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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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 ...

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