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>
	<li>Charras, G., Sahai, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+influences+of+the+extracellular+environment+on+cell+migration.">Physical influences of the extracellular environment on cell migration.</a> Nature Reviews Molecular Cell Biology. 15 (12), 813-824 (2014).</li><li>Friedl, P., Wolf, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasticity+of+cell+migration:+A+multiscale+tuning+model.">Plasticity of cell migration: A multiscale tuning model.</a> Journal of Cell Biology. 188 (1), 11-19 (2010).</li><li>Friedl, P., Gilmour, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Collective+cell+migration+in+morphogenesis,+regeneration+and+cancer.">Collective cell migration in morphogenesis, regeneration and cancer.</a> Nature reviews. 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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 : JoVE. (29), (2009).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Barry, D. J., Durkin, C. H., Abella, J. 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.

analyzing-vivo-cell-migration-using-cell-transplantations-time-lapse

Research

JoVE Journal

Developmental Biology

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

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 ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

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 approaches to determine protein half-life are either limited to population averages from lysed cells or require the use of protein synthesis inhibitors. This protocol describes a method to measure protein half-lives in single living adherent cells, using SNAP-tag fusion proteins in combination with fluorescence time-lapse microscopy. Any protein of interest fused to a SNAP-tag can be covalently bound by a fluorescent, cell permeable dye that is coupled to a benzylguanine derivative, and the decay of the labeled protein population can be monitored after washout of the residual dye. Subsequent cell tracking and quantification of the integrated fluorescence intensity over time results in an exponential decay curve for each tracked cell, allowing for determining protein degradation rates in single cells by curve fitting. This method provides an estimate for the heterogeneity of half-lives in a population of cultured cells, which cannot easily be assessed by other methods. The approach presented here is applicable to any type of cultured adherent cells expressing a protein of interest fused to a SNAP-tag. Here we use mouse embryonic stem (ES) cells grown on E-cadherin-coated cell culture plates to illustrate how single cell degradation rates of proteins with a broad range of half-lives can be determined.

This protocol describes a method to determine protein half-lives in single living adherent cells, using pulse labeling and fluorescence time-lapse imaging of SNAP-tag fusion proteins.

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 permeability, thereby allowing different molecules to be introduced to the cell. In this paper, electroporation is used to introduce plasmids to ependymoglial cells, which line the ventricular zone of the adult zebrafish telencephalon. A fraction of these cells shows stem cell properties and generates new neurons in the zebrafish brain; therefore, studying their behavior is essential to determine their roles in neurogenesis and regeneration. The introduction of plasmids via electroporation enables long-term labeling and tracking of a single ependymoglial cell. Furthermore, plasmids such as Cre recombinase or Cas9 can be delivered to single ependymoglial cells, which enables gene recombination or gene editing and provides a unique opportunity to assess the cell&#8217;s autonomous gene function in a controlled, natural environment. Finally, this detailed, step-by-step electroporation protocol is used to obtain successful introduction of plasmids into a large number of single ependymoglial cells.

Presented here is an electroporation method for plasmid DNA delivery and ependymoglial cell labeling in the adult zebrafish telencephalon. This protocol is a quick and efficient method to visualize and trace individual ependymoglial cells and opens up new possibilities to apply electroporation to a broad range of genetic manipulations.

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 or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

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 vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

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