Name: analyzing in vivo cell migration using cell transplantations and time-lapse imaging in zebrafish embryos
Uploaded: 2016-04-29T14:00:00
Description: Watch this Scientific Journal Video about Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos at JoVE.com

We thank F. Bouallague and the IBENS animal facility for excellent zebrafish care. Research reported in this publication was supported by the Fondation ARC pour la recherche sur le cancer, grants N&#176; SFI20111203770 and N&#176; PJA 20131200143.

Note: Figure 1 presents the outline of the protocol.
1. Preparation of the Needles for Injection and Transplantation
Note: Needles can be prepared at any time and stored. Keep them in a Petri dish, on a band of modeling clay. Seal the dish with parafilm to protect from dust.
<ol>
	<li>For injection needles, pull a glass capillary (outside diameter 1.0 mm, inside diameter 0.58 mm, without filament (see list of Materials)) with a mi...

<ol>
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V., Way, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Open+source+software+for+quantification+of+cell+migration,+protrusions,+and+fluorescence+intensities.">Open source software for quantification of cell migration, protrusions, and fluorescence intensities.</a> The Journal of Cell Biology. 209 (1), 163-180 (2015).</li><li>Gorelik, R., Gautreau, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+and+unbiased+analysis+of+directional+persistence+in+cell+migration.">Quantitative and unbiased analysis of directional persistence in cell migration.</a> Nature Protocols. 9 (8), 1931-1943 (2014).</li><li>Sacan, A., Ferhatosmanoglu, H., Coskun, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CellTrack:+An+open-source+software+for+cell+tracking+and+motility+analysis.">CellTrack: An open-source software for cell tracking and motility analysis.</a> Bioinformatics. 24 (14), 1647-1649 (2008).</li><li>Tahinci, E., Symes, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Distinct+functions+of+Rho+and+Rac+are+required+for+convergent+extension+during+Xenopus+gastrulation.">Distinct functions of Rho and Rac are required for convergent extension during Xenopus gastrulation.</a> Developmental biology. 259 (2), 318-335 (2003).</li><li>Zhang, T., Yin, 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=Stat3-Efemp2a+modulates+the+fibrillar+matrix+for+cohesive+movement+of+prechordal+plate+progenitors.">Stat3-Efemp2a modulates the fibrillar matrix for cohesive movement of prechordal plate progenitors.</a> Development. 141 (22), 4332-4342 (2014).</li><li>Riedl, J., Crevenna, A. H., 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=Lifeact:+a+versatile+marker+to+visualize+F-actin.">Lifeact: a versatile marker to visualize F-actin.</a> Nature methods. 5 (7), 605-607 (2008).</li><li>Burkel, B. M., Von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell Motility and the Cytoskeleton. 64 (11), 822-832 (2007).</li><li>Yoo, S. K., Deng, Q., Cavnar, P. J., Wu, Y. I., Hahn, K. M., Huttenlocher, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+regulation+of+protrusion+and+polarity+by+PI3K+during+neutrophil+motility+in+live+zebrafish.">Differential regulation of protrusion and polarity by PI3K during neutrophil motility in live zebrafish.</a> Developmental cell. 18 (2), 226-236 (2010).</li><li>Spracklen, A. J., Fagan, T. N., Lovander, K. E., Tootle, T. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+pros+and+cons+of+common+actin+labeling+tools+for+visualizing+actin+dynamics+during+Drosophila+oogenesis.">The pros and cons of common actin labeling tools for visualizing actin dynamics during Drosophila oogenesis.</a> Developmental biology. 393 (2), 209-226 (2014).</li><li>Johnson, H. W., Schell, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neuronal+IP3+3-kinase+is+an+F-actin-bundling+protein:+role+in+dendritic+targeting+and+regulation+of+spine+morphology.">Neuronal IP3 3-kinase is an F-actin-bundling protein: role in dendritic targeting and regulation of spine morphology.</a> Molecular biology of the Cell. 20 (24), 5166-5180 (2009).</li><li>Yi, J., Wu, X. S., Crites, T., Hammer, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Actin+retrograde+flow+and+actomyosin+II+arc+contraction+drive+receptor+cluster+dynamics+at+the+immunological+synapse+in+Jurkat+T+cells.">Actin retrograde flow and actomyosin II arc contraction drive receptor cluster dynamics at the immunological synapse in Jurkat T cells.</a> Molecular Biology of the Cell. 23 (5), 834-852 (2012).</li></ol>

This protocol presents an easy way to study the role of a candidate gene in cell migration in vivo, by combining the creation of chimeric embryos using cell transplantation with live imaging.
Creation of mosaic embryos
Studying the dynamics of a cell requires the visualization of its contour to analyze cytoplasmic extensions. This can be achieved by labeling isolated cells in an otherwise unlabeled&#160;&#8211; or differently labeled - environment, thus off...

In multicellular organisms, cell migration is essential both for the development of the embryo where it ensures the organization of cells into tissues and organs, and for adult life, where it takes part to tissue homeostasis (wound healing) and immunity. In addition to these physiological functions, cell migration is also involved in diverse pathological situations, including, in particular, cancer metastasis.
Cell migration has been analyzed in vitro for decades, providing an overall understanding of the molecular mechanisms ensuring cell movements on flat surfaces. In vivo however, cells are confronted by a more complex environment. It clearly appeared in the past years that migration within an organism may be influenced by external cues such as the extracellular matrix, neighboring cells or secreted chemokines guiding migration, and that the mechanisms driving cell migration may vary from what has been described in vitro1,2. The mechanisms ensuring in vivo cell migration have received less attention so far, mainly because of the increased technical difficulty, compared to in vitro studies. In vivo analysis of cell migration in particular requires direct optical access to migrating cells, techniques to label unique cells in order to see their dynamics and morphology, as well as gain or loss of function approaches to test the role of candidate genes. So far, only a few model systems harboring these characteristics have been used to dissect in vivo cell migration3.
We recently used the migration of the prospective prechordal plate in early zebrafish embryos as a new convenient model system to assess the function of candidate genes in controlling in vivo cell migration4,5. Prospective prechordal plate (also known as anterior mesendoderm) is a group of cells forming at the onset of gastrulation on the dorsal side of the embryo. During gastrulation this group collectively migrates towards the animal pole of the embryo6-8, to form the prechordal plate, a mesendodermal thickening, anterior to the notochord, and underlying the neural plate. The anterior part of the prechordal plate will give rise to the hatching gland, while its posterior part likely contributes to head mesoderm9. Thanks to the external development and optical clarity of the fish embryo, cell migration can be directly and easily observed in this structure.
Cell transplantation is a very potent technique that allows for the rapid and easy creation of mosaic embryos10. Expressing fluorescent cytoskeletal markers in transplanted cells results in the labeling of isolated cells, the morphology and dynamics of which can be easily observed. Combining this to loss or gain of function approaches permits the analysis of cell-autonomous functions of a candidate gene.
The presented protocol describes how we assessed the function of the TORC2 component Sin1 in controlling cell migration and actin dynamics in vivo5. But, as mentioned in the results and further discussed, it could be used to analyze the potential implication of any candidate gene in controlling cell migration in vivo.

<table border="0" cellpadding="0" cellspacing="0" width="679">
	<tbody>
		<tr height="63">
			<td height="63" style="height:63px;width:323px;">Glass capillaries (outside diameter 1.0 mm, inside diameter 0.58&#160;mm)</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td align="right" style="width:119px;">300085</td>
			<td style="width:167px;">standard thickness</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;">Glass capillaries (outside diameter 1.0 mm, inside diameter 0.78 mm)</td>
			<td style="width:71px;">Harvard Apparatus</td>
			<td align="right">300085</td>
			<td>thin-walled</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Penicillin-Streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td>P4333</td>
			<td>10&#160;000&#160;units penicillin and 10&#160;mg streptomycin per ml</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">fine tweezers</td>
			<td style="width:71px;">Dumont Fine Science Tools</td>
			<td style="width:119px;">11254-20</td>
			<td>5F</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">glass bottom dishes</td>
			<td style="width:71px;">MatTek</td>
			<td>P35G-0-10-C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:323px;">Air transjector</td>
			<td style="width:71px;">Eppendorf</td>
			<td align="right" style="width:119px;">5246</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Micro-forge</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">MF-900</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Microgrinder</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">EG-44</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Micromanipulator (for injection)</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">MN-151</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:323px;">Micromanipulator (for cell transplantation)</td>
			<td style="width:71px;">Leica</td>
			<td style="width:119px;">Leica Micromanipulator</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Hammilton Syringe</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">IM-9B</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:323px;">Micropipette puller</td>
			<td style="width:71px;">David Kopf Instruments</td>
			<td style="width:119px;">Model 720</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:323px;">Transplantation mold</td>
			<td style="width:71px;">Adapative Science Tools</td>
			<td style="width:119px;">PT-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Needle holder</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">HI-7</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">Tube connector</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">CI-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:323px;">PTFE tubing</td>
			<td style="width:71px;">Narishige</td>
			<td style="width:119px;">CT-1</td>
			<td></td>
		</tr>
	</tbody>
</table>

analyzing in vivo cell migration using cell transplantations and time-lapse imaging in zebrafish embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration have been thoroughly analyzed in vitro. However, in vivo cell migration somehow differs from in vitro migration, and has proven more difficult to analyze, being less accessible to direct observation and manipulation. This protocol uses the migration of the prospective prechordal plate in the early zebrafish embryo as a model system to study the function of candidate genes in cell migration. Prechordal plate progenitors form a group of cells which, during gastrulation, undergoes a directed migration from the embryonic organizer to the animal pole of the embryo. The proposed protocol uses cell transplantation to create mosaic embryos. This offers the combined advantages of labeling isolated cells, which is key to good imaging, and of limiting gain/loss of function effects to the observed cells, hence ensuring cell-autonomous effects. We describe here how we assessed the function of the TORC2 component Sin1 in cell migration, but the protocol can be used to analyze the function of any candidate gene in controlling cell migration in vivo.

The presented technique was used to analyze the role of Sin1, one of the core components of the Tor complex 2 (TORC2), in controlling in vivo cell migration. The use of cell transplantation permits labeling of isolated cells and analysis of cell-autonomous effects. Movie S1 shows the migration of transplanted prechordal plate progenitor cells. Actin labeling with ABP140 allows the easy visualization of actin-rich cytoplasmic protrusions. We measured their frequency and orientatio...

Watch this Scientific Journal Video about Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos at JoVE.com

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Combining cell transplantation, cytoskeletal labeling and loss/gain of function approaches, this protocol describes how the migrating zebrafish prospective prechordal plate can be used to analyze the function of a candidate gene in in vivo cell migration.

Analyzing In Vivo Cell Migration using Cell Transplantations and Time-lapse Imaging in Zebrafish Embryos

Cell migration is key to many physiological and pathological conditions, including cancer metastasis. The cellular and molecular bases of cell migration ...

The overall goal of this procedure is to analyze the function of a candidate gene in controlling in vivo cell migration using cell transplantation combined with live imaging in the Zebrafish embryo. This method can help address key questions in the cell migration field such as determining the role and importance of new candidate genes in in vivo cell migration. The main advantage of this technique is that it creates mosaic embryos which allows good imaging and cell adenomyosis.To prepare cell transplantation needles use a micro pipette puller to pull a glass capilary. Then, with a microforge, cut the tip of the needle at the point where the inside diameter is 35 micrometers. Using a microgrinder bevel the cut end of the capillary at an angle of 45 degrees.To eliminate glass residues insert the needle into a needle holder mounted on a syringe then use 2%hydrofluoric acid to rinse the inside of the needle three times by drawing the solution in and out. Follow by rinsing the needle with acetone three times. To prepare dishes for injection or cell transplantation prepare 1%agarose by heating 50 milliliters of embryo medium containing 0.5 grams of agarose in a microwave for one minute at full power.Cool the agarose gel to about 60 degrees Celsius and pour the entire volume into a 90 millimeter petri dish. Insert a mold to create lines for injection or to create wells for cell transplantation. When the agarose gel is set use forceps to remove the mold.To inject donor embryos, under a stereomicroscope use forceps to gently squeeze collected transgenic embryos into the lines of an injection dish filled with embryo medium. With forceps orient the embryos with the cells toward the top then incubate the dish at 28 degrees Celsius until the embryos have reach the four cell stage. Next, load an injection needle with two microliters of a previously prepared injection solution containing the actin-binding domain of the yeast actin-binding protein 140 bound to mcherry.Then insert the needle into a needle holder attached to a micromanipulator and connected to an air transjector. Open the needle by using fine forceps to gently touch the needle tip or by pinching it's extremity. Then insert the needle into one of the four cells and inject the solution to fill 70%of the cell volume.Inject 30 embryos in a similar manner. When the embryos have reached sphere stage use a plastic pasture pipette to transfer host transgenic goosecoid GFP embryos and previously injected donor embryos into seperate 35 millimeter petri dishes coated with one millimeter agarose in embryo medium and filled with Pen/Strep embryo medium. Using fine tweezers manually extract the embryos from their chorions.Then place the embryos into the incubator at 28 degrees Celsius. When the embryos have reached shield stage under a flourescent microscope select donor embryos expressing ABP140 mcherry within the green shield. Using a fire polished pasture pipette transfer the host embryos into a row of wells in the cell transplantation dish filled with Pen/Strep EM.Then place the donor embryos in the neighboring row.Next with an eyelash carefully orient the embryos with the shield up. Then using a needle holder attached to a syringe rinse the tip of the needle by drawing in and expelling out 70%ethanol. Dry the needle by drawing air into it.When the needle is dry insert it into the needle holder connected to the Hamilton syringe with the bevel facing upwards. To perform shield to shield transplantation enter the shield of a donor embryo with the transplantation needle and draw up about 10 to 20 cells labeled with ABP140 mcherry. Carefully avoid drawing yolk.Transplant the cells into the shield of the host embryo and repeat the cell transfer until all host embryos have been transplanted. When transplantation is complete use a fire polished pasture pipette to remove any damaged embryos. Incubate the transplanted embryos at 28 degrees Celsius for about 30 minutes to allow them to recover.To transplant single prechordal plate pregenitor cells after dechorionating embryos as demonstrated earlier use a fire polished pasture pipette to transfer three donor embryos into a cell transplantation dish filled with Pen/Strep calcium free ringers. With fine tweezers for each embryo dissect an explant containing the injected ABP140 mcherry labeled cells and rapidly discard the rest of the embryos. Using an eyelash gently stir the explants until the cells dissociate.Then draw up a single, isolated cell in the transplantation needle and transplant it into the shield of a host embryo as demonstrated earlier in the video. For optimal control of the aspiration and transplantation keep the air/water separation at about 20 centimeters from the end of the needle. To mount the embryos fill a glass bottom vial with one millimeter of hot 0.2%agarose in EM.Place the vial at 42 degrees Celsius in a heat block.Transfer a selected embryo into the agarose solution and discard the excess embryo medium from the pipette. Then draw the embryo into the pipette with agarose and transfer it to a previously prepared imaging chamber along with a drop of agarose. Before the agarose is set use an eyelash to orient the embryo by placing the perspective prechordal plate upwards for imaging with an upright microscope, or on the glass bottom for imaging with an inverted microscope.There will be about 40 seconds to orient the embryo before the agarose gel sets depending on the room temperature. Orient the embryo rapidly by manipulating the blastoderm rather than the yolk which is very fragile. After all embryos are mounted and the agarose is set use Pen/Strep EM to fill the chamber to prevent the embryos from drying.Carry out live cell imaging and cell dynamics experiements according to the text protocol. The technique demonstrated in this video was used to analyze the role of Sin1, one of the core components of TOR complex two in controlling in vivo cell migration. This movie shows the migration of transplanted prechordal plate pregenitor cells.Actin labeling allows visualize of actin rich cytoplasmic protrusions. Through analysis of frequency and orientation it was observed that wild type cells frequently produce large cytoplasmic protrusions, oriented in the direction of the animal pole, and of migration. As seen in this figure, loss of function of Sin1 leads to a drastic reduction in the number of protrusions and randomization of the remaining protrusions demonstrating the importance of Sin1 in controlling actin rich protrusion formation.Interestingly the Sin1 phenotype can be rescued by expression of a constitutively active form of Rac1 strongly suggesting that TOR C2 controls actin dynamics and cell protrusion formation through Rac1. This technique was also used to analyze the role of arpin, an inhibitor of the ARP2/3 complex in cell migration. As shown here, loss of function of arpin leads to an increase in protrusion frequency, due either to more frequent protrusion formation, or an increase in protrusion stability.Measuring protrusion lifetime in the absence of arpin revealed that the temporal persistence of protrusions doubled indicating an increase in stability. Once mastered the transplantation of about 30 embryos can be done in 45 minutes. While attempting this procedure it's important to keep an eye on the development of the embryos as all the steps are critically timed.Following this procedure, other methods like live confocal microscopy can be performed to provide better imaging and address questions regarding the sub cell organization of cytoscotic elements, polarity markers, addition proteins, or any other cell constituent. After watching this video you should have a good understanding of how to use transplants of prechordal plate pregenitors to analyze the role of any candidate gene in in vivo cell migration.

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

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Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Single-Cell Quantification of Protein Degradation Rates by Time-Lapse Fluorescence Microscopy in Adherent Cell Culture

Proteins are in a dynamic state of synthesis and degradation and their half-lives can be adjusted under various circumstances. However, most commonly used ...

Electroporation Method for In Vivo Delivery of Plasmid DNA in the Adult Zebrafish Telencephalon

Electroporation is a transfection method in which an electrical field is applied to cells to create temporary pores in a cell membrane and increase its ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...