DP est récipiendaire du Prix Ruth Kirschstein 1F32-01A1 DA021977 du National Institute on Drug Abuse. Ce travail a été soutenu par la Fondation Margaret M. Alkek au RHF.

<html> I. Schéma Aperçu: <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="Figure 1" width="460" height="384" /> Cette vidéo montre les différentes étapes de test pour l&#39;induction neurale dans l&#39;embryon de poulet. Tout d&#39;abord, l&#39;embryon hôte est explanté dans la culture Nouveaux [NI1]. Ensuite, l&#39;embryon n&#39;est Donnor explantés dans une solution saline et le nœud de Hensen (l &#39;«organisateur» dans chiches) est étiqueté avec fluorescentes DiI colorant [NI2]. Nœud de Hensen est excisé de l&#39;embryon Donnor [NI3] et transplantés dans l&#39;embryon hôte [NI4, NI5]. L&#39;embryon hôte est ensuite mis en culture pendant 22 heures, période après laquelle le tissu Donnor a induit un axe ectopique dans la zone d&#39;accueil opaca [NI6]. Après la culture, l&#39;embryon d&#39;accueil est fixe et traitées pour photooxydation en présence du DAB: Par cette réaction chimique, les cellules dérivées Donnor, qui apparaissent en rouge sous la fluorescence FITC avant photooxydation [NI7], deviennent bruns réaction suivante avec DAB [NI8]. L&#39;embryon est ensuite traitée par hybridation in situ avec un marqueur neuronal [NI8]; tissu hôte dérivés a formé le tissu neural, en présence de la greffe Donnor. Ce tissu apparaît en bleu. 1: Préparation de l&#39;embryon hôte pour la culture néo- <ol><li> Ce protocole induction neurale test commence avec des oeufs qui ont été incubés à la Hamburger &amp; Hamilton étape 3 + (ou HH3 +). Ces œufs ont été incubés mis de leur côté dans un incubateur humide pendant 13 heures. </li><li> Préparer l&#39;embryon hôte pour nouvelle culture (étapes 3.1. À 5,5 9). </li><li> Couvrez la surface de l&#39;embryon hôte avec 200 ul salines. Cela facilitera le transfert tard et le positionnement de la greffe Donnor. </li><li> Enfin, la couverture de l&#39;assemblée avec un Falcon 35 inversé mm boîte de culture. </li></ol> 2: explantation de l&#39;embryon dans une solution saline Donnor <ol><li> Ouvrez l&#39;œuf en tapant sur la coquille avec une pince et enlever des morceaux de la coquille, tout autour de la coquille. Retirer haut de coquille et jetez-les. </li><li> Retirez l&#39;albumine épaisse avec une pince, et de l&#39;inclinaison du sac vitellin avec des pinces grossières pour que l&#39;embryon tournée vers le haut. </li><li> Avec des ciseaux fins, découpez un carré de vitellus autour de l&#39;embryon. Retirez l&#39;embryon du jaune avec une cuillère, et les placer dans un plat contenant du PBS. </li><li> En utilisant des pinces, détacher l&#39;embryon de la zone pellucide Donnor. </li><li> Transfert Donnor embryon à un plat recouvert Sylgard contenant du PBS. </li></ol> 3: L&#39;étiquetage, l&#39;excision et la transplantation de la greffe Donnor <ol><li> Utiliser un extracteur de microélectrodes, tirer 50 pipettes en verre ul microcapillaire. Sous le microscope, couper la pointe de la micropipette l&#39;aide de pinces fines. Micropipette Placer dans un plat de Pétri bordée d&#39;un ruban de plastiline. </li><li> Préparer la solution de teinture qui sera utilisé pour étiqueter le greffon avant la transplantation: carbocyanine colorant DII (1,1 &#39;-dioctadécyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine perchlorate): Premièrement, préparer un stock de DiI de 1ml l&#39;éthanol absolu à 0,5%. Ce stock peut être conservé à-20C dans le noir. Dans un bain-marie réglé à 42 ° C, le mélange préchauffé 40 ul d&#39;actions DiI à 360μl 0.3M sucrose. La température du bain d&#39;eau est nécessaire afin d&#39;éviter la précipitation du DII et la formation de cristaux insolubles. Centrifuger brièvement la solution sur un Spectrafuge mini (pour les 5-10 secondes). La solution Dii est maintenant prêt à utiliser. </li><li> Utilisant des broches d&#39;insectes, sécuriser l&#39;embryon Donnor sur le fond du plat Sylgard couverts. </li><li> Retour-remplir la micropipette avec DII. En appliquant une légère pression en utilisant un tube aspirateur, d&#39;appliquer un petit bolus d&#39;DiI au nœud de Hensen. Assurez-vous que le nœud de Hensen ensemble est étiqueté. </li><li> En utilisant une pipette ou un couteau microcapillaire microdissecting, couper le nœud de Hensen («organisateur», greffe Donnor). </li><li> Marquer la face ventrale et l&#39;extrémité postérieure de la greffe avec du carmin. Cela permettra d&#39;assurer que la greffe est positionnée avec bonne orientation dorso-ventrale à l&#39;étape 4.3. </li><li> En utilisant une pipette 200 pl Gilson, le transfert de la greffe à l&#39;embryon hôte. </li></ol> 4: Sécurisation de la greffe et la culture de l&#39;embryon hôte <ol><li> Retirer la solution saline de l&#39;embryon hôte, en veillant à la greffe reste positionné à proximité avec le site hôte. </li><li> En utilisant une pipette ou un couteau microcapillaire microdissecting, faire une petite incision (80-100 microns) dans la zone pellucide / aire région frontière opaca, au niveau du nœud de Hensen de l&#39;embryon hôte. </li><li> En utilisant une pipette ou un couteau microcapillaire microdissecting, la position de la greffe de telle sorte que la face ventrale dirigée vers vous et l&#39;extrémité postérieure est parallèle à la région équivalent dans l&#39;embryon hôte. </li><li> Retirer salines restants, et le processus de l&#39;embryon hôte pour nouvelle culture de 81 à 22 h (étapes 6.1 à 6.6 9). </li><li> L&#39;embryon hôte est alors fixé une nuit à 4 ° C (étapes 7.1 à 7.7 9). </li></ol>e &quot;&gt; 5: Photooxidise la greffe en fluorescence <ol><li> Sous une hotte, préparer une solution DAB travaillant dans un tampon Tris: Dissoudre substrat DAB (3,3 &#39;-diaminobenzidine tétrachlorhydrate) dans un tampon Tris (Tris-HCl 100 mM, pH 7,4) à 500μg/ml; garder solution dans l&#39;obscurité, sur la glace. </li><li> Laver l&#39;embryon à deux reprises dans du PBS pendant 5 mn chacun et une fois dans un tampon Tris pendant 5 mn. </li><li> Transfert de l&#39;embryon à une diapositive cavité en verre contenant 1 ml de tampon Tris. </li><li> Remplacer la solution tampon Tris avec 1,5 ml DAB solution. Eliminer Eppendorf pointe dans un seau contenant une solution javellisée à 10% pour décontaminer DAB. Placer une lamelle sur le dessus de la diapositive cavité. </li><li> Sous FITC (isothiocyanate de fluorescéine) de fluorescence, concentrer l&#39;objectif du microscope sur le greffon (cellules Donnor sera apparaissent en rouge fluorescent) et d&#39;exposer à la fluorescence FITC jusqu&#39;à ce que toutes les cellules fluorescentes rouges sont devenues brunes (ce qui arrive comme un résultat de DII fluorochrome étant photoconverted d&#39;insolubles cristaux bruns, par une exposition à onde d&#39;excitation FITC (488nm), en présence de DAB. Ce processus prend entre 20 mn et 2 heures. </li><li> Après l&#39;achèvement de la réaction de photooxydation, enlever la lamelle à l&#39;aide de pinces fines. Transfert embryon à un flacon à scintillation en verre de 20 ml contenant PBTw (ou PBS avec 0,1% de Tween-20). </li><li> Désactiver DAB à partir lamelle de verre et faites glisser la cavité par une incubation dans une solution javellisée à 10%. </li></ol> 6: Processus de l&#39;embryon pour l&#39;hybridation, la photographie in situ et de sectionnement <ol><li> Procéder à l&#39;hybridation in situ (étapes 03.07 à 06.05 9), l&#39;incubation de l&#39;embryon pour seulement DIG sonde marquée. </li><li> Procéder à photographier (étape 8.2 9) et de sectionnement (étapes 8.3 et 8.4 9). </li></ol> Les résultats représentatifs Dans l&#39;exemple suivant de dosage induction neurale, la greffe est indiquée Donnor induire l&#39;expression de l&#39;Anourie marqueur de neurones à partir de tissus de l&#39;hôte dérivés. Dans cet exemple particulier, nous avons évalué l&#39;acquisition de la capacité de provoquer neurones des régions embryonnaires qui normalement n&#39;ont pas «organisateur» des capacités: la greffe Donnor provient d&#39;un embryon dans lequel l &#39;«organisateur» région a été enlevée chirurgicalement de l&#39;embryon au stade Donnor 3 +. Après l&#39;ablation, l&#39;embryon Donnor on laisse se développer dans la nouvelle culture, jusqu&#39;à ce que le trou a guéri et une structure ressemblant à nœud de Hensen a formé. Cette structure est ensuite étiqueté avec DII, excisés de l&#39;embryon Donnor et greffé à la zone pellucide / zone de la frontière opaca d&#39;un embryon hôte. Suite à la nouvelle culture, un axe miniature est formé à côté de l&#39;embryon hôte: cet axe miniatures se compose d&#39;une tige de globules rouges (Donnor dérivés) et une structure de tête de type (hôte dérivés): Dans (A), Donnor dérivés DiI étiquetés cellules sont présentés avant la fixation sous microscopie à fluorescence FITC. Ces cellules Donnor dérivés ont proliféré pour former une miniature notochorde, qui apparaît comme la tige de globules rouges. (B) photoconversion suite de DII, ces cellules dérivées Donnor apparaissent brun. La greffe a Donnor induit la formation de tissu neural de l&#39;hôte: ce tissu est constitué de précurseurs pour le cerveau, tel que révélé par leur expression dans les cas suivants marqueur Anourie hybridation in situ [tiré à part de 6]. <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="Figure 2" width="230" height="154" /> Remarque: Dans cette procédure, nous avons utilisé des cellules DiI étiquetés afin de différencier les tissus neuraux induisant Donnor (rouge / marron) de l&#39;hôte dérivés &quot;neurones&quot; tissu (bleu). Au lieu d&#39;utiliser un poussin dérivés, Dii greffons étiquetés Donnor, certains auteurs utilisent aussi des tissus provenant d&#39;embryons Donnor cailles au lieu d&#39;chiches [par exemple, 6]. Cette méthode alternative permet également de différencier Donnor du tissu hôte. Dans ce cas, la greffe (tissu Donnor) provient d&#39;oeufs de caille au lieu d&#39;œufs de poule. Après l&#39;étape 4.5, l&#39;hôte est transformé pour l&#39;immunohistochimie monter l&#39;ensemble avec un anticorps spécifique à la caille des tissus (QCP1) et les étapes 3.1 à 3.4 et 5.1 à 5.7 sont omises de ce protocole. Un exemple de cette procédure alternative est indiqué ci-dessous: Dans ce cas, nous avons utilisé la même procédure expérimentale que dans A, B, mais nous avons utilisé la caille dérivées des tissus Donnor (comme indiqué par l&#39;expression d&#39;anticorps QCPN [brun]): Le régénérée noeud provenant d&#39;embryons de caille a induit l&#39;expression de marqueurs pan-neuronal Sox2 dans l&#39;hôte [tiré à part de 6]: <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="Figure 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>

Cette vidéo montre les différentes étapes à effectuer un essai pour l&#39;induction neurale; Ce dosage est essentiellement utilisé pour la caractérisation de molécules induisant putatifs neurale chez le poulet, et peut donc être utilisé pour une large variété d&#39;applications, allant de micromanipulations embryologique 1-4; 6 à démêler nouvelles cascades de signalisation 7,8, toutes visant à la compréhension de l&#39;étape initiale de la formation du cerveau et du reste du systè...

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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>
		</div>

assay for neural induction in the chick embryo

L&#39;embryon de poulet est un outil précieux dans l&#39;étude du développement embryonnaire précoce. Sa transparence, l&#39;accessibilité et la facilité de manipulation, en font un outil idéal pour étudier la formation et la structuration initiale du système nerveux. Cette vidéo montre comment tissu organisateur du greffon dans un hôte, une méthode par laquelle le nœud de Hensen s (l&#39;organisateur de l&#39;embryon de poulet) est greffé à un ectoderme compétente d&#39;accueil. La greffe organisateur instruit recouvrant na avez tissus d&#39;adopter un sort via des signaux neuronaux induisant neurones. Ce mécanisme est appelé l&#39;induction neurale, et constitue la première étape dans la formation du cerveau et la moelle épinière chez les amniotes. Cette méthode est essentiellement utilisée pour la caractérisation des molécules induisant putatifs neurale chez le poulet. Cette vidéo montre les différentes étapes de l&#39;essai pour l&#39;induction neurale; d&#39;abord, l&#39;embryon n&#39;est Donnor explantés et épinglé sur un plat. Ensuite, l&#39;embryon d&#39;accueil est préparé pour une nouvelle culture. Le greffon est excisée et transplanté à la marge d&#39;hôte zone pellucide. L&#39;hôte est cultivée pendant 18 à 22 heures. L&#39;ensemble est fixé et traitées pour d&#39;autres applications (par exemple, hybridation in situ). Cette méthode a été initialement conçu par Waddington<sup&gt; 1,2</sup&gt; Et 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

L&#39;induction neurale est la première étape dans la formation du cerveau. C&#39;est un mécanisme par lequel le nœud de Hensen (organisateur), instruit les tissus adjacents à adopter un destin neuronal, c&#39;est à dire de donner naissance au système nerveux. Cette vidéo montre un essai pour l&#39;induction neurale dans l&#39;embryon de poulet.

Dosage de l&#39;induction neurale dans l&#39;embryon de poulet

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

Research

JoVE Journal

Biology

17.1K vues.  Texas A&M University (TAMU). L'induction neurale est la première étape dans la formation du cerveau. C'est un mécanisme par lequel le nœud de Hensen (organisateur), instruit les tissus adjacents à adopter un destin neuronal, c'est à dire de donner naissance au système nerveux. Cette vidéo montre un essai pour l'induction neurale dans l'embryon de poulet.

Vidéo: Assay for Neural Induction in the Chick Embryo

Double Whole Mount in situ Hybridization of Early Chick Embryos

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  gene expression in brain, neural tube, somite and heart primordia formation. This video demonstrates the different steps in 2-color whole mount in situ hybridization; First, the embryo is dissected from the egg and fixed in paraformaldehyde. Second, the embryo is processed for prehybridization. The embryo is then hybridized with two different probes, one coupled to DIG, and one coupled to FITC.  Following overnight hybridization, the embryo is incubated with DIG coupled antibody. Color reaction for DIG substrate is performed, and the region of interest appears blue. The embryo is then incubated with FITC coupled antibody. The embryo is processed for color reaction with FITC, and the region of interest appears red. Finally, the embryo is fixed and processed for phtograph and sectioning. A troubleshooting guide is also presented.

This video demonstrates 2-color whole mount in situ hybridization, a method by which the spatial and temporal expression pattern of 2 different genes can be visualized in young chick embryos. This method was originally introduced by David Wilkinson, Domingos Henrique, Phil Ingham and David Ish -Horowicz.

Double Mont entier hybridation in situ d&#39;embryons de poulet début

Recherche

Biologie

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.

Méthode de coloration Mont anticorps entier de Chick

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.

Enregistrement électrophysiologique dans le Drosophile Embryon

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.

Techniques de microinjection pour l&#39;étude de la mitose dans les Drosophila melanogaster Embryon syncytial

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.

Poussin<em&gt; Ovo ex</em&gt; Culture et<em&gt; Ovo ex</em&gt; CAM Test: Comment il fonctionne vraiment

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.

Décrypter Pathways axonale des groupes génétiquement définis de neurones dans le tube neural Chick Utilisant In ovo L&#39;électroporation

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.

La xénotransplantation de cellules souches humaines dans l&#39;embryon de poulet

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.

Test clonogénique: cellules adhérentes

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.

Mise à jour embryonnaires de souris des cellules souches basée Assay pour quantifier l&#39;efficacité d&#39;induction cardiogénique

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.

Suivi des déformations des tissus morphogénétiques dans l&#39;embryon de poulet Early

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