Wir möchten Paul Poirier, der Medienproduzent, am Mount St. Vincent Universität für seine Arbeit in Filmen und Bearbeiten der Videoteil des Manuskripts danken. Wir sind uns der Naturwissenschaft und Technik Research Council of Canada für die Finanzierung.

Hinweis: Alle Lieferungen sind in Tabelle 1 aufgeführt. 1. Speichern der Hühnerembryonen <ol><li> Inkubieren Hühnereier des Stammes Gallus gallus horizontal bei 37 ° C mit ca. 40% Luftfeuchtigkeit und schalten Eier einmal oder zweimal täglich. Drehen Eier ist wichtig, den Embryo aus auf die Eierschale haftet. </li><li> Sie nicht, das Ei in den 24 Stunden vor der Einrichtung der Kultur, da sonst der Embryo ventral der Dottermasse entfernt werden und wird beim Öffnen der Eier in Schritt 3 Zusätzlich beschädigt schalten, halten Sie die Eier bei 4 o C Grad für nicht mehr als eine Woche vor der Inkubation auf &quot;halt&quot; Entwicklung, aber das ist nicht ideal. </li></ol> 2. Staging Hühnerembryonen <ol><li> Stufe die Hühnerembryonen mit dem Hamburger und Hamilton 12 Staging-Tabelle. Die ideale Bühne für die Einrichtung des Ex-ovo-Kultivierung ist HH Stadium 19-20 (ca. 3 Tagen Inkubation) because dies kurz nach der Kopf dreht sich mit 53 HPF. Hinweis: HH Stadium 19-20 wird durch folgende morphologische Merkmale gekennzeichnet: Somiten in die Mehrheit der Schwanz verlängert, aber die ganz am Ende der Schwanz bleibt unsegmentierten die Schwanzknospe gewellt ist, ist die Allantois klein und hat Gefäßsystem beschränkt, die Beinknospen sind größer als die Flügelknospen und die Augen sind nicht pigmentierten oder haben eine gräuliche Farbe. </li></ol> 3. Entfernen der Embryo von der Shell <ol><li> Vor dem Entfernen der Embryo aus der Schale, sprühen die Shell mit 70% Ethanol und lassen Sie ihn trocknen. Das Ei Schalten Sie nicht schnell, da dies den Embryo schädigen. </li><li> Darauf achten, daß die Ausrichtung des Eier verändern, vorsichtig knacken das Ei an der Unterseite (dh der Seite, die während der Inkubation ventralen war) und lassen den Embryo in eine sterile wiegen Boot (88 x 88 x 23 mm). Wischen Sie wiegen Boote mit 70% Ethanol. Untersuchen Sie den Embryo, um sicherzustellen, dass der Dottersack nicht beschädigt wird. Wenn das Eigelb hgebrochen, entsorgen Sie den Embryo, da es nicht überlebt. </li><li> Beachten Sie die Embryos zu gewährleisten, dass es machbar ist; das Herz schlägt, erscheint Gefäßsystem normal und keine offensichtlichen Anomalien vorhanden sind. Sicherzustellen, dass die Ränder des Wägeschiffchen trocken sind. </li><li> Mit einer Pipette fallen 40 ul Penicillin / Streptomycin (5.000 Einheiten Penicillin, 5 mg Streptomycin pro ml) auf der Oberseite des Albumin zur Verhinderung einer Infektion. </li></ol> 4. Vorbereiten der Feuchtekammer <ol><li> Eine kleine Stapel von Kim-Wipes und / oder ein absorbierendes Kissen aus Baumwolle in den Boden einer sterilen Kunststoffbehälter (12 x 12 x 6 cm) abgewischt und mit 70% Ethanol. </li><li> In sterilem destilliertem Wasser, um die kim-Wischtücher und / oder Baumwolle (ca. 150 ml Wasser) befeuchten. </li></ol> 5. Montage der Ex Ovo Kultur <ol><li> Legen Sie die wiegen Boot, das die Embryos auf der Oberseite des feuchten Polsterung. Dann Ort Hälfte eines quadratischen sterile Petrischale (9.5 x 9.5 cm) oben auf ter Boot wiegen, um eine lose Deckel zu bilden. </li><li> Decken Sie die Kunststoffbehälter mit Deckel. Drücken Sie zwei Ecken des Deckels, eine Teildichtung, die noch ermöglicht eine gute Luftzirkulation in der Kammer gewährleistet. </li><li> Legen Sie das Setup in das 37 ° C-Inkubator, bis die gewünschte Stufe. </li><li> Platzieren Behälter Wasser in den Inkubator zur Kontrolle der Feuchtigkeit, wenn ein kontrollierter Luftfeuchtigkeit Inkubator nicht verfügbar ist. Sterilisieren Sie das gesamte Wasser in den Feuchtekammern und in den Inkubator und füllt als während der Inkubationszeit benötigt. 40% Luftfeuchtigkeit ist ideal. </li></ol>

<ol>
	<li>Auerbach, R., Kubai, L., Knighton, D., Folkman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+procedure+for+the+long-term+cultivation+of+chicken+embryos.">A simple procedure for the long-term cultivation of chicken embryos.</a> Dev. Biol. 41, 391-394 (1974).</li><li>Gennaro, L. D., Packard, D. S., Stach, R. W., Wagner, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+and+differentiation+of+chicken+embryos+in+simplified+shell-less+cultures+under+ordinary+conditions+of+incubation.">Growth and differentiation of chicken embryos in simplified shell-less cultures under ordinary conditions of incubation.</a> Growth. 44, 343-354 (1980).</li><li>Tufan, C. A., Akdogan, I., Adiguzel, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+culture+of+the+chick+embryo+as+a+model+system+in+the+study+of+developmental+neurobiology.">Shell-less culture of the chick embryo as a model system in the study of developmental neurobiology.</a> Neuroanat. 3, 8-11 (2004).</li><li>Duench, K., Franz-Odendaal, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BMP+and+Hedgehog+signaling+during+the+development+of+scleral+ossicles.">BMP and Hedgehog signaling during the development of scleral ossicles.</a> Dev. Biol. 365 (1), 251-258 (2012).</li><li>Datar, S., Bhonde, R. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shell-less+Chick+Embryo+Culture+as+an+Alternative+in+vitro+Model+to+Investigate+Glucose-Induced+Malformation+in+Mammalian+Embryos.">Shell-less Chick Embryo Culture as an Alternative in vitro Model to Investigate Glucose-Induced Malformation in Mammalian Embryos.</a> Rev Diabet Stud. 2 (4), 221-227 (2005).</li><li>Fisher, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chick+embryos+in+shell-less+culture.+Tested+studies+for+laboratory+teaching.">Chick embryos in shell-less culture. Tested studies for laboratory teaching.</a> , (1983).</li><li>Dunn, B. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technique+for+shell-less+culture+of+the+72-hour+avian+embryo).">Technique for shell-less culture of the 72-hour avian embryo).</a> Poultry Science. 53, 409-412 (1974).</li><li>Yalcin, H., Shekhar, A., Rane, A. A., Butcher, J. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+ex-ovo+chicken+embryo+culture+system+suitable+for+imaging+and+microsurgery+applications.">An ex-ovo chicken embryo culture system suitable for imaging and microsurgery applications.</a> J. Vis. Exp. (44), (2010).</li><li>Silver, P. H. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Special+problems+of+experimenting+in+ovo+on+the+early+chick+embryo,+and+a+solution.">Special problems of experimenting in ovo on the early chick embryo, and a solution.</a> J Embryol Exp Morph. 8 (4), 369-375 (1960).</li><li>Spurlin, J., Lwigale, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+technique+to+increase+accessibility+to+late-stage+chick+embryos+for+in+ovo+manipulations.">A technique to increase accessibility to late-stage chick embryos for in ovo manipulations.</a> Dev. Dyn. 242 (2), 148-154 (2012).</li><li>Dorrell, M. I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ex+ovo+model+for+directly+visualizing+chicken+embryo+development.">Ex ovo model for directly visualizing chicken embryo development.</a> American Biology Teacher. 74 (9), (2012).</li><li>Hamburger, V., Hamilton, H. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+series+of+normal+stages+in+the+development+of+the+chick+embryo.">A series of normal stages in the development of the chick embryo.</a> J. Morph. 88, 49-92 (1951).</li><li>Drossopoulou, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+model+for+anteroposterior+patterning+of+the+vertebrate+limb+based+on+sequential+long-and+short-range+Shh+signaling+and+Bmp+signaling.">A model for anteroposterior patterning of the vertebrate limb based on sequential long-and short-range Shh signaling and Bmp signaling.</a> Development. , 127-1337 (2000).</li><li>Sys, G. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+in+ovo+CAM-assay+as+a+xenograft+model+for+sarcoma.">The in ovo CAM-assay as a xenograft model for sarcoma.</a> J. Vis. Exp. (77), e50522 (2013).</li><li>Franz-Odendaal, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Towards+understanding+the+development+of+scleral+ossicles+in+chicken,+Gallus+gallus.">Towards understanding the development of scleral ossicles in chicken, Gallus gallus.</a> Dev. Dyn. 237, 3240-3251 (2008).</li></ol>

Die Autoren haben keine konkurrierenden finanziellen Interessen in Bezug auf die in diesem Manuskript bereitgestellten Informationen.

Ex Ovo Kultivierung und Fenster beide haben Vorteile und Herausforderungen. Hier vergleichen wir die Vorteile und Herausforderungen der Styroporbecher ex ovo-Methode und der Fenster Methode, um unsere verbesserte ex ovo Methode angezeigt. Unsere Methode ermöglicht Manipulation und einfache Beobachtung des Hühnerembryos in späten Entwicklungsstadien und unsere Verbesserungen der traditionellen ex ovo Verfahren 1, 2, 3 machen es zudem sehr einfach, in grundständigen Lehre Labor Klassen verwenden. Obwohl viele Forscher bevorzugen die Fenstermethode Huhn Entwicklung zu studieren, weil es einfach durchzuführen und hat eine ausgezeichnete Überlebensfähigkeit zu späten Entwicklungsstadien (HH 41-42), hat es Einschränkungen. Die größte ist die begrenzte Fähigkeit, den gesamten Embryo nach HH Stufe 30 zu sehen Nach Stadium HH 30, muss das Fenster in der Eierschale sehr groß, um den wachsenden Embryo bewegen sehen sein. Das große Fenster ist difficult zur Abdichtung vollständig (was zu Problemen mit der Sterilität und Überlebensfähigkeit) und erhalten eine gute Beleuchtung im Inneren des Eis kann eine Herausforderung sein. Darüber hinaus sind fortgeschritten Embryonen großen und schwer zugänglichen (oder zu manipulieren) durch das Fenster. Die ex ovo hier beschriebene Verfahren bietet volle ungehinderten Zugang zum Embryo. Sobald die Wägeschiffchen in der Feuchtigkeitskammer gelegt, ist die gesamte Aufbau sehr stabil. Der gesamte Feuchtigkeitskammer kann unter einem Präpariermikroskop angebracht werden und eine hohe Vergrßerung verwendet werden, um den gesamten Embryo beobachten. Im Vergleich dazu hat der Styroporbecher Verfahren eine feuchte Kammer, die groß ist (Tassenhöhe) und das macht es sehr schwierig, unter dem Mikroskop Ziele zu setzen. Der Becherentwurf ist auch weit weniger stabil als die Flach wiegen Boot und diese Becher kann leicht umgestoßen werden. Unsere Verbesserungen des ex ovo Verfahren machen diese Methode einfach, in einem Klassenzimmer oder Labor-Einstellung zu verwenden und ermöglicht die student um vollen Zugriff auf Embryonen aus Stadium HH 19 bis zu HH 40 haben; inszeniert, wenn wichtige Organentwicklung auftritt. Der größte Nachteil der ex ovo Methode ist Sterilität. Um Infektionen zu vermeiden, umfaßt unsere Methode die Zugabe von Antibiotika. Eine weitere Dosis des Antibiotikums können für den Embryo in späteren Phasen gegeben werden. Die während der Kultur-Setup verabreichten Antibiotika auch scheint nicht die Entwicklung des Embryos beeinflussen. Sterilisieren von Behältern mit 70% igem Ethanol vor der Anwendung drastisch erhöhen die Überlebensfähigkeit. Insgesamt ist die ex ovo Verfahren ideal für die Anzeige und Bearbeitung von Embryonen ohne Zugangsbeschränkungen oder Entwicklungsphase. Frühere Untersuchungen haben gezeigt, dass die Kultivierung der Embryonen ex ovo nicht negativ beeinflusst Strukturierung 4,15. Zum Beispiel haben wir keine Veränderungen in der Strukturierung oder Induktion des Skeletts sehen trotz der Abwesenheit von der Eierschale. Unsere verbesserte Verfahren löst mehrere cERAUSFORDERUNGEN die mit der aktuellen traditionellen ex ovo-Methode 1,2, 3 existieren.

Ex ovo culturing has played an important role in the study of development of the chicken1, 2. This culturing method has been used to study neurological diseases, limb development, craniofacial development, and as a model to investigate malformations associated with diabetes 3, 4, 5.
There are many variations to the ex ovo technique. The most common approach is to use a Styrofoam cup6,7,8 or a glass bowl5. In these methods, the cup or bowl is lined with plastic wrap to cradle the embryo, a lid is placed on the cup, and the embryo is then placed in an incubator with appropriate humidity6. This set up however, can be technically challenging. The first challenge is the plastic wrap that is used to cradle the embryo. This wrap is difficult to work with and often does not adhere to the cup very well. To solve this problem, an elastic band is placed around the cup to hold the wrap in place. Despite this, the wrap can still slip, which is fatal to the embryo. The plastic wrap has the potential to tear or get punctured by forceps or needles that may be used during embryo manipulations and observations. Finally, this set-up is not very stable and students can easily knock the cups over. The height of the cups also makes it very difficult to place the embryo under a stereomicroscope, which has a limited objective to stage height. These challenges make it difficult for undergraduate students to work with live chick embryos in teaching labs, such as advanced developmental biology courses. 
The above challenges in the ex ovo method has meant that researchers turn to the windowing method 9,10 to view embryonic chick development. In this technique, a hole or &#8220;window&#8221; is made in the eggshell overlying the embryo. The hole can be re-sealed with tape or wax9 to allow for further embryonic development. Although the windowing method has some advantages, such as the ability to view embryonic development and easy maintenance, this method also has several limitations. The first is that the window needs to be fairly large in order to view the entire embryo (especially at late stages). Secondly, large windows are difficult to seal; an improper seal will lead to sterility and survivability problems. Using molten wax as a sealant adds another inconvenient and messy step to the protocol. Therefore, although the windowing method may be ideal for chick embryos at young stages (HH 11 &#8211; HH 27), viewing the entire embryo at late stages is not easily accomplished. 
Here, we describe an improved and simple ex ovo culturing technique11 that avoids the need for high tech equipment, is easy to handle under a stereomicroscope, gives the embryo enough support to perform microscopic manipulations, and enables researchers to view the growth of the embryo in its entirety well into the later stages of development (up to HH 40-41). With these advances in the ex ovo technique, individuals gain access to a more complete understanding of embryonic development. For instance, growth into later stages allows individuals to observe developmental processes that do not occur until this time point, such as ossification, feather development, and advanced limb and eye development. The entire embryo and extraembryonic membranes and vasculature are clearly visible. More advanced research can also be performed, such as, embryonic manipulations (i.e., implanting beadssoaked in inhibitors or inserting barriers between tissue layers), and researchers are then able to observe the effect of the manipulations in later stage embryos.

<table border="0" cellpadding="0" cellspacing="0" width="658">
	<tbody>
		<tr height="78">
			<td height="78" style="height:79px;width:177px;">Penicillin/Streptomycin</td>
			<td style="width:136px;">Sigma</td>
			<td style="width:152px;">P4458</td>
			<td style="width:192px;">Make small aliquots to avoid freeze/thaw events</td>
		</tr>
		<tr height="22">
			<td height="61" rowspan="3" style="height:61px;width:177px;">Square Petri Dish</td>
			<td rowspan="3" style="width:136px;">N/A</td>
			<td rowspan="3" style="width:152px;">N/A</td>
			<td rowspan="3" style="width:192px;">9.5 cm x 9.5 cm</td>
		</tr>
		<tr height="19">
		</tr>
		<tr height="20">
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Weigh Boat</td>
			<td style="width:136px;">Fischer Scientific</td>
			<td align="right" style="width:152px;">8732113</td>
			<td style="width:192px;">88 x 88 x 23 mm</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:177px;">Ziplock container</td>
			<td style="width:136px;">Ziplock</td>
			<td style="width:152px;">N/A</td>
			<td style="width:192px;">12 cm x 12 cm x 6 cm</td>
		</tr>
	</tbody>
</table>

optimized ex-ovo culturing of chick embryos to advanced stages of development

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos model organisms, such as the chicken, are often used. The chicken is an excellent model organism due to its low cost and minimal maintenance, however they present observational challenges because they are enclosed in an opaque eggshell. In order to properly view the embryo as it develops, the shell must be windowed or removed. Both windowing and ex ovo techniques have been developed to assist researchers in the study of embryonic development. However, each of the methods has limitations and challenges. Here, we present a simple, optimized ex ovo culture technique for chicken embryos that enables the observation of embryonic development from stage HH 19 into late stages of development (HH 40), when many organs have developed. This technique is easy to adopt in both undergraduate classes and more advanced research laboratories where embryo manipulations are conducted.

Diese ex ovo Verfahren erlaubt die Beobachtung von Embryonen aus einem frühen Entwicklungsstadium (HH 19/20) zum fortgeschrittenen Stadium der Entwicklung (HH 40-41) (1A und 1B). Einrichten der Kultur bei HH 19-20 erhöht die Überlebensfähigkeit der Embryonen in der Kultur. Vor dem Drehen des Kopfes (vor 53 HPF) Überlebensfähigkeit in Kultur und nach der Stufe 21 sehr niedrig ist, neigt der Embryo mehr an die Shell auf Entfernung bleiben so weniger intakte Embryonen gewonnen werden. Im Allgemeinen ist die Überlebensfähigkeit des Embryos in ex ovo Kultur hoch bis zu HH 35-36 (90-100%), aber es Tropfen in den fortgeschrittenen Stadien der Entwicklung (ca. 40% überleben HH 40-41). Dazu kommt, obwohl es hat sich gezeigt, dass die Entwicklung normal erscheint bei diesen späten Stadien und Strukturierung des Skeletts ist unempfindlich 4,15 haben wir beobachtet, dass die Schenkel an diesen späten Stadien darauf hindeutet, dass das Ausmaß und Zeitpunkt der Knochenbildung beeinträchtigt werden gebogen. Dies istnicht überraschend angesichts der Bedarf an Kalzium aus der Schale zur Knochenbildung. Ein detaillierter Vergleich der Entwicklung des Embryos im Vergleich zu Kontrollen ist im Gange und geht über den Umfang dieser Methoden Papier. Den Zugang zu den Embryo während der Entwicklung ist aus zwei Hauptgründen. Erstens sind die Forscher in der Lage, die Entwicklung auf einer täglichen Basis, ohne zu entsiegeln und verschließen ein Fenster anzuzeigen. Zum Beispiel sind Menschen in der Lage, des Körpers und der Augenentwicklung in mehreren Stufen (1C und 1D) zu beobachten. Zweitens ex ovo Kultivierung ermöglicht eine größere (uneingeschränkt) Zugriff auf den Embryo, wodurch ein einfacher Manipulationen oder Operationen. Embryonale Manipulationen häufig, müssen mit einem Stereomikroskop durchgeführt werden. Mit der ex ovo hier beschriebenen, im Gegensatz zu dem Styropor / Plastikfolie Methode 6,7,8 Kultivierungsverfahren der Embryo Set-up ist einfach und platzierter das Mikroskop ist weniger tief und nicht umkippt. Dies ermöglicht es dem Forscher, die Feuchtigkeitskammer genau zu positionieren, wie benötigt, und um es zu drehen, um den Zugriff auf den Embryo zu optimieren, da es keine Hindernisse (Eischale) Verschleiern der Embryo und da die Kammer ist sehr stabil. Insgesamt ist die Unterstützung der Embryo empfängt von dem Wägeschiffchen ermöglicht sanften Druck auf den Embryo während der Handhabung ohne die Einrichtung Umkippen angewendet werden. Mehrere Entwicklungsbiologie Methoden können auf die Embryonen in diesem Kultursystem durchgeführt werden. Dazu gehören detaillierte Beobachtungen der Orgel (zB Auge, Gehirn usw.) oder Gewebeentwicklung (zB Wachstum von Blutgefäßen) oder Anwendung teratogene Mittel. Manipulationen umfassen Pfropfung von Geweben auf die Chorioallantoismembran um die Wirkung der Kombination von verschiedenen Gewebeschichten zu studieren oder Tumorwachstum und Metastasierung 14 zu studieren. Eine andere beliebte Manipulation während der Entwicklung durchgeführt wird bead oder Barriere Implantation. Bead Implantation ermöglicht ein Forscher, einen Inhibitor oder Faktor auf den Wulst örtlich tränken und dann implantieren den Wulst zu inhibieren oder induzieren Gene lokal in dem Bereich von Interesse. Dies wird oft mittels Affigel oder Heparin-Kügelchen (2A) durchgeführt. So kann beispielsweise bone morphogenetic proteins (BMPs) lokal bei der Induktion der Neuralleiste Knochen 4, 15 gesperrt werden, oder in der sich entwickelnden Extremitätenknospe 13. Nachdem eine Wulst implantiert, kann der Embryo in den Inkubator gegeben werden, und es wird weiter zu entwickeln. Dies ermöglicht es den Forschern, die stromabwärts Wirkungen der Hemmung eines bestimmten Gens (in diesem Fall BMP), wie beispielsweise die Hemmung der skleralen Ossikel Bildung im Skleralring (2B und 2C) 15 anzuzeigen. Auch Hindernisse können einfach zwischen Gewebeschichten, um Gewebe zu Gewebe-Signalisierung und die Mikroinjektion von Farbstoffen können auch einfach durchgeführt werden studieren eingefügt werden. </p&gt; <img alt="Figur 1" src="/files/ftp_upload/52129/52129fig1.jpg" /> Abbildung 1. Anzeigen des Embryos ex ovo. (A) HH Stufe 20 Hühnerembryo nach der Entnahme aus der Schale, Pfeil zeigt auf den Embryo mit vaskularisierten embryonalen Membranen. (B) A HH Stufe 40 Embryos in einem offenen feuchten Kammer. (C) HH Stufe 35 Embryo zeigt frühe Extremitätenknospen (Pfeil). (D) Stadium HH 41 Embryos, die später Entwicklung von Strukturen, wie das Auge (Pfeil). <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig1large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a> <img alt="Abbildung 2" src="/files/ftp_upload/52129/52129fig2.jpg" /> Abbildung 2. Beispiel für Noggin getränkt Wulst Implantation experiment. 4 (A) Embryo HH Stufe 35 durch ein kleines Loch in den übereinanderliegenden Membranen, um einen Zugang zu der darunter liegenden Auge für Korn Implantation gewinnen zerrissen ausgesetzt. Hohe Vergrößerung der Affigel Wulst neben einem Binde Papille in Leiste dargestellt. Maßstabsbalken in C ist 75 um. (B) Die alkalische Phosphatase nach Noggin Wulst Implantation (Pfeil), die Lücke im Ring der Gehörknöchelchen, HH 38 (C) AP gefärbten linke Auge zeigt keine Störung an den Ring der Gehörknöchelchen (Steuer befleckt rechten Auge ), HH 38. Einzelheiten dieses Experiments sind in Duench und Franz-Odendaal 4 zur Verfügung gestellt. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52129/52129fig2large.jpg" target="_blank">Bitte klicken Sie hier, um eine größere Version dieses Bild anzuzeigen.</a>

Watch this Scientific Journal Video about Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development at JoVE.com

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40).

Optimierte<em&gt; Ex-ovo</em&gt; Züchtung von Hühnerembryonen zu fortgeschrittenen Entwicklungsstadium

Research in anatomy, embryology, and developmental biology has largely relied on the use of model organisms. In order to study development in live embryos ...

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Nanyang Technological University

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JoVE Journal

Developmental Biology

Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

13.5K Aufrufe.  Mount Saint Vincent University. Viewing and accessing the chicken embryo during development can be challenging. We have developed an ex ovo method that is simple, cost effective, and can easily be used in a classroom or research setting. This method provides access to the embryo into late stages of embryonic development (HH 40). 

Serie: Optimized Ex-ovo Culturing of Chick Embryos to Advanced Stages of Development

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying the biology of retinal neurons, particularly photoreceptors. The applications of this system go beyond basic research, as they can easily be adapted to high throughput technologies for drug development. However, genetic manipulation of retinal photoreceptors in these cultures has proven to be very challenging, posing an important limitation to the usefulness of the system. We have recently developed and validated an ex&#160;ovo plasmid electroporation technique that increases the rate of transfection of retinal cells in these cultures by five-fold compared to other currently available protocols1. In this method embryonic chick eyes are enucleated at stage 27, the RPE is removed, and the retinal cup is placed in a plasmid-containing solution and electroporated using easily constructed custom-made electrodes. The retinas are then dissociated and cultured using standard procedures. This technique can be applied to overexpression studies as well as to the downregulation of gene expression, for example via the use of plasmid-driven RNAi technology, commonly achieving transgene expression in 25% of the photoreceptor population. The video format of the present publication will make this technology easily accessible to researchers in the field, enabling the study of gene function in primary retinal cultures. We have also included detailed explanations of the critical steps of this procedure for a successful outcome and reproducibility.

Efficient Gene Transfer in Chick Retinas for Primary Cell Culture Studies: An Ex-ovo Electroporation Approach

Embryonic chick retinal cell cultures constitute valuable tools for the study of photoreceptor biology. We have developed an efficient gene transfer technique based on ex&#160;ovo plasmid electroporation of the retinas prior to culture. This technique considerably increases transfection efficiencies over currently available protocols, making genetic manipulation feasible for this system.

Effizienten Gentransfer in Küken Netzhäute für primäre Zellkulturstudien: Eine<em&gt; Ex-ovo</em&gt; Elektroporation Ansatz

The cone photoreceptor-enriched cultures derived from embryonic chick retinas have become an indispensable tool for researchers around the world studying ...

Forschung

Entwicklungsbiologie

Forward Genetic Approach to Uncover Stress Resistance Genes in Mice &#8212; A High-throughput Screen in ES Cells

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely limits its potential. We describe here the use of mouse embryonic stem (ES) cells as surrogate cells to screen for a phenotype of interest and subsequently introduce these cells into a host embryo to develop into a living mouse carrying the phenotype. This method provides (1) a cost effective, high-throughput platform for genetic screen in mammalian cells; (2) a rapid way to identify the mutated genes and verify causality; and (3) a short-cut to develop mouse mutants directly from these selected ES cells for whole animal studies. We demonstrated the use of paraquat (PQ) to select resistant mutants and identify mutations that confer oxidative stress resistance. Other stressors or cytotoxic compounds may also be used to screen for resistant mutants to uncover novel genetic determinants of a variety of cellular stress resistance.

Stress resistance is one of the hallmarks for longevity and is known to be genetically governed. Here, we developed an unbiased high-throughput method to screen for mutations that confer stress resistance in ES cells with which to develop mouse models for longevity studies.

Vorwärts genetische Ansatz zur Stressresistenz-Gene in Mäuse Entdecken - Hochdurchsatz-Bildschirm in ES-Zellen

Phenotype-driven genetic screens in mice is a powerful technique to uncover gene functions, but are often hampered by extremely high costs, which severely ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Ein Roman<em&gt; Ex Ovo</em&gt; Banding-Technik intrakardialer Hämodynamik in einem embryonalen Hühner System ändern

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of morphogenetic events, such as gastrulation2, neurulation3-5, somitogenesis6, heart bending7,8, and brain formation9-13, during early embryogenesis. Key to understanding morphogenetic processes is to follow them dynamically by time-lapse imaging. The acquisition of time-lapse movies of chick embryogenesis ex ovo has been limited either to short time windows or to the need for an incubator to control temperature and humidity around the embryo14. Here, we present a new technique to culture chick embryos ex ovo for high-resolution time-lapse imaging using transmitted light microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique (EC-culture)1 and allows for the culturing of chick embryos without the need for a climate chamber. The embryo is sandwiched between two identical filter paper carriers and is kept fully submerged in a simple, temperature-controlled medium covered by a layer of light mineral oil. Starting from the primitive streak stage (Hamburger-Hamilton stage 5, HH5)15 up to at least the 28-somite stage (HH16)15, embryos can be cultured with either their ventral or dorsal side up. This allows the acquisition of time-lapse movies covering about 30 hr of embryonic development. Representative time-lapse frames and movies are shown. Embryos are compared morphologically to an embryo cultured in the standard EC-culture.
The submerged filter paper sandwich provides a stable environment to study early dorsal and ventral morphogenetic processes. It also allows for live fluorescence imaging and micromanipulations, such as microsurgery, bead implantation, microinjection, gene silencing, and electroporation, and has a strong potential to be combined with immersion objectives for laser-based imaging (including light-sheet microscopy).

A Submerged Filter Paper Sandwich for Long-term Ex Ovo Time-lapse Imaging of Early Chick Embryos

Here, we present a new technique to culture chick embryos ex ovo for time-lapse imaging using transmitted light and fluorescence microscopy. The submerged filter paper sandwich is a variant of the well-established filter paper carrier technique1 that allows for the culturing of chick embryos fully-submerged in a liquid culture medium.

Ein Abgetaucht Filterpapier Sandwich für Langzeit<em&gt; Ex Ovo</em&gt; Zeitraffer-Imaging von frühen Hühnerembryonen

Due to its availability, low cost, flat geometry, and transparency, the ex ovo chick embryo has become a major vertebrate animal model for the study of ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Induktion von Hypoxie im lebenden Frosch und Zebrafisch Embryonen

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Multi-Photon Time Lapse Imaging to Visualize Development in Real-time: Visualization of Migrating Neural Crest Cells in Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to numerous cell types throughout the body. Understanding the biology of the neural crest has been limited, reflecting a lack of genetically tractable models that can be studied in vivo and in real-time. Zebrafish is a particularly important developmental model for studying migratory cell populations, such as the neural crest. To examine neural crest migration into the developing eye, a combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time videos of the developing eye in transgenic zebrafish embryos, namely Tg(sox10:EGFP) and Tg(foxd3:GFP), as sox10 and foxd3 have been shown in numerous animal models to regulate early neural crest differentiation and likely represent markers for neural crest cells. Multi-photon time-lapse imaging was used to discern the behavior and migratory patterns of two neural crest cell populations contributing to early eye development. This protocol provides information for generating time-lapse videos during zebrafish neural crest migration, as an example, and can be further applied to visualize the early development of many structures in the zebrafish and other model organisms.

A combination of the advanced optical techniques of laser scanning microscopy with long wavelength multi-photon fluorescence excitation was implemented to capture high-resolution, three-dimensional, real-time imaging of neural crest migration in Tg(sox10:EGFP) and Tg(foxd3:GFP) zebrafish embryos.

Multi-Photonen-Time Lapse Imaging Entwicklung in Echtzeit zu visualisieren: Visualisierung der Migration Neuralleisten-Zellen in den Zebrafish Embryos

Congenital eye and craniofacial anomalies reflect disruptions in the neural crest, a transient population of migratory stem cells that give rise to ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Entwicklung eines In-vitro- Assay, hämatopoetischen Stammzellen und Vorläuferzellen Quantitate Zellen (HSPCs) bei der Entwicklung von Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Ganz-Mount-In-Situ-Hybridisierung in Zebrafisch-Embryonen und Tube-Formations-Assay in iPSC-ECs zur Untersuchung der Rolle von Endoglin in der Gefäßentwicklung

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

Vorbereitung kleiner RNA-Bibliotheken für die Sequenzierung von frühen Mausembryonen

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

A Simple and Effective Transplantation Device for Zebrafish Embryos

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex developmental processes. Zebrafish embryos are ideally suited for these manipulations since they are easily accessible, relatively large in size, and transparent. However, previously developed devices for cell removal and transplantation are cumbersome to use or expensive to purchase. In contrast, the transplantation device presented here is economical, easy to assemble, and simple to use. In this protocol, we first introduce the handling of the transplantation device as well as its assembly from commercially and widely available parts. We then present three applications for its use: generation of ectopic clones to study signal dispersal from localized sources, extirpation of cells to produce size-reduced embryos, and germline transplantation to generate maternal-zygotic mutants. Finally, we show that the tool can also be used for embryological manipulations in other species such as the Japanese rice fish medaka.

Embryological manipulations such as extirpation and transplantation of cells are important tools to study early development. This protocol describes a simple and effective transplantation device to perform these manipulations in zebrafish embryos.

Ein einfaches und effektives Transplantationsgerät für Zebrafischembryonen

Classical embryological manipulations, such as removing cells and transplanting cells within or between embryos, are powerful techniques to study complex ...