Name: dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation
Uploaded: 2014-03-04T17:00:00
Description: Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

The authors thank Adeline Dolly for the pictures of explant on fibronectin, Eric Theveneau for helpful discussions and the Animal Facility of the Institut Curie. C.M. is a postdoctoral fellow of Region Ile de France (Domaine d'Interet Majeur Stem Pole), Universite Paris Sud (Attache Temporaire d'Enseignement et de Recherche), and Agence Nationale de la Recherche. This work was funded by the Universite Paris Sud (Attractivite 2011), the Centre National de la Recherche Scientifique (Action Thematique et Incitative sur Programme), Association pour la Recherche contre le Cancer Grant SFI20101201882, Ligue contre le Cancer, and Agence Nationale de la Recherche (Agence Nationale de la Recherche Programme Blanc).

Experiments comply with National and European regulation on the protection of animals used for scientific purposes and with internationally established principles of replacement, reduction and refinement.
1. Preparation of Fibronectin-coated Dishes14
<ol>
	<li>Pipette 500 ml of 10 mg/ml culture grade fibronectin diluted in 1x&#160;Phosphate Buffered Saline (PBS) into a standard plastic Petri dish (&#216; 40 x 11 mm). Incubate at 37 &#176;C for 1 hr. Note: if using glass dishes or ...

<ol>
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This protocol describes an easy technique to explant premigratory cranial NC in X. laevis embryos. The embryos used for such experiments need to be robust and heal well. Discard any batch of unhealthy embryos. In addition, grow embryos at various temperatures (from 12 to 18-20 &#176;C), in order to excise neural crest at stage 17 and not later. After stage 17, neural crest may be mixed with cephalic mesoderm and cannot be removed completely. Xenopus tissues heal quickly and then loose their adh...

The neural crest (NC) is a transient embryonic cell population that emerges from the neural tube at the end of neurulation in vertebrate embryos. The signaling and genetic events that control NC specification start as early as gastrulation. The NC is specified at the border between the neural and non-neural ectoderm by signals from surrounding dorsal tissues. At the end of neurulation, NC cells undergo an epithelium-to-mesenchyme transition (EMT) and migrate extensively in the embryo following stereotyped routes&#160;by responding to surrounding guiding cues. Once they have reached their final destination, they differentiate into a vast array of derivatives, e.g. neurons, glia, bone, cartilage, and pigmented cells1-5. Because of their contribution to many cell types and embryonic tissues, defects at any step of NC cells development, from induction to final differentiation, can cause congenital syndromes named neurocristopathies6. Experimental manipulation of the developing NC at different stages - specification, EMT, migration, and differentiation - will improve our understanding of neurocristopathies and allow design of potential therapeutic strategies.
The Xenopus laevis embryo is a model of choice to study NC development. Large numbers of embryos are easy to obtain, and external fertilization gives access to the very first steps of development. Many tools are available to experimentally manipulate X. laevis embryonic development. Gene gain-of-function and knockdown are easy to perform by microinjecting individual cells of early blastulas. Embryonic tissues can be cut for in vitro reassociation 7-11 or back-grafting assays12,13.
In this protocol, we describe how to dissect out premigratory cranial NC in X. laevis late neurulas, prior to migration. These explants can be cultured on fibronectin-coated plates to study migration and differentiation in controlled in vitro experimental conditions. NC explants can also be grafted into normal or manipulated host embryos to study their migration and differentiation in vivo.

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			<td height="20" style="height:20px;width:244px;">Fibronectin from bovine plasma</td>
			<td style="width:71px;">Sigma</td>
			<td style="width:119px;">F4759-1MG</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:244px;">Bovine Serum Albumine. Fraction V</td>
			<td style="width:71px;">Euromedex</td>
			<td style="width:119px;">04-100-811-C</td>
			<td>Lower quality grade may&#160; be suitable for this application</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:244px;">Stainless Steel Insect Pins. Size 000</td>
			<td style="width:71px;">FST</td>
			<td align="right" style="width:119px;">26001</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:244px;">Dumont #5 forceps 0.05 mm x 0.02 mm</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">11252-20</td>
			<td></td>
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dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

When plated on fibronectin, the neural crest explants attach rapidly (15-30 min) and the explant spreads flat within 2 hr (Figure 1A). After 3-6 hr cells start to scatter. At 24 hr at 15 &#176;C, many cells have started to migrate away from the explant (Figures 1B and 1C). Yolk makes cells very bright under phase contrast (Figure 1B). Cell protrusions (filopodes and lamellipodes) are clearly visible after phalloidin staining of the actin cytoskeleton (<s...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

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