat blastula and yolk chopping for all embryos, and then leave the dish undisturbed for 30 minutes while the embryos recover. Then, use a glass pipette to transfer the embryos to the previously prepared 35 millimeter glass dish containing fresh one-third Ringer's solution and place them in a 28.5 degree Celsius incubator to allow for complete recovery before continuing with imaging. After performing two step chopping with blastula and yolk, embryos showed seemingly normal overall morphology throughout the developmental stages compared to the control embryos, other than size difference.On the other hand, blastula-only chopped embryos showed a peculiar morphology, especially at earlier stages. During epiboly, the embryos had a constricted and indented appearance. At the following somite stage, midline structures were found to be flattened at many axial levels.At later stages, the body structures adjacent to the yolk, such as the mid and hindbrain still showed a relatively flattened shape, possibly due to increased tension from the relatively larger yolk. Time lapse imaging of somite formation showed that in both control and chopped embryos, the sizes of somites that were formed at later stages were smaller compared to the ones from earlier stages. Also, throughout the somite formation stages, chopped embryos had smaller somites than the ones in control embryos.When the two step chopping technique was performed on membrane mCherry injected embryos, imaging of their spinal cords at 20 hours post fertilization showed reduction in neural tube heights due to size reduction. Two step chopping is very important to obtain smaller but otherwise normal embryos with high efficiency. It is important to remember not to chop the yolk while chopping off cells from the blastula.For yolk, make a tiny wounding on it for the content to ooze out. And the yolk membrane will heal up naturally in the proper buffer. Because the chopped embryos can be recovered quickly, any methods or analysis can be applied to normal embryos can be performed following the size reduction method.The simplicity and versatility of this technique is very powerful as anyone could introduce this technique to study scaling problem in any tissue or organ in zebrafish. We believe with this technique researchers can tackle scaling problems in a variety of systems that have been untouched before. We have already made important discoveries in scaling of somite and neural tube patterning.

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Research

JoVE Journal

Developmental Biology

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress liver regeneration.

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

A Method for Characterizing Embryogenesis in Arabidopsis

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, early stage plant embryos are small and contain few cells, making them difficult to observe and analyze. A method is described here for characterizing pattern formation in plant embryos under a microscope using the model organism Arabidopsis. Following the clearance of fresh ovules using Hoyer's solution, the cell number in and morphology of embryos could be observed, and their developmental stage could be determined by differential interference contrast microscopy using a 100X oil immersion lens. In addition, the expression of specific marker proteins tagged with Green Fluorescent Protein (GFP) was monitored to annotate cell identity specification during embryo patterning by confocal laser scanning microscopy. Thus, this method can be used to observe pattern formation in wild-type plant embryos at the cellular and molecular levels, and to characterize the role of specific genes in embryo patterning by comparing pattern formation in embryos from wild-type plants and embryo-lethal mutants. Therefore, the method can be used to characterize embryogenesis in Arabidopsis.

A Method for Characterizing Embryogenesis in Arabidopsis

This protocol outlines a method for observing embryogenesis in Arabidopsis via ovule clearance followed by the inspection of embryo pattern formation under a microscope.

Given the highly predictable nature of their development, Arabidopsis embryos have been used as a model for studies of morphogenesis in plants. However, ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...