DP ist Empfänger von Ruth Kirschstein-Award 1F32 DA021977-01A1 aus dem National Institute on Drug Abuse. Diese Arbeit wurde von der Margaret M. Alkek Stiftung RHF unterstützt.

<html> I. Schematische Übersicht: <img src="/files/ftp_upload/old/1027_2009_1_22_2/Figure1.png" alt="Abbildung 1" width="460" height="384" /> Dieses Video zeigt die verschiedenen Schritte in Assay für neuronale Induktion in Hühnerembryo. Zuerst wird der Host-Embryo in New Kultur explantierten [NI1]. Dann wird die donnor Embryo ist in Kochsalzlösung und Hensen den Knoten (der &quot;Veranstalter&quot; in chick) explantiert wird mit einem Fluoreszenzfarbstoff DiI markierten [Ni2]. Hensen den Knoten aus der donnor Embryo herausgeschnitten [NI3] und transplantiert werden, um den Host-Embryo [NI4, Ni5]. Der Host-Embryo wird dann für bis zu 22 Stunden, kultiviert nach welchem ​​Zeitraum die donnor Gewebe hat eine ektopische Achse in den Host-Bereich induziert opaca [Ni6]. Nach Kultur, ist der Host-Embryo fixiert und für die Photooxidation in Gegenwart von DAB: Mit dieser chemischen Reaktion, donnor abgeleiteten Zellen, die rot erscheinen unter FITC Fluoreszenz vor Photooxidation [Ni7], braun folgende Reaktion mit DAB geworden [NI8]. Der Embryo wird dann in situ Hybridisierung verarbeitet mit einem neuronalen Marker [NI8]; Host abgeleitete Gewebe Nervengewebe in die Gegenwart des donnor Transplantat gebildet. Dieses Gewebe blau erscheint. 1: Vorbereiten des Host-Embryo für Neue Kultur <ol><li> Diese neuronale Induktion Assay-Protokoll beginnt mit Eiern, die Hamburger und Hamilton Stufe 3 inkubiert wurden + (oder HH3 +). Diese Eier wurden bebrütet legte auf ihrer Seite in einem feuchten Inkubator für 13 Stunden. </li><li> Bereiten Sie den Host-Embryo für Neue Kultur (Schritte 3.1. Bis 5,5 9). </li><li> Bedecken Sie die Oberfläche des Host Embryo mit 200 ul Kochsalzlösung. Dies erleichtert die spätere Übertragung und die Positionierung der donnor Transplantats. </li><li> Schließlich decken die Montage mit einer invertierten Falcon 35 mm Kulturschale. </li></ol> 2: Explantation der donnor Embryo in Kochsalzlösung <ol><li> Öffnen Sie das Ei, indem Sie auf die Schale mit einer Pinzette und Entfernen von Teilen der Schale, rund um die Schale. Entfernen Sie die obere von Shell und entsorgen. </li><li> Entfernen Sie die dicken Albumin mit einer Pinzette, und kippen Sie den Dottersack mit groben Pinzette so, dass der Embryo nach oben zeigt. </li><li> Mit einer feinen Schere, schneiden Sie ein Quadrat der Dottersack rund um den Embryo. Entfernen Sie den Embryo aus dem Eigelb mit einem Löffel, und in eine Schale mit PBS. </li><li> Mit einer Pinzette, nehmen Sie den donnor Embryo aus dem Bereich pellucida. </li><li> Transfer donnor Embryo zu einem Sylgard abgedeckt Schale mit PBS. </li></ol> 3: Labeling, Ausschneiden und Transplantation der donnor graft <ol><li> Mit einer Mikroelektrode Abzieher, ziehen 50 pl Glas Mikrokapillare Pipetten. Unter dem Mikroskop, schneiden Sie die Spitze der Mikropipette mit feinen Pinzetten. Legen Sie Mikropipette in eine Petrischale ausgekleidet mit einem Band von Plastilin. </li><li> Bereiten Sie die Farbstofflösung, die verwendet werden, um das Transplantat vor der Transplantation Label werden: Carbocyanin Farbstoff DiI (1,1 &#39;-Dioctadecyl-3, 3,3&#39;, 3&#39;-tetramethylindocarbocyanine Perchlorat): Zuerst bereiten einen Bestand von DiI in 1ml absolutem Ethanol bei 0,5%. Diese Aktie kann bei -20 ° C im Dunkeln gelagert werden. In einem Wasserbad bei 42 ° C eingestellt, vorgewärmte Mischung 40 ul DiI Lager zu 360μl 0,3 M Saccharose. Das Wasserbad Temperatur ist erforderlich, um Niederschläge von DiI und Bildung unlöslicher Kristalle zu verhindern. Kurz Spin die Lösung auf einer Mini-Spectrafuge (5-10 Sekunden). Die DiI-Lösung ist jetzt einsatzbereit. </li><li> Mit Insektennadeln, sichern Sie die donnor Embryo auf der Unterseite des Sylgard bedeckten Teller. </li><li> Back-füllen die Mikropipette mit DiI. Mit leichtem Druck mit einem Ansaugrohr Montage, wenden Sie einen kleinen Bolus DiI zu Hensen den Knoten. Achten Sie darauf, die gesamte Hensen den Knoten beschriftet ist. </li><li> Mit einem Mikrokapillare Pipette oder einer microdissecting Messer, schnitt Hensen den Knoten (&quot;Veranstalter&quot;, donnor graft). </li><li> Mark der Bauchseite und hinteren Ende des Transplantats mit Carmin. Dadurch wird sichergestellt, dass das Transplantat mit der richtigen dorso-ventrale Orientierung in Schritt 4.3 positioniert ist. </li><li> Mit einem 200 ul Gilson Pipette das Transplantat an den Host-Embryo. </li></ol> 4: Sicherung des Transplantats und die Kultivierung der Host-Embryo <ol><li> Entfernen Kochsalzlösung vom Host Embryo, so dass Sie sicher, dass das Transplantat positioniert bleibt in der Nähe mit dem Gastgeber vor Ort. </li><li> Mit einem Mikrokapillare Pipette oder einer microdissecting Messer einen kleinen Einschnitt (80-100 um) im Bereich pellucida / Bereich opaca Grenzbereich, auf der Ebene der Hensen den Knoten von Host-Embryo. </li><li> Mit einem Mikrokapillare Pipette oder einer microdissecting Messer, Position der Prothese, so dass Bauchseite zugewandt Ihnen und dem hinteren Ende ist parallel zur entsprechenden Region in Host-Embryo. </li><li> Entfernen Sie verbleibende Salzlösung, und verarbeiten die Host-Embryo für New Kultur für 81-22 h (Schritte von 6,1 bis 6,6 9). </li><li> Der Host-Embryo wird dann über Nacht bei 4 ° C (Schritte von 7,1 bis 7,7 9) fixiert. </li></ol>e &quot;&gt; 5: Photooxidise das Transplantat unter Fluoreszenz <ol><li> Unter Abzug, bereiten Sie einen DAB funktionierende Lösung in Tris-Puffer: Lösen DAB Substrat (3,3 &#39;-Diaminobenzidintetrahydrochlorid) in Tris-Puffer (100 mM Tris-HCl, pH 7,4) bei 500μg/ml; halten Lösung in dunklen, auf Eis. </li><li> Waschen Sie den Embryo zweimal in PBS für 5 Minuten jeder und einmal in Tris-Puffer für 5 Minuten. </li><li> Übertragung des Embryos zu einem Glas Hohlraum Folie mit 1 ml Tris-Puffer. </li><li> Ersetzen Sie die Tris-Puffer-Lösung mit 1,5 ml DAB-Lösung. Entsorgen Eppendorfspitze in Eimer mit einer 10% igen Bleichlösung um DAB zu dekontaminieren. Legen Sie ein Deckglas auf der Hohlraum schieben. </li><li> Unter FITC (Fluoresceinisothiocyanat) Fluoreszenz, Schwerpunkt das Ziel des Mikroskops auf dem Transplantat (donnor Zellen fluoreszierend rot erscheinen) und aussetzen FITC Fluoreszenz, bis alle rot-fluoreszierenden Zellen geworden sind braun (in diesem Fall als Ergebnis der DiI Fluorochrom wird photokonvertiertem zu unlöslichen braunen Kristallen, durch Bestrahlung mit FITC Anregungswellenlänge (488 nm) in Gegenwart von DAB. Dieser Vorgang dauert zwischen 20 Minuten und 2 Stunden. </li><li> Nach Abschluss der Photooxidation Reaktion, entfernen Sie das Deckglas mit feinen Pinzetten. Transfer-Embryo zu Glas eine 20 ml Szintillations Fläschchen mit PBTw (oder PBS mit 0,1% Tween-20). </li><li> Deaktivieren DAB von Deckglas und Glas Hohlraum slide durch Inkubation in 10% Bleichmittel-Lösung. </li></ol> 6: Prozess der Embryo in-situ-Hybridisierung, Fotografie und Schneiden <ol><li> Fahren Sie mit in-situ-Hybridisierung (Schritte von 3,7 bis 6,5 9), Inkubation der Embryonen nur für DIG markierten Sonde. </li><li> Fahren Sie mit dem Fotografieren (Schritt 8.2 9) und Schneiden (Schritte 8.3 und 8.4 9). </li></ol> Repräsentative Ergebnisse Im folgenden Beispiel der neuralen Induktion Assay wird die donnor Transplantat gezeigt induzieren die Expression des neuronalen Marker Schwanzlosigkeit vom Host abgeleitete Gewebe. In diesem Beispiel untersuchten wir die Akquisition von neuralen Induktion Fähigkeit embryonaler Regionen, die normalerweise nicht über &quot;Veranstalter&quot; Fähigkeiten: die donnor Transplantat stammt aus einem Embryo, in denen die &quot;Veranstalter&quot; Region operativ aus dem donnor Embryo im Stadium entfernt 3 +. Nach Ablation ist die donnor Embryo darf in New Kultur zu entwickeln, bis das Loch verheilt ist und eine Struktur ähnlich Hensen den Knoten gebildet hat. Diese Struktur wird dann mit DiI markiert, ausgeschnitten aus der donnor Embryo und gepfropft Bereich pellucida / Bereich opaca Grenze eines Host-Embryo. Nach New Kultur, ist ein Miniatur-Achse angrenzend an den Host-Embryo: das Miniatur-Achse besteht aus einem Stab von roten Blutkörperchen (donnor abgeleitet) und einem Kopf-artige Struktur (Host abgeleitet): In (A), donnor-derived DiI markierten Zellen vor der Fixierung unter FITC Fluoreszenz-Mikroskopie gezeigt. Diese donnor-abgeleiteten Zellen haben vermehrt auf eine Miniatur Chorda, die als Stab der roten Blutkörperchen scheint zu bilden. (B) Nach Photokonversion DiI erscheinen diese donnor-abgeleiteten Zellen braun. Die donnor Transplantat hat die Bildung von Nervengewebe aus dem Host-induzierte: dieses Gewebe besteht aus Vorstufen für Gehirn, wie sie ihren Ausdruck für den Marker Schwanzlosigkeit folgenden in situ Hybridisierung zeigte [ab 6 abgedruckt]. <img src="/files/ftp_upload/old/1027_2009_1_22_3/Figure 2.png" alt="Abbildung 2" width="230" height="154" /> Hinweis: In diesem Verfahren haben wir DiI markierten Zellen verwendet werden, um neuronale Induktion donnor Gewebe (rot / braun) von Host-derived &quot;neuronalen&quot; Gewebe (blau) unterscheiden. Anstelle der Verwendung eines chick abgeleitet, DiI markierten donnor Transplantat, einige Autoren auch donnor Gewebe von Embryonen Wachtel statt chick abgeleitet [zB 6]. Diese alternative Methode ermöglicht es auch, um donnor aus Wirtsgewebe zu unterscheiden. In diesem Fall stammt das Transplantat (donnor Gewebe) aus Wachteleier statt Hühnereiern. Nach dem Schritt 4.5, der Host für ganze Berg Immunhistochemie mit einem Antikörper spezifisch für Gewebe (QCP1) und Schritte von 3,1 bis 3,4 und von 5,1 bis 5,7 Wachteln verarbeitet werden vom Protokoll weggelassen. Ein Beispiel für diese alternative Vorgehensweise ist wie folgt dar: In diesem Fall haben wir die gleichen Versuchsanordnung wie in A, B, aber wir werden Wachtel-derived donnor Gewebe (wie durch die Expression von QCPN Antikörper [braun] dargestellt): Das regenerierte Knoten aus Wachtel Embryo abgeleitet hat der Ausdruck pan-neuronalen Marker Sox2 in den Host-induzierte [nachgedruckt von 6]: <img src="/files/ftp_upload/old/1027_2009_1_22_4/Figure 3.png" alt="Abbildung 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>

Dieses Video zeigt die verschiedenen Schritte bei der Durchführung eines Assays für neuronale Induktion; Dieser Test ist im Wesentlichen für die Charakterisierung von putativen neuralen Induktion Moleküle in chick verwendet, und kann somit für eine Vielzahl von Anwendungen, angefangen von embryonalen Mikromanipulationen 1-4 verwendet werden; 6 bis entwirren neue Signalkaskaden 7,8, alle mit dem Ziel, das Verständnis der erste Schritt bei der Bildung des Gehirns und des restlichen N...

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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>
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		</div>

assay for neural induction in the chick embryo

Der Hühnerembryo ist ein wertvolles Werkzeug in der Studie der frühen Embryonalentwicklung. Seine Transparenz, Zugänglichkeit und einfache Handhabung machen ihn zu einem idealen Werkzeug für die Erforschung der Entstehung und ersten Strukturierung des Nervensystems. Dieses Video zeigt, wie Graft Veranstalter Gewebe in einen Wirt, ein Verfahren, mit dem Hensen s Knoten (der Veranstalter im Hühnerembryo) an einen Host zuständige Ektoderm aufgepfropft wird. Der Veranstalter Transplantat weist darüber liegenden na Gewebe bereits in ein neuronales Schicksal über neurale Induktion Signale anzunehmen. Dieser Mechanismus wird als neuronale Induktion bezeichnet und stellt den ersten Schritt in die Bildung von Gehirn und Rückenmark in Amnioten. Diese Methode ist wesentlich für die Charakterisierung von putativen neuralen Induktion Moleküle in chick verwendet. Dieses Video zeigt die verschiedenen Schritte in der Assay für neuronale Induktion; Erste, der donnor Embryo explantiert und merken sich auf einem Teller. Dann wird die Host-Embryo für Neue Kultur vorbereitet. Das Transplantat wird herausgeschnitten und transplantiert werden, um den Host-Bereich pellucida Marge. Der Gastgeber ist für 18-22 Stunden kultiviert. Die Montage ist fixiert und für weitere Anwendungen (zB in-situ-Hybridisierung). Diese Methode wurde ursprünglich von Waddington entwickelt<sup&gt; 1,2</sup&gt; Und 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

Neuronale Induktion ist der erste Schritt bei der Bildung des Gehirns. Es ist ein Mechanismus, durch den Hensen den Knoten (Veranstalter), weist benachbartes Gewebe ein neuronales Schicksal anzunehmen, dh zu steigen, um das Nervensystem zu geben. Dieses Video zeigt ein Test für neuronale Induktion in Hühnerembryo.

Assay für Neural Induction im Hühnerembryo

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 Aufrufe.  Texas A&M University (TAMU). Neuronale Induktion ist der erste Schritt bei der Bildung des Gehirns. Es ist ein Mechanismus, durch den Hensen den Knoten (Veranstalter), weist benachbartes Gewebe ein neuronales Schicksal anzunehmen, dh zu steigen, um das Nervensystem zu geben. Dieses Video zeigt ein Test für neuronale Induktion in Hühnerembryo.

Serie: 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.

Verfahren zur Whole Mount Antikörper-Färbung in Küken

Forschung

Biologie

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.

Elektrophysiologische Aufzeichnung in der 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.

Mikroinjektion Techniken zur Untersuchung Mitose in der 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 ...

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.

Küken Ex ovo Kultur und Ex ovo CAM-Assay: Wie es wirklich funktioniert

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.

Die Entzifferung Axonal Pathways von genetisch definierte Gruppen von Neuronen in der Küken Neuralrohr Verwendung In ovo Elektroporation

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.

Xenotransplantation von menschlichen Stammzellen in die Hühnerembryo

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.

Klonogene Test: adhärenter Zellen

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.

Geändert Maus embryonalen Stammzellen basierte Assay zur Quantifizierung von Kardiogener Induction Efficiency

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.

Tracking Morphogenetic Tissue Verformungen in der frühen Hühnerembryo

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.

Maus embryonalen Entwicklung in einem Serum-freien 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 ...