DP è destinatario del Premio Ruth Kirschstein DA021977 1F32-01A1 dal National Institute on Drug Abuse. Questo lavoro è stato sostenuto dal M. Margaret Alkek Fondazione RHF.

<html> I. Panoramica schematica: <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="Figura 1" width="460" height="384" /> Questo video mostra le varie fasi di analisi per l&#39;induzione neurale in embrione di pollo. In primo luogo, l&#39;embrione host è espiantati nella cultura New [NI1]. Poi, l&#39;embrione è donnor espiantati in soluzione salina e nodo di Hensen (l &#39;&quot;organizzatore&quot; in pulcino) è marcato con colorante fluorescente Dii [NI2]. Nodo di Hensen è asportato dall&#39;embrione donnor [NI3] e trapiantati per l&#39;embrione host [NI4, NI5]. L&#39;embrione host è poi coltivate per un massimo di 22 ore, periodo dopo il quale il tessuto donnor ha indotto un asse ectopica nell&#39;area ospite opaca [NI6]. In seguito la cultura, l&#39;embrione host è fisso e trattati per fotoossidazione alla presenza del DAB: In questa reazione chimica, le cellule derivate donnor, che appaiono rossi sotto fluorescenza FITC prima fotoossidazione [NI7], diventano marroni seguente reazione con DAB [NI8]. L&#39;embrione viene quindi elaborato per l&#39;ibridazione in situ con un pennarello neurale [NI8]; tessuto ospite derivato si è formato tessuto neurale in presenza del trapianto donnor. Questo tessuto appare blu. 1: Preparare l&#39;embrione di accoglienza per la cultura di New <ol><li> Questo protocollo di test di induzione neurale inizia con le uova che sono state incubate per Hamburger &amp; Hamilton fase 3 + (o HH3 +). Queste uova sono state incubate previste dalla loro parte in un umido incubatore per 13 ore. </li><li> Preparare l&#39;embrione di accoglienza per la cultura Nuovo (passi 3.1. A 5.5 9). </li><li> Coprire la superficie dell &#39;embrione ospite con 200 microlitri soluzione salina. Ciò faciliterà il trasferimento più tardi e il posizionamento dell&#39;innesto donnor. </li><li> Infine, comprende il montaggio con un piatto capovolto Falcon 35 millimetri cultura. </li></ol> 2: espianto l&#39;embrione donnor in soluzione fisiologica <ol><li> Aprire l&#39;uovo toccando il guscio con una pinza e rimuovere i pezzi del guscio, in tutto il guscio. Rimuovere superiore del guscio e scartare. </li><li> Rimuovere l&#39;albumina spessore con una pinza, e inclinare il sacco vitellino con una pinza grossolana in modo che l&#39;embrione verso l&#39;alto. </li><li> Utilizzando forbici, tagliare un quadrato di tutto il sacco vitellino dell&#39;embrione. Rimuovere l&#39;embrione dal tuorlo con un cucchiaio, e mettere in un piatto contenente PBS. </li><li> Utilizzando pinze, staccare l&#39;embrione donnor da zona pellucida. </li><li> Trasferimento donnor embrione di un piatto coperto Sylgard contenente PBS. </li></ol> 3: Etichettatura, asportare e trapiantare l&#39;innesto donnor <ol><li> Utilizzando un estrattore microelettrodo, tirare 50 pipette di vetro microlitri microcapillare. Sotto il microscopio, tagliare la punta della micropipetta con una pinza sottile. Micropipetta posto in una capsula di Petri rivestito con un nastro di plasteline. </li><li> Preparare la soluzione colorante che saranno utilizzati per etichettare l&#39;innesto precedente al trapianto: carbocyanine colorante Dii (1,1 &#39;-dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perclorato): in primo luogo, preparare uno stock di DII in 1 ml etanolo assoluto a 0,5%. Questo stock possono essere conservati a-20C nel buio. In un bagnomaria regolato a 42 ° C, mescolare preriscaldata 40 microlitri magazzino DII a 360μl 0.3M saccarosio. La temperatura del bagno l&#39;acqua è necessaria per evitare la precipitazione di DII e la formazione di cristalli insolubili. In breve giro la soluzione su un spectrafuge mini (per 5-10 secondi). La soluzione DII è ora pronto all&#39;uso. </li><li> Utilizzando perni insetti, fissare l&#39;embrione donnor sul fondo del piatto Sylgard coperto. </li><li> Back-riempiono la micropipetta con DII. Applicando una leggera pressione con un gruppo del tubo aspiratore, applicare un piccolo bolo di DII al nodo di Hensen. Assicurarsi che l&#39;intero nodo di Hensen è etichettato. </li><li> Utilizzando una pipetta microcapillare o un coltello microdissecting, tagliare il nodo di Hensen (&quot;organizzatore&quot;, innesto donnor). </li><li> Contrassegnare il lato ventrale e la fine posteriore dell&#39;innesto con carminio. Questo farà sì che l&#39;innesto è posizionato con adeguata dorso-ventrale orientamento al punto 4.3. </li><li> Usando una pipetta Gilson 200μl, trasferire l&#39;innesto per l&#39;embrione di accoglienza. </li></ol> 4: Garantire l&#39;innesto e la coltura l&#39;embrione ospite <ol><li> Rimuovere salina dall&#39;embrione ospite, assicurandosi che il trapianto rimane posizionato in prossimità con il sito ospitante. </li><li> Utilizzando una pipetta microcapillare o un coltello microdissecting, fare una piccola incisione (80-100μm) nella zona pellucida / area regione di confine opaca, a livello del nodo di Hensen di embrione di accoglienza. </li><li> Utilizzando una pipetta microcapillare o un microdissecting coltello, la posizione in modo che l&#39;innesto lato ventrale volti verso di voi e la fine posteriore è parallelo a regione equivalente in embrione ospitante. </li><li> Rimuovere salina rimanenti, e l&#39;embrione di processo host per nuova cultura per 81-22 ore (passi 6,1-6,6 9). </li><li> L&#39;embrione host è poi fissata la notte a 4 ° C (passi 7,1-7,7 9). </li></ol>e &quot;&gt; 5: Photooxidise l&#39;innesto in fluorescenza <ol><li> Sotto cappa, preparare una soluzione DAB di lavoro in tampone Tris: Sciogliere substrato DAB (3,3 &#39;-diaminobenzidina tetraidrocloruro) in Tris buffer (100mM Tris-HCl, pH 7,4) a 500μg/ml; tenere soluzione nel buio, sul ghiaccio. </li><li> Lavare l&#39;embrione due volte in PBS per 5 minuti ciascuno e una volta in tampone Tris per 5 mn. </li><li> Trasferire l&#39;embrione in una diapositiva cavità di vetro contenente 1 ml di tampone Tris. </li><li> Sostituire la soluzione tampone tris con 1,5 ml di soluzione DAB. Smaltire Eppendorf punta nel secchio contenente una soluzione di candeggina al 10% per decontaminare DAB. Collocare un coprioggetto sopra scivolo cavità. </li><li> Sotto FITC (isotiocianato di fluorescina) fluorescenza, messa a fuoco l&#39;obiettivo del microscopio sul trapianto (cellule donnor apparirà rosso fluorescente) e di esporre a fluorescenza FITC fino a quando tutte le cellule fluorescenti rosse sono diventate marroni (questo accade a causa della DII fluorocromo essere photoconverted di cristalli insolubili marrone, con esposizione a lunghezza d&#39;onda di eccitazione FITC (488 nm) in presenza del DAB. Questo processo richiede tra 20 minuti e 2 ore. </li><li> In seguito al completamento della reazione fotoossidazione, rimuovere il coprioggetto utilizzando una pinza sottile. Il trasferimento di embrioni al vetro una fiala di scintillazione da 20 ml contenente PBTw (o PBS con 0,1% Tween-20). </li><li> Disattivare DAB da coprioggetto e far scorrere cavità di vetro tramite l&#39;incubazione in una soluzione di candeggina al 10%. </li></ol> 6: l&#39;embrione di processo di ibridazione in situ, la fotografia e sezionamento <ol><li> Procedere con ibridazione in situ (passi 3,7-6,5 9), incubando l&#39;embrione solo per DIG sonda marcata. </li><li> Procedere con fotografare (passo 8,2 9) e sezionamento (punti 8.3 e 8.4 9). </li></ol> Rappresentante Risultati Nel seguente esempio di test di induzione neurale, l&#39;innesto donnor è mostrato indurre l&#39;espressione del marcatore Scudatu neurali derivate da tessuto ospite. In questo esempio particolare, abbiamo valutato l&#39;acquisizione della capacità di indurre neurali delle regioni embrionale che normalmente non hanno &quot;organizzatore&quot; abilità: l&#39;innesto donnor proviene da un embrione in cui la regione &quot;organizzatore&quot; è stato chirurgicamente rimosso dall&#39;embrione allo stadio donnor 3 +. A seguito di ablazione, l&#39;embrione donnor è permesso di sviluppare la cultura nuova, fino a quando il foro è guarito e una struttura simile a nodo di Hensen si è formata. Questa struttura è poi etichettato con DII, escisse dall&#39;embrione donnor e innestati a zona pellucida / zona di confine opaca di un embrione di accoglienza. A seguito di nuova cultura, un asse in miniatura si forma adiacente l&#39;embrione host: questo asse in miniatura è costituito da una verga di globuli rossi (donnor derivati) e una testa come la struttura (host derivati): In (A), donnor di derivazione DII etichetta le cellule vengono mostrati prima fissazione sotto FITC microscopia a fluorescenza. Questi donnor cellule derivate hanno proliferato a formare una miniatura notocorda, che appare come verga di globuli rossi. (B) fotoconversione seguito di DII, questi donnor cellule derivate dal appaiono marrone. L&#39;innesto donnor ha indotto la formazione di tessuto neurale dall&#39;host: questo tessuto è composto da precursori per il cervello, come rivelato da loro espressione per le seguenti marcatore Scudatu ibridazione in situ [ristampato da 6]. <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="figura 2" width="230" height="154" /> Nota: In questa procedura, abbiamo usato cellule Dii etichettati al fine di differenziare il tessuto neurale inducendo donnor (rosso / marrone) da host derivato tessuto &quot;neurale&quot; (blu). Invece di usare un pulcino deriva, Dii innesto etichettati donnor, alcuni autori anche l&#39;uso dei tessuti donnor derivate da embrioni di quaglia al posto di pulcino [ad esempio 6]. Questo metodo alternativo permette inoltre di differenziare donnor dal tessuto ospite. In questo caso, l&#39;innesto (tessuto donnor) nasce da uova di quaglia al posto delle uova di gallina. Seguendo passo 4.5, l&#39;ospite viene elaborato per l&#39;immunoistochimica montare tutto con un anticorpo specifico per quaglia tessuto (QCP1) e passi 3,1-3,4 e 5,1-5,7 vengono omessi dal protocollo. Un esempio di questa procedura alternativa è la seguente: In questo caso, abbiamo utilizzato la stessa procedura sperimentale come in A, B, ma abbiamo usato quaglia derivati ​​tessuto donnor (come dimostra l&#39;espressione di anticorpi QCPN [brown]): Il rigenerato nodo derivato da embrioni di quaglia ha indotto l&#39;espressione del marcatore pan-neurale Sox2 nel paese ospitante [ristampato da 6]: <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="Figura 3" width="154" height="230" />

<ol>
	<li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Experiments+on+the+development+of+chick+and+duck+embryos,+cultivated+in+vitro.">Experiments on the development of chick and duck embryos, cultivated in vitro.</a> Phil. Trans. R. Soc. Lond. B. 221, 179-230 (1932).</li><li>Waddington, C. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Induction+by+the+primitive+streak+and+its+derivatives+in+the+chick.">Induction by the primitive streak and its derivatives in the chick.</a> J. Exp. Biol. 10, 38-46 (1933).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Excision+et+transplantation+des+diff&amp;eacute;rentes+r&amp;eacute;gions+de+la+ligne+primitive+chez+le+poulet.">Excision et transplantation des diff&amp;eacute;rentes r&amp;eacute;gions de la ligne primitive chez le poulet.</a> C. R. Ass. Anat. 49, 632-639 (1964).</li><li>Gallera, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Primary+induction+in+birds.">Primary induction in birds.</a> Adv. Morph. 9. , 149-180 (1971).</li><li>Serbedzija, G. N., Fraser, S. E., Bronner-Fraser, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathways+of+neural+crest+cell+migration+in+the+mouse+embryo+as+revealed+by+vital+dye+labeling.+Development+108.">Pathways of neural crest cell migration in the mouse embryo as revealed by vital dye labeling. Development 108.</a> , 605-612 (1990).</li><li>Psychoyos, D., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Restoration+of+the+organizer+after+radical+ablation+of+Hensen's+node+and+the+anterior+primitive+streak+in+the+chick+embryo.+Development+122.">Restoration of the organizer after radical ablation of Hensen's node and the anterior primitive streak in the chick embryo. Development 122.</a> , 3263-3273 (1996).</li><li>Joubin, K., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+interactions+continuously+define+the+organizer+during+the+cell+movements+of+gastrulation.">Molecular interactions continuously define the organizer during the cell movements of gastrulation.</a> Cell. 98, 559-571 (1999).</li><li>Streit, A., Berliner, A. J., Papanayotou, C., Sirulnik, A., Stern, C. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Initiation+of+neural+induction+by+FGF+signaling+before+gastrulation.">Initiation of neural induction by FGF signaling before gastrulation.</a> Nature. , 406-474 (2000).</li><li>Psychoyos, D., Finnell, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+Culture+of+Early+Chick+Embryos+ex+vivo+(New+Culture.">Method for Culture of Early Chick Embryos ex vivo (New Culture.</a> JoVE. 20, 10-3791 (2008).</li></ol>

Questo video mostra le varie fasi di esecuzione di un test per l&#39;induzione neurale; Questo test è essenzialmente utilizzato per la caratterizzazione di molecole che inducono putativo neurali in pulcino, e quindi possono essere utilizzati per una vasta gamma di applicazioni, che vanno dalla micromanipolazioni embriologica 1-4; da 6 a svelare nuove cascate di segnalazione 7,8, tutti volti alla comprensione del primo passo nella formazione del cervello e del restante sistema nervoso.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Eggs</td>
<td>Animal</td>
<td>Charles River Laboratories</td>
<td>Premium Fertile</td>
<td>Fertilized, HH3+ (14 hr)</td>
</tr>
<tr>
<td>Stereomicroscope</td>
<td>Microscope</td>
<td>Leica Microsystems</td>
<td>MZ9.5 or similar</td>
<td>&nbsp;</td>
<tr>
<td>Hybridization Incubator</td>
<td>Equipment</td>
<td>Robbins Scientific</td>
<td>M1000</td>
<td>Use with inverted Pyrex dish and 500 ml ddH2O beaker</td>
</tr>
<tr>
<td>Marsh Automatic Incubator</td>
<td>Equipment</td>
<td>Lyon</td>
<td>RX</td>
<td>&nbsp;</td>
<tr>
<td>Pyrex dish </td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Watchmaker's glass 50mm</td>
<td>Tool</td>
<td>VWR</td>
<td>66112-060</td>
<td>&nbsp;</td>
<tr>
<td>Glass rings</td>
<td>Tool</td>
<td>Physical Plant facility</td>
<td>&nbsp;</td>
<td>cut 4 mm thick sections of glass tubing (27 mm outer diam, 25 mm inner diam). Do not fine polish.</td>
</tr>
<tr>
<td>Curved Forceps (1)</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>72991-4C</td>
<td>&nbsp;</td>
<tr>
<td>Forceps (2)</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>11002-13</td>
<td>blunt ended using sharpening Stone and 100ul mineral oil</td>
</tr>
<tr>
<td>Fine scissors</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>14161-10</td>
<td>&nbsp;</td>
<tr>
<td>Plastic dishes</td>
<td>Tool</td>
<td>Falcon</td>
<td>353001</td>
<td>&nbsp;</td>
<tr>
<td>Rubber Bulb</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70980</td>
<td>&nbsp;</td>
<tr>
<td>DiI</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>D-282</td>
<td>&nbsp;</td>
<tr>
<td>Aspirator tube assembly</td>
<td>Tool</td>
<td>Sigma</td>
<td>A5177-5EA</td>
<td>&nbsp;</td>
<tr>
<td>Microelectrode puller</td>
<td>Equipment</td>
<td>Sutter Instruments</td>
<td>Sutter Instruments	P-97 Flaming/Brown Micropipette</td>
<td>&nbsp;</td>
<tr>
<td>Pasteur Capillary Pipette</td>
<td>Tool</td>
<td>Electron Microscopy Sciences</td>
<td>70950-12</td>
<td>round edge under flame</td>
</tr>
<tr>
<td>Culture chamber</td>
<td>Tool</td>
<td>Pioneer Plastics</td>
<td>030C</td>
<td>&nbsp;</td>
<tr>
<td>Microcapillary tube</td>
<td>Tool</td>
<td>Sigma</td>
<td>P1049-1PAK</td>
<td>&nbsp;</td>
<tr>
<td>Microdissecting knife</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>10056-12</td>
<td>Use to puncture cavities prior to in situ hybridization</td>
</tr>
<tr>
<td>Minuten pins 0.2mm diam</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26002-20</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>Reagent</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>
<tr>
<td>Sylgard 184 Silicon Elastomer Curing Agent and Base</td>
<td>&nbsp;</td>
<td>Dow Corning</td>
<td>0001986475</td>
<td>Mix 1 part Curing Agent, 9 parts Base; set O/N at 37C</td>
</tr>
<tr>
<td>Diethylpyrocarbonate (depc)</td>
<td>&nbsp;</td>
<td>Acros Organics</td>
<td>10025025</td>
<td>Add 1ml depc to 1l PBS; shake; autoclave</td>
</tr>
<tr>
<td>16% PFA</td>
<td>&nbsp;</td>
<td>Electron Microscopy Sciences</td>
<td>15710</td>
<td>&nbsp;</td>		</tbody>
		</table>
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assay for neural induction in the chick embryo

L&#39;embrione di pollo è uno strumento prezioso per lo studio dello sviluppo embrionale precoce. La sua trasparenza, accessibilità e facilità di manipolazione, lo rendono uno strumento ideale per studiare la formazione ed il modello iniziale del sistema nervoso. Questo video mostra come tessuto organizzatore innesto in un ospite, un metodo attraverso il quale nodo di Hensen s (l&#39;organizzatore in embrione di pollo) si innesta in un ectoderma competente del paese ospitante. L&#39;innesto organizzatore istruisce sovrastante na ve tessuto di adottare un destino neurale inducendo tramite segnali neurali. Questo meccanismo viene definito come l&#39;induzione neurale, e costituisce il primo passo nella formazione del cervello e del midollo spinale in amnioti. Questo metodo è utilizzato essenzialmente per la caratterizzazione di molecole putativo neurale inducendo in pulcino. Questo video mostra le varie fasi del test per l&#39;induzione neurale; In primo luogo, l&#39;embrione è donnor espiantati e appuntato su un piatto. Poi, l&#39;embrione host è pronto per la cultura Nuovo. L&#39;innesto viene asportato e trapiantato nella zona pellucida margine ospite. L&#39;host è in coltura per 18-22 ore. L&#39;assemblea è fisso e trattati per le applicazioni più (ad esempio di ibridazione in situ). Questo metodo è stato originariamente ideato da Waddington<sup&gt; 1,2</sup&gt; E Gallera<sup&gt; 3,4</sup&gt;.

Watch this Scientific Journal Video about Assay for Neural Induction in the Chick Embryo at JoVE.com

Assay for Neural Induction in the Chick Embryo

Induzione neurale è il primo passo nella formazione del cervello. Si tratta di un meccanismo attraverso il quale nodo di Hensen (organizzatore), istruisce tessuti adiacenti ad adottare un destino neurale, cioè di dare origine al sistema nervoso. Questo video dimostra un test per l&#39;induzione neurale in embrione di pollo.

Test di induzione neurale nell&#39;embrione Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ...

assay-for-neural-induction-in-the-chick-embryo

Nanyang Technological University

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JoVE Journal

Biology

17.1K Views.  Texas A&M University (TAMU). Induzione neurale è il primo passo nella formazione del cervello. Si tratta di un meccanismo attraverso il quale nodo di Hensen (organizzatore), istruisce tessuti adiacenti ad adottare un destino neurale, cioè di dare origine al sistema nervoso. Questo video dimostra un test per l'induzione neurale in embrione di pollo.

Video: Assay for Neural Induction in the Chick Embryo

Method for Whole Mount Antibody Staining in Chick

The chick embryo is a valuable tool in the study of early embryonic development. Its transparency, accessibility and ease of manipulation, make it an ideal tool for studying antibody expression in developing brain, neural tube and somite. This video demonstrates the different steps in whole-mount antibody staining using HRP conjugated secondary antibodies; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, endogenous peroxidase is inactivated; The embryo is then exposed to primary antibody. After several washes, the embryo is incubated with secondary antibody conjugated to HRP. Peroxidase activity is revealed using reaction with diaminobenzidine substrate. Finally, the embryo is fixed and processed for photography and sectioning. The advantage of this method over the use of fluorescent antibodies is that embryos can be processed for wax sectioning, thus enabling the study of antigen sites in cross section. This method was originally introduced by Jane Dodd and Tom Jessell 1.

This video demonstrates whole mount immunohistochemistry, a method by which the spatial and temporal expression pattern of an antigen can be visualized in young chick embryos. This method was originally introduced by Jane Dodd and Tom Jessell.

Metodo per la colorazione intero anticorpi Monte a Chick

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Biologia

Electrophysiological Recording in the Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite isolated from each other, with largely independent histories and scientific communities. However, the interface between these usually disparate fields is the developmental programs underlying acquisition of functional electrical signaling properties and differentiation of functional chemical synapses during the final phases of neural circuit formation. This interface is a critically important area for investigation. In Drosophila, these phases of functional development occur during a period of &lt;8 hours (at 25&deg;C) during the last third of embryogenesis. This late developmental period was long considered intractable to investigation owing to the deposition of a tough, impermeable epidermal cuticle. A breakthrough advance was the application of water-polymerizing surgical glue that can be locally applied to the cuticle to enable controlled dissection of late-stage embryos. With a dorsal longitudinal incision, the embryo can be laid flat, exposing the ventral nerve cord and body wall musculature to experimental investigation. Whole-cell patch-clamp techniques can then be employed to record from individually-identifiable neurons and somatic muscles. These recording configurations have been used to track the appearance and maturation of ionic currents and action potential propagation in both neurons and muscles. Genetic mutants affecting these electrical properties have been characterized to reveal the molecular composition of ion channels and associated signaling complexes, and to begin exploration of the molecular mechanisms of functional differentiation. A particular focus has been the assembly of synaptic connections, both in the central nervous system and periphery. The glutamatergic neuromuscular junction (NMJ) is most accessible to a combination of optical imaging and electrophysiological recording. A glass suction electrode is used to stimulate the peripheral nerve, with excitatory junction current (EJC) recordings made in the voltage-clamped muscle. This recording configuration has been used to chart the functional differentiation of the synapse, and track the appearance and maturation of presynaptic glutamate release properties. In addition, postsynaptic properties can be assayed independently via iontophoretic or pressure application of glutamate directly to the muscle surface, to measure the appearance and maturation of the glutamate receptor fields. Thus, both pre- and postsynaptic elements can be monitored separately or in combination during embryonic synaptogenesis. This system has been heavily used to isolate and characterize genetic mutants that impair embryonic synapse formation, and thus reveal the molecular mechanisms governing the specification and differentiation of synapse connections and functional synaptic signaling properties.

Electrophysiological Recording in the Drosophila Embryo

Electrophysiological recordings from Drosophila embryos allow analyses of developing muscle and neuron electrical properties, as well as characterization of functional synaptogenesis at the glutamatergic neuromuscular junction and central cholinergic and GABAergic synapses.

Registrazione elettrofisiologiche in Drosophila Embryo

Drosophila is a premier genetic model for the study of both embryonic development and functional neuroscience. Traditionally, these fields are quite ...

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of the Drosophila melanogaster syncytial embryo for studying mitosis1. Drosophila has useful genetics with a sequenced genome, and it can be easily maintained and manipulated. Many mitotic mutants exist, and transgenic flies expressing functional fluorescently (e.g. GFP) - tagged mitotic proteins have been and are being generated. Targeted gene expression is possible using the GAL4/UAS system2. 

The Drosophila early embryo carries out multiple mitoses very rapidly (cell cycle duration, &asymp;10 min). It is well suited for imaging mitosis, because during cycles 10-13, nuclei divide rapidly and synchronously without intervening cytokinesis at the surface of the embryo in a single monolayer just underneath the cortex. These rapidly dividing nuclei probably use the same mitotic machinery as other cells, but they are optimized for speed; the checkpoint is generally believed to not be stringent, allowing the study of mitotic proteins whose absence would cause cell cycle arrest in cells with a strong checkpoint. Embryos expressing GFP labeled proteins or microinjected with fluorescently labeled proteins can be easily imaged to follow live dynamics (Fig. 1). In addition, embryos can be microinjected with function-blocking antibodies or inhibitors of specific proteins to study the effect of the loss or perturbation of their function3. These reagents can diffuse throughout the embryo, reaching many spindles to produce a gradient of concentration of inhibitor, which in turn results in a gradient of defects comparable to an allelic series of mutants. Ideally, if the target protein is fluorescently labeled, the gradient of inhibition can be directly visualized4. It is assumed that the strongest phenotype is comparable to the null phenotype, although it is hard to formally exclude the possibility that the antibodies may have dominant effects in rare instances, so rigorous controls and cautious interpretation must be applied. Further away from the injection site, protein function is only partially lost allowing other functions of the target protein to become evident.

Microinjection Techniques for Studying Mitosis in the Drosophila melanogaster Syncytial Embryo

This protocol describes the use of microinjection and high resolution imaging in the Drosophila melanogaster syncytial embryo to study mitosis.

Tecniche di microiniezione per Studiare Mitosi in Drosophila melanogaster Embryo sinciziale

This protocol describes the use of the Drosophila melanogaster syncytial embryo  for studying mitosis1.  Drosophila has useful genetics with a sequenced ...

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study angiogenesis but also to study tumorigenesis. After the chick chorioallantoic membrane (CAM) has developed, its blood vessel network can be easily accessed, manipulated and observed and therefore provides an optimal setting for angiogenesis assays. Since the lymphoid system is not fully developed until late stages of incubation, the chick embryo serves as a naturally immunodeficient host capable of sustaining grafted tissues and cells without species-specific restrictions. In addition to nurturing developing allo- and xenografts, the CAM blood vessel network provides a uniquely supportive environment for tumor cell intravasation, dissemination, and vascular arrest and a repository where arrested cells extravasate to form micro metastatic foci.
For experimental purposes, in most of the recent studies the CAM was exposed by cutting a window through the egg shell and experiments were carried out in ovo, resulting in significant limitations in the accessibility of the CAM and possibilities for observation and photo documentation of effects. When shell-less cultures of the chick embryo were used1-4, no experimental details were provided and, if published at all, the survival rates of these cultures were low. We refined the method of ex ovo culture of chick embryos significantly by introducing a rationally controlled extrusion of the egg content. These ex ovo cultures enhance the accessibility of the CAM and chick embryo, enabling easy in vivo documentation of effects and facilitating experimental manipulation of the embryo. This allows the successful application to a large number of scientific questions: (1) As an improved angiogenesis assay5,6, (2) an experimental set up for facilitated injections in the vitreous of the chick embryo eye7-9, (3) as a test environment for dissemination and intravasation of dispersed tumor cells from established cell lines inoculated on the CAM10-12, (4) as an improved sustaining system for successful transplantation and culture of limb buds of chicken and mice13 as well as (5) for grafting, propagation, and re-grafting of solid primary tumor tissue obtained from biopsies on the surface of the CAM14.
In this video article we describe the establishment of a refined chick ex ovo culture and CAM assay with survival rates over 50%. Besides we provide a step by step demonstration of the successful application of the ex ovo culture for a large number of scientific applications. 
Daniel S. Dohle, Susanne D. Pasa, and Sebastian Gustmann contributed equally to this study.

Chick ex ovo Culture and ex ovo CAM Assay: How it Really Works

The chick chorioallantoic membrane (CAM) is a unique, naturally immunodeficient supportive culture environment to study angiogenesis and tumorigenesis. This video article demonstrates the different steps in chick ex ovo culture, application of potentially angiogenic substances and successful inoculation of tumor cells and tissues on the surface of the CAM.

Pulcino Ex ovo Cultura e Ex ovo CAM saggio: Come funziona veramente

Chicken eggs in the early phase of breeding are between in vitro and in vivo systems and provide a vascular test environment not only to study ...

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking axonal projection of spinal interneurons in vertebrates, germ line-targeted reporter genes yield bilaterally symmetric labeling. Therefore, it is hard to distinguish between the ipsi- and contra-laterally projecting axons. Unilateral electroporation into the chick neural tube provides a useful means to restrict expression of a reporter gene to one side of the central nervous system, and to follow axonal projection on both sides 1 ,2-5. This video demonstrates first how to handle the eggs prior to injection. At HH stage 18-20, DNA is injected into the sacral level of the neural tube, then tungsten electrodes are placed parallel to the embryo and short electrical pulses are administered with a pulse generator. The egg is sealed with tape and placed back into an incubator for further development. Three days later (E6) the spinal cord is removed as an open book preparation from embryo, fixed, and processed for whole mount antibody staining. The stained spinal cord is mounted on slide and visualized using confocal microscopy.

Deciphering Axonal Pathways of Genetically Defined Groups of Neurons in the Chick Neural Tube Utilizing in ovo Electroporation

This video demonstrates how to visualize axonal pathways of genetically defined groups of neurons in the embryonic chick spinal cord utilizing in ovo electroporation of reporter genes under the control of specific enhancer elements.

Decifrare Percorsi assonale di gruppi geneticamente definito di neuroni nel tubo neurale Chick Utilizzando In ovo Elettroporazione

Employment of enhancer elements to drive expression of reporter genes in neurons is a widely used paradigm for tracking axonal projection. For tracking ...

Xenotransplantation of Human Stem Cells into the Chicken Embryo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the behavior of cells in a standardized in vivo environment. The main advantages of the chicken embryo include low cost, high accessibility, ease of surgical manipulation and lack of a fully developed immune system. Xenotransplantation into chicken embryos can provide valuable information about cell proliferation, differentiation and behavior, the responses of cells to signals in defined embryonic tissue niches, and tumorigenic potential. Transplanting cells into chicken embryos can also be a step towards transplantation experiments in other animal models. Recently the chicken embryo has been used to evaluate the neurogenic potential of human stem and progenitor cells following implantation into neural anlage1-6. In this video we document the entire procedure for transplanting human stem cells into the developing central nervous system of the chicken embryo. The procedure starts with incubation of fertilized eggs until embryos of the desired age have developed. The eggshell is then opened, and the embryo contrasted by injecting dye between the embryo and the yolk. Small lesions are made in the neural tube using microsurgery, creating a regenerative site for cell deposition that promotes subsequent integration into the host tissue. We demonstrate injections of human stem cells into such lesions made in the part of the neural tube that forms the hindbrain and the spinal cord, and into the lumen of the part of the neural tube that forms the brain. Systemic injections into extraembryonic veins and arteries are also demonstrated as an alternative way to deliver cells to vascularized tissues including the central nervous system. Finally we show how to remove the embryo from the egg after several days of further development and how to dissect the spinal cord free for subsequent physiological, histological or biochemical analyses.

In this paper we present a method for transplanting human stem cells into various regions of the central nervous system of the chicken embryo. This provides an in vivo model for assessing the proliferation and differentiation of various types of human stem cells in embryonic tissue environments.

Lo xenotrapianto di cellule staminali umane in embrioni di pollo

The chicken embryo is a classical animal model for studying normal embryonic and fetal development and for xenotransplantation experiments to study the ...

Clonogenic Assay: Adherent Cells

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. Apart from documenting the method, the initial landmark study generated the first radiation-dose response curve for X-ray irradiated mammalian (HeLa) cells in culture1. Basically, the clonogenic assay enables an assessment of the differences in reproductive viability (capacity of cells to produce progeny; i.e. a single cell to form a colony of 50 or more cells) between control untreated cells and cells that have undergone various treatments such as exposure to ionising radiation, various chemical compounds (e.g. cytotoxic agents) or in other cases genetic manipulation. The assay has become the most widely accepted technique in radiation biology and has been widely used for evaluating the radiation sensitivity of different cell lines. Further, the clonogenic assay is commonly used for monitoring the efficacy of radiation modifying compounds and for determining the effects of cytotoxic agents and other anti-cancer therapeutics on colony forming ability, in different cell lines. A typical clonogenic survival experiment using adherent cells lines involves three distinct components, 1) treatment of the cell monolayer in tissue culture flasks, 2) preparation of single cell suspensions and plating an appropriate number of cells in petri dishes and 3) fixing and staining colonies following a relevant incubation period, which could range from 1-3 weeks, depending on the cell line. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cell lines with the use of an immortalized human keratinocyte cell line (FEP-1811)2. Also, our aims are to describe common features of clonogenic assays including calculation of the plating efficiency and survival fractions after exposure of cells to radiation, and to exemplify modification of radiation-response with the use of a natural antioxidant formulation.

The applicability of the clonogenic assay for evaluating reproductive viability has been established for more than 50 years. Here we demonstrate the general procedure for performing the clonogenic assay with adherent cells.

Clonogenica saggio: cellule aderenti

The clonogenic (or colony forming) assay has been established for more than 50 years; the original paper describing the technique was published in 19561. ...

Modified Mouse Embryonic Stem Cell based Assay for Quantifying  Cardiogenic Induction Efficiency

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways. One of the hurdles to its understanding has been the varied methods in correlating changes of key signaling events to differentiation efficiency. We describe here the use of a mouse embryonic stem (ES) cell based assay to identify critical time windows for Wnt/&beta;-catenin and BMP signal activation during cardiogenic induction. By scoring for contracting embryonic bodies (EBs) in a 96-well plate format, we can quickly quantify cardiogenic efficiency and identify crucial time windows for Wnt/&beta;-catenin and BMP signal activation in a time course following specific modulator treatments. The principal outlined here is not limited to cardiac induction alone, and can be applied towards the study of many other cell lineages. In addition, the 96-well format has the potential to be further developed as a high throughput, automated assay to allow for the testing of more sophisticated experimental hypotheses.

We describe the use of a mouse ES cell based assay to identify critical time windows for Wnt/&#946;-catenin and BMP signal activation during cardiogenic induction. The method provides a standardized platform that reliably quantifies cardiogenic efficiency, and it is applicable to the study of other cell lineages.

Modificato il mouse sulle cellule staminali Saggio base per quantificare l&#39;efficienza di induzione cardiogeno

Differentiation of pluripotent stem cells is tightly controlled by temporal and spatial regulation of multiple key signaling pathways.  One of the hurdles ...

Tracking Morphogenetic Tissue Deformations in the Early Chick Embryo

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. Tracking fiducial markers on the surfaces of these cellular sheets is a well-established method for estimating morphogenetic quantities such as growth, contraction, and shear. However, not all surface labeling techniques are readily adaptable to conventional imaging modalities and possess different advantages and limitations. Here, we describe two labeling methods and illustrate the utility of each technique. In the first method, hundreds of fluorescent labels are applied simultaneously to the embryo using magnetic iron particles. These labels are then used to quantity 2-D tissue deformations during morphogenesis. In the second method, polystyrene microspheres are used as contrast agents in non-invasive optical coherence tomography (OCT) imaging to track 3-D tissue deformations. These techniques have been successfully implemented in our lab to studythe physical mechanisms of early head fold, heart, and brain development, and should be adaptable to a wide range morphogenetic processes.

This article describes surface labeling and ex ovo tissue culture in the early chick embryo. Techniques amenable to time-lapse bright field, fluorescence, and optical coherence tomography imaging are presented. Tracking surface labels with high spatiotemporal resolution enables kinematic quantities such as morphogenetic strains (deformations) to be calculated in both two and three dimensions.

Monitoraggio Deformazioni tessuto morfogenetica in embrione di pollo primi

Embryonic epithelia undergo complex deformations (e.g. bending, twisting, folding, and stretching) to form the primitive organs of the early embryo. ...

Mouse Embryonic Development in a Serum-free Whole Embryo Culture System

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in an oxygenated rolling bottle culture system. Mouse embryos at E10.5 were carefully isolated from the uterus with intact yolk sac and in a process involving precise surgical maneuver the embryos were gently exteriorized from the yolk sac while maintaining the vascular continuity of the embryo with the yolk sac. Compared to embryos prepared with intact yolk sac or with the yolk sac removed, these embryos exhibited superior survival rate and developmental progression when cultured under similar conditions. We show that these mouse embryos, when cultured in a defined medium in an atmosphere of 95% O2 / 5% CO2 in a rolling bottle culture apparatus at 37 &#176;&#8203;C for 16-40 hr, exhibit morphological growth and development comparable to the embryos developing in utero. We believe this method will be useful for investigators needing to utilize whole embryo culture to study signaling interactions important in embryonic organogenesis.

Serum utilized in embryo cultures contains unknown components that can affect the outcome of experiments especially in studies involving signaling interactions. Here we utilized a serum-free oxygenated culture system and show that mid-gestation mouse embryos cultured for 16-40 hr&#160;exhibit morphological development comparable to embryos developing in utero.

Embrionali di topo di sviluppo in un embrione di tutta la cultura di sistema privo di siero

Mid-gestation stage mouse embryos were cultured utilizing a serum-free culture medium prepared from commercially available stem cell media supplements in ...