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

Gli autori ringraziano Adeline Dolly per le foto di espianto di fibronectina, Eric Theveneau per utili discussioni e il Fondo Animal dell&#39;Institut Curie. CM è un borsista postdottorato della Regione Ile de France (Domaine d&#39;interet Majeur Stelo Pole), Université Paris Sud (addetto Temporaire d&#39;Enseignement et de Recherche), e Agence Nationale de la Recherche. Questo lavoro è stato finanziato dalla Université Paris Sud (Attractivite 2011), il Centre National de la Recherche Scientifique (Programma d&#39;azione thématique et sur fonti di incentivi), Association pour la Recherche contre le Cancer di Grant SFI20101201882, Ligue contre le Cancer, e Agence Nationale de la Recherche (Agence Nationale de la Recherche programma Blanc).

Gli esperimenti sono conformi alla normativa nazionale ed europea sulla protezione degli animali utilizzati a fini scientifici e con i principi sanciti a livello internazionale di sostituzione, riduzione e perfezionamento. 1. Preparazione di piatti fibronectina rivestite 14 <ol><li> Pipettare 500 ml di 10 mg / ml grado fibronectina cultura diluito in 1x tampone fosfato (PBS) in un piatto di plastica standard di Petri (Ø 40 x 11 mm). Incubare a 37 ° C per 1 ora. Nota: se si uti...

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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=Directional+migration+of+neural+crest+cells+in+vivo+is+regulated+by+Syndecan-4/Rac1+and+non-canonical+Wnt+signaling/RhoA.">Directional migration of neural crest cells in vivo is regulated by Syndecan-4/Rac1 and non-canonical Wnt signaling/RhoA.</a> Development. 135 (10), 1771-1780 (2008).</li><li>Kashef, J., K&#246;hler, A., Kuriyama, S., Alfandari, D., Mayor, R., Wedlich, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cadherin-11+regulates+protrusive+activity+in+Xenopus+cranial+neural+crest+cells+upstream+of+Trio+and+the+small+GTPases.">Cadherin-11 regulates protrusive activity in Xenopus cranial neural crest cells upstream of Trio and the small GTPases.</a> Genes Dev. 23 (12), 1393-1398 (2009).</li><li>Hwang, Y. -. 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Questo protocollo descrive una tecnica facile da espiantare premigratory cranica NC in X. laevis embrioni. Gli embrioni utilizzati per tali esperimenti devono essere robusti e guarire bene. Eliminare ogni partita di embrioni sani. Inoltre, crescere embrioni a diverse temperature (da 12 a 18-20 ° C), al fine di asportare cresta neurale nella fase 17 e non più tardi. Dopo stadio 17, cresta neurale può essere miscelata con mesoderma cefalico e non può essere rimosso completamente. Tessuti Xenopus guar...

La cresta neurale (NC) è una popolazione di cellule embrionali transitoria che emerge dal tubo neurale a fine neurulazione negli embrioni dei vertebrati. Gli eventi di segnalazione e genetici che controllano specifiche NC iniziano già nel gastrulazione. La NC è specificato al confine tra la neurale e ectoderma neurale non da segnali dai tessuti dorsali circostanti. Alla fine del neurulazione, celle NC subiscono una transizione epitelio-to-mesenchima (EMT) e migrano ampiamente nell&#39;embrione seguendo percorsi stereotipati rispondendo al circostante spunti guida. Una volta che hanno raggiunto la loro destinazione finale, si differenziano in una vasta gamma di derivati, ad esempio, i neuroni, glia, ossa, cartilagine e cellule pigmentate 1-5. A causa del loro contributo a molti tipi di cellule e tessuti embrionali, difetti in ogni fase di sviluppo celle NC, dalla induzione alla differenziazione finale, può causare sindromi congenite denominate neurocristopathies 6. Emanipolazione Xperimental del CN ​​sviluppare in fasi diverse - specifica, EMT, migrazione e differenziamento - permetterà di migliorare la nostra comprensione di neurocristopathies e permettere la progettazione di potenziali strategie terapeutiche. L&#39;embrione laevis Xenopus è un modello di scelta per studiare lo sviluppo NC. Un gran numero di embrioni sono facili da ottenere, e la fecondazione esterna dà accesso alle prime fasi di sviluppo. Molti strumenti sono a disposizione per manipolare sperimentalmente X. laevis sviluppo embrionale. Gene guadagno-di-funzione e atterramento sono facili da eseguire da microinjecting singole cellule dei primi blastulas. Tessuti embrionali possono essere tagliati per riassociazione in vitro 7-11 o saggi di back-innesto 12,13. In questo protocollo, si descrive come sezionare fuori premigratory cranica NC in X. laevis neurulas fine, prima della migrazione. Tali espianti possono essere coltivate su fibronectina-coated piastre per studiare la migrazione e la differenziazione in vitro controllati in condizioni sperimentali. Espianti NC possono anche essere innestati in normali o manipolato gli embrioni di accoglienza per studiare la loro migrazione e differenziamento in vivo.

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	<tbody>
		<tr height="20">
			<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

La cresta neurale (NC) è una popolazione cellulare dorsale tubo neurale transitoria che subisce una transizione epitelio-to-mesenchima (EMT) alla fine di neurulazione, migra verso estesamente vari organi, e si differenzia in molti tipi di derivati ​​(neuroni, glia, cartilagine e osso, le cellule pigmentate ed endocrine). In questo protocollo, si descrive come sezionare il premigratory cranica NC da Xenopus laevis embrioni, al fine di studiare lo sviluppo NC in vivo e in vitro. Il modello di rana offre molti vantaggi per studiare lo sviluppo precoce; lotti abbondanti sono disponibili, gli embrioni si sviluppano rapidamente, il guadagno in vivo e la perdita di strategie funzionali consentono la manipolazione dell&#39;espressione genica prima di NC dissezione del donatore e / o embrioni di accoglienza. Gli espianti NC possono essere piastrate su fibronectina e utilizzati per studi in vitro. Essi possono essere coltivate per diversi giorni in un mezzo definito privo di siero. Abbiamo anche descritto come innestare espianti NC indietroin embrioni di accoglienza per lo studio della migrazione NC e la differenziazione in vivo.

Quando placcato in fibronectina, gli espianti cresta neurale attaccano rapidamente (15-30 min) e l&#39;espianto si diffonde piatta entro 2 ore (Figura 1A). Dopo 3-6 cellule hr cominciano a disperdersi. A 24 ore a 15 ° C, molte cellule hanno iniziato a migrare dalla espianto (Figure 1B e 1C). Tuorlo rende le cellule molto luminoso in contrasto di fase (Figura 1B). Sporgenze cellulari (filopodes e lamellipodes) sono chiaramente visibili dopo falloidina c...

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

Questo protocollo descrive come sezionare premigratory cresta neurale cranica (NC) da Xenopus laevis neurulas. Tali espianti possono essere placcati in fibronectina e coltivate in vitro, o innestate di nuovo in embrioni di accoglienza. Questa tecnica consente di studiare i meccanismi di NC epitelio a mesenchima transizione, migrazione e differenziazione.

Dissezione dei<em&gt; Xenopus laevis</em&gt; Cresta neurale per<em&gt; In vitro</em&gt; Espianto Cultura o<em&gt; In vivo</em&gt; Trapianto

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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JoVE Journal

Biology

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

Dissection of Organizer and Animal Pole Explants from Xenopus laevis Embryos and Assembly of a Cell Adhesion Assay

Dissezione di organizzatore e Espianti Polo animali da embrioni di Xenopus laevis e montaggio di un saggio Adesione Cellulare

Ricerca

Biologia

Plastic Embedding and Sectioning of Xenopus laevis Embryos

Incorporare plastica e Sezionamento embrioni di Xenopus laevis

Obtaining Eggs from Xenopus laevis Females

Ottenere uova da Xenopus laevis Femmine

The eggs of Xenopus laevis intact, lysed, and/or fractionated are useful for a wide variety of experiments. This protocol shows how to induce egg laying, ...

Preparation and Fractionation of Xenopus laevis Egg Extracts

Preparazione e frazionamento di Xenopus laevis Egg Estratti

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Microinjection of Xenopus Laevis Oocytes

Microiniezione degli ovociti di Xenopus Laevis

Microinjection of Xenopus laevis oocytes followed by thin-sectioning electron microscopy (EM) is an excellent system for studying nucleocytoplasmic ...

Tissue Determination Using the Animal Cap Transplant (ACT) Assay in Xenopus laevis

Tissue Determination Using the Animal Cap Transplant ACT Assay in Xenopus laevis

Determinazione del tessuto Utilizzando il trapianto Cap animali (ACT) Assay in Xenopus laevis</em

Many proteins play a dual role in embryonic development. Those that regulate cell fate determination in a specific tissue can also affect the development ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Live-cell imaging e analisi quantitativa della embrionali in cellule epiteliali Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Production of Transgenic Xenopus laevis by Restriction Enzyme Mediated Integration and Nuclear Transplantation

Produzione di transgenici Xenopus laevis Integrazione mediata da enzima di restrizione e trapianto nucleare

Stable integration of cloned gene products into the Xenopus genome is necessary to control the time and place of expression, to express genes at later ...

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch Clamp and Perfusion Techniques for Studying Ion Channels Expressed in Xenopus oocytes

Patch clamp e Tecniche di perfusione per la valutazione dei canali ioni Espresso in Xenopus Ovociti

The protocol presented here is designed to study the activation of the large conductance, voltage- and Ca2+-activated K+ (BK) channels. The protocol may ...

Facial Transplants in Xenopus laevis Embryos

Facial Transplants in Xenopus laevis Embryos

Trapianti viso a<em&gt; Xenopus laevis</em&gt; Embrioni

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...