Die Autoren möchte Gregory Haller (Mount Saint Vincent Universität) zu danken, für seine Vorarbeiten bei der Entwicklung des Protokolls. Die Autoren auch möchte Nicholas Jones (Mount Saint Vincent University) danken für seine technische Expertise und Unterstützung bei den Dreharbeiten und Produktion von Audio/visuellen Teil des Manuskripts. Daniel Andrews wurde unterstützt durch die Finanzierung von MSVU und naturwissenschaftlich-technischen Forschung Rat von Kanada (NSERC) über ein Undergraduate Student Research Award. Tamara A. Franz-Odendaal wird durch eine NSERC Discovery Grant unterstützt.

Hinweis: Für Embryo Bühnen, nutzen Sie die Hamburger und Hamilton12 (HH) Inszenierung Tabelle.
(1) Embryo Inkubation
<ol><li>Inkubieren Sie befruchteten Hühnereiern in einem sterilen, temperaturgeführte Inkubator bei 37 ° C ± 1 ° C und ~ 40 % Luftfeuchtigkeit.</li>
	<li>Drehen Sie die Eier 1 X pro Tag und es ihnen ermöglichen, zu HH34 inkubieren (8 Tage nach Befruchtung).</li>
</ol>2. Vorbereitung und Sterilisation von Materialien
<ol><li>Für 12 Embryonen, Autoklaven, 2 L destilliertes Wasser, 1 L 0,85 % Küken Kochsalzlösung, 12 glaspipetten, 1 Karton Papiertaschentuch, 24 2,5 x 2,5 cm Quadrate der halbdurchlässigen Filterpapier und 24 2,5 x 2,5 cm Quadrate aus Stahldraht mesh mit den Kanten nach unten gebogen. Um die Materialien zu sterilisieren Autoklaven bei 121 ° C für mindestens 15 Minuten. Hinweis: Das Autoklavieren der Materialien kann im Voraus erfolgen.</li>
	<li>Den Inkubator mit 70 % Ethanol durch Abwischen der Seiten, die Regale und die Tür zu sterilisieren. Platz 2-Kunststoff-Behälter mit autoklaviert destilliertes Wasser in die brutmaschine Feuchtigkeit zu halten. Prewarm Kultur-Inkubator auf 37 ° C ± 1 ° C und ~ 40 % Luftfeuchtigkeit. Sicherzustellen Sie, dass der Inkubator ist völlig dunkel; Wenn die Tür transparent ist, mit Alufolie bedecken.</li>
	<li>Prewarm 100 mL Nährmedium und 1 mL Penicillin, indem man sie in der Kultur-Inkubator.</li>
	<li>Decken Sie den Arbeitsbereich mit einem Papiertuch und Sterilisieren der Bank durch Besprühen mit 70 % igem Ethanol. 2 Paar Zangen, Dissektion Schere, einer Rasierklinge und einem Plastiklöffel mit 70 % Ethanol in ähnlicher Weise zu sterilisieren.</li>
	<li>Platz 12 steril 100 mm Petrischalen und 24 sterile 35 mm Petrischalen auf der Bank für den Einsatz. Stellen Sie sicher, dass die Gerichte bis zum Gebrauch in einem sterilen Beutel bleiben.</li>
</ol>(3) Embryo Vorbereitung
Hinweis: Ab diesem Zeitpunkt tragen Sie eine schützende Staub Gesichtsmaske zur Vermeidung einer Kontamination der Kulturen. Eine bakterielle oder pilzartige Infektion wird die Kultur ruinieren und besteht die Gefahr der Ausbreitung schnell auf alle Kulturen im Inkubator.
<ol><li>Crack öffnen Sie das Ei und den Embryo zu einem 100 mm Petrischale mit 0,85 % Küken Kochsalzlösung zu übertragen. Stadium des Embryos nach den anatomischen Merkmalen in der Hamburger und Hamilton Inszenierung Leitlinien12 beschrieben und bestätigen den Embryo ist am HH34.</li>
	<li>Schneiden Sie den Hals und dann halbieren Sie den Kopf des Embryos an der Mittellinie mit einer sterilisierten Rasierklinge. Mit steriler Pinzette entfernen Sie das Gehirn. Lassen Sie den Schnabel intakt.</li>
</ol>4. Kultur-Setup
<ol><li>Platz 2 der 35 mm Petrischalen in einem 100 mm Petrischale.</li>
	<li>Legen Sie ein Stahl Drahtgeflecht im Inneren jedes 35 mm Petrischale.</li>
	<li>Mit Pinzette, 1 der halbierten Köpfe auf ein Stück 2,5 x 2,5 cm halbdurchlässigen Filterpapier mit dem Auge nach oben übertragen. Stellen Sie sicher, das Gewebe ist sicher und rutscht nicht von durch leicht kippen.</li>
	<li>Mit Pinzette, sorgfältig legen Sie das Augengewebe und das Filterpapier auf das Stahl Drahtgeflecht in 1 der 35 mm Petrischalen, erstellen eine erhöhte Bühne mit dem Augengewebe an der Spitze.</li>
	<li>Wiederholen Sie 4.3 und 4.4 mit der anderen Hälfte Kopf.</li>
	<li>Mit einer sterilen Glaspipette, vorsichtig hinzugeben Sie Nährmedium direkt in jede Petrischale von 35 mm bis die Stufe des Filterpapiers. Tauchen Sie der halbe Kopf im Nährmedium nicht.</li>
	<li>Das Nährmedium in jede Petrischale 50 µL von 10.000 U/mL Penicillin-Streptomycin hinzufügen.</li>
	<li>Falten Sie ein Stück Seidenpapier in ein kleines Quadrat und legen Sie sie innen 100 mm Petrischale. Mit autoklaviert destilliertes Wasser befeuchten.</li>
	<li>Legen Sie die Kulturschale in den sterilen, dunklen Brutschrank bei 37 ° C ± 1 ° C und ~ 40 % Luftfeuchtigkeit für bis zu 4 Tage.</li>
	<li>Wiederholen Sie die Schritte 3 und 4 für jedes Embryo. Hinweis: Die Anzahl der Embryonen benötigt von der spezifischen Experiment abhängen; Hier beschreiben wir das Protokoll für 12 Embryonen.</li>
</ol>5. Kultur-Wartung
<ol><li>Einmal pro Tag die Kunststoff-Behälter in den Inkubator mit frischem, autoklaviert destilliertes Wasser nachfüllen.</li>
	<li>Täglich prüfen Sie alle Kulturen für bakterielle oder Pilzinfektionen. Kulturen, die aufgelöst haben oder in denen das Medium Farbe verändert hat, sind infiziert. Entsorgen Sie alle Kulturen, die infiziert sind.</li>
</ol>6. Fixierung
<ol><li>Nach der Kultivierung, entfernen Sie alle Gerichte aus dem Inkubator.</li>
	<li>Entfernen Sie mit einer Zange, vorsichtig das Auge aus Filterpapier, kümmert sich nicht um das Gewebe reißen.</li>
	<li>Befestigen Sie das Augengewebe 4 % Paraformaldehyd in 1 x Phosphat-gepufferte Kochsalzlösung über Nacht bei 4 ° C oder in 10 % Neutral gepufferte Formalin über Nacht bei Raumtemperatur.</li>
	<li>Das Gewebe bei 4 ° C in 1 x Phosphat-gepufferte Kochsalzlösung aufbewahren.</li>
</ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functions+of+motor+proteins+in+echinoderm+embryos:+an+argument+in+support+of+antibody+inhibition+experiments.">Functions of motor proteins in echinoderm embryos: an argument in support of antibody inhibition experiments.</a> Cell Motility and the Cytoskeleton. 39 (4), 257-260 (1998).</li><li>Horakova, D., 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=Effect+of+FGFR+inhibitors+on+chicken+limb+development.">Effect of FGFR inhibitors on chicken limb development.</a> Development, Growth &amp; Differentiation. 56 (8), 555-572 (2014).</li><li>Gerlach, G. F., Morales, E. E., Wingert, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbead+Implantation+in+the+Zebrafish+Embryo.">Microbead Implantation in the Zebrafish Embryo.</a> Journal of Visualized Experiments. (101), e52943 (2015).</li><li>Luz-Madrigal, A., Grajales-Esquivel, E., Del Rio-Tsonis, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electroporation+of+Embryonic+Chick+Eyes.">Electroporation of Embryonic Chick Eyes.</a> Bio-protocol. 5 (12), e1498 (2015).</li><li>Roach, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Long-term+organ+culture+of+embryonic+chick+femora:+a+system+for+investigating+bone+and+cartilage+formation+at+an+intermediate+level+of+organization.">Long-term organ culture of embryonic chick femora: a system for investigating bone and cartilage formation at an intermediate level of organization.</a> Journal of Bone and Mineral Research. 5 (1), 85-100 (1990).</li><li>Jung, H. S., 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=Local+inhibitory+action+of+BMPs+and+their+relationships+with+activators+in+feather+formation:+implications+for+periodic+patterning.">Local inhibitory action of BMPs and their relationships with activators in feather formation: implications for periodic patterning.</a> Developmental Biology. 196 (1), 11-23 (1998).</li><li>McKinnell, I. W., Turmaine, M., Patel, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sonic+Hedgehog+functions+by+localizing+the+region+of+proliferation+in+early+developing+feather+buds.">Sonic Hedgehog functions by localizing the region of proliferation in early developing feather buds.</a> Developmental Biology. 272 (1), 76-88 (2004).</li><li>Smith, E. L., Kanczler, J. M., Oreffo, R. 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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> Journal of Morphology. 88 (1), 49-92 (1951).</li><li>Yu, 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+developmental+biology+of+feather+follicles.">The developmental biology of feather follicles.</a> The International Journal of Developmental Biology. 48, 181-191 (2004).</li><li>Jabalee, J., Hillier, S., Franz-Odendaal, T. 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K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Toward+an+understanding+of+the+epithelial+requirement+for+osteogenesis+in+scleral+mesenchyme+of+the+embryonic+chick.">Toward an understanding of the epithelial requirement for osteogenesis in scleral mesenchyme of the embryonic chick.</a> Journal of Experimental Zoology. 259 (1), 92-108 (1991).</li><li>Mbiene, J. P., MacCallum, D. K., Mistretta, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Organ+cultures+of+embryonic+rat+tongue+support+tongue+and+gustatory+papilla+morphogenesis+in+vitro+without+intact+sensory+ganglia.">Organ cultures of embryonic rat tongue support tongue and gustatory papilla morphogenesis in vitro without intact sensory ganglia.</a> Journal of Comparative Neurology. 377 (3), 324-340 (1997).</li><li>Cloney, K., Franz-Odendaal, T. 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Die Autoren haben nichts preisgeben.

Dieses Protokoll nutzt etablierten Gewebe züchten Techniken um ein Huhn Auge aus embryonalen Tag 8 (HH34) wachsen in Vitro für 4 Tage. Diese Raster-Kultivierung Methode wurde ursprünglich von Trowell15beschrieben. Wir optimieren ein Protokoll von Pinto und Halls 1991 Studie16 unter Verwendung einer halbdurchlässigen Membran mit dem Gitter, induktive Signalübertragung zwischen getrennten Gewebeschichten der embryonalen Auge15zu studieren...

Das embryonale Huhn (Gallus Gallus) ist ein hervorragendes Modellorganismus häufig verwendet im Bereich der Biologie. Die Inkubationszeit beträgt etwa 21 Tage und viele Eier können inkubiert werden gleichzeitig, Experimente zu machen, schnell und effizient. Vielleicht ist vor allem der Embryo auch leicht manipuliert ermöglicht die umfassende Studie der wichtigsten Entwicklungsprozesse und der Gene und Proteine, die diese Prozesse steuern.
Das embryonale Huhn Auge ist ein komplexes Organ, das durch das Zusammenspiel einer Reihe von verschiedenen Geweben ähnlich wie viele andere Körpersysteme entwickelt. Diese Methode ermöglicht die Untersuchung der Entwicklung dieser Gewebe, besonders in fortgeschrittenen Stadien der Entwicklung. Z. B. möglicherweise die mehrschichtige Netzhaut von besonderem Interesse für alle, die die Entwicklung des Nervensystems. Alternative Methoden, die das Studium der anderen Augengewebe zu ermöglichen, wie z. B. die Hornhaut, Glaskörperblutung Körper, der Linse, der Sklera und die Augenlider für Forscher von Nutzen sind. Das Huhn embryonalen Auge enthält auch eine Reihe von flachen Knochen, der skleralen Gehörknöchelchen, die als Modell für die Untersuchung von intramembranous Knochen Induktion und Verknöcherung in Wirbeltieren1verwendet werden können.
Derzeit gibt es eine Reihe von Methoden zur Untersuchung der embryonalen Entwicklung. Mikroinjektionen von inhibitorischen Antikörpern oder anderen hemmenden Moleküle2,3, chirurgisch implantiert Microbeads Inhibitor4trieft und Elektroporation5 sind alle Methoden, die zum Downregulate Gene verwendet werden können oder in einem Embryo interessierender Proteine. Ähnliche Methoden sind an Upregulate Proteine verwendet. Diese Methoden sind nicht ohne ihre Grenzen. Beispielsweise wenn Sie Chemikalien verwenden, um die embryonale Entwicklung zu ändern, die tödlichen Auswirkungen auf den Embryo ausgewertet werden müssen, und dies schränkt die Verwendung der oben genannten Methoden, um lokalisierte Websites Anwendung bei Dosen niedrig genug, um die Überlebensfähigkeit der gewährleisten die Embryo.
In-vitro- Gewebekulturen wurde in einer Vielzahl von Organismen verwendet, um Entwicklung zu studieren und kann verwendet werden, um einige der oben genannten Einschränkungen zu umgehen. Beispielsweise sind die Oberschenkelknochen6, Feder Knospen7,8und Gliedmaßen9 des Huhns alle untersucht worden mit Gewebe züchten Methoden, wie die Hoden der Maus10 und die Wurzeln und stengel der Pflanzen11. Diese Methoden Wissenschaftler gewähren ein hohes Maß an Kontrolle über die Gewebeentwicklung, wie z. B. die Fähigkeit, die Temperatur schwanken und verändern die nährstoffverfügbarkeit. Die Isolierung des Gewebes aus der gesamte Embryo macht es auch weit weniger anfällig gegen die tödlichen Auswirkungen von Chemikalien, wodurch es Manipulation Studien auf globaler Ebene bei höheren Konzentrationen. Ein weiterer bemerkenswerter Vorteil der in-vitro- Kultivierung ist der Erhalt der zellulären Umgebung das Gewebe; die Anordnung der Gewebe relativ unverändert bleibt, die es ermöglichen, die Interaktionen zwischen verschiedenen Gewebe Arten9zu untersuchen. In-vitro- Kultivierung öffnet Türen zu weiteren experimentellen nähert sich somit in in Vivo oder in Ovo Modelle nicht verfügbar.
Derzeit ist die Entwicklung des embryonalen Auges mit Chemikalien besonders anspruchsvoll. Eine Reihe von extraembryonic Membranen abdecken des Embryos erschwert Microbeads oder Chemikalien anzuwenden; der Embryo ist auch sehr aktiv in der Eizelle, als es älter, wird eine ohnehin schwierige Methode weiter zu erschweren. Dieses Protokoll ermöglicht einen einfachen Zugang für das Auge und seine umgebenden Gewebe, diese Hindernisse zu beseitigen, und gleichzeitig neue Möglichkeiten, die Entwicklungsprozesse im Auge zu untersuchen. Dieses Protokoll wurde gegründet, um die Induktion der skleralen Gehörknöchelchen innerhalb der embryonalen Auges zu studieren.

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tinfoil</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
	</tbody>
</table>

organotypic culture method to study the development of embryonic chicken tissues

Das embryonale Huhn wird häufig als zuverlässige Modellorganismus für vertebrate Entwicklung verwendet. Seine Erreichbarkeit und kurze Inkubationszeit macht Zeit es ideal für Experimente. Derzeit ist die Studie dieser Entwicklungsstörungen Wege im Huhn Embryo mittels Inhibitoren und Drogen an lokalisierten Standorten und bei niedrigen Konzentrationen mit einer Vielzahl von Methoden. In-vitro- Gewebekulturen ist eine Technik, die es die Untersuchung von Gewebe getrennt vom Wirtsorganismus ermöglicht, während gleichzeitig viele der körperlichen Einschränkungen vorhanden, bei der Arbeit mit ganzen Embryonen, wie die Anfälligkeit der Embryonen zu umgehen hohe Dosen von potenziell tödlichen Chemikalien. Hier präsentieren wir Ihnen eine organotypischen Kultivierung Protokoll für die Kultivierung der embryonalen halben Kopf in Vitro, präsentiert neue Möglichkeiten für die Prüfung von Entwicklungsprozessen über den derzeit etablierten Verfahren.

Mit dieser Methode kann Auge embryonalen Huhn vom 8. Tag seiner Entwicklung (HH34) in Vitro für 4 Tage kultiviert werden. Vier Tage in Ovo Entwicklungsstand entspricht HH38.
Diese Kultivierung Methode unterstützt die Entwicklung der Feder Knospen rund um das Auge und an den Augenlidern (Abbildung 1 b). Diese Feder Knospen sind nicht vorhanden in Ovo im HH34 vor der Kulti...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Organotypischen Kultur-Methode, die Entwicklung der embryonalen Gewebe zu studieren

Hier präsentieren wir eine organotypischen Kultivierung Protokoll zur embryonalen Organe wachsen in-vitro-. Mit dieser Methode kann die Entwicklung der embryonalen Gewebe untersucht werden und gleichzeitig ein hohes Maß an Kontrolle über die Kultur Umwelt.

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ...

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K Aufrufe.  Mount Saint Vincent University. Hier präsentieren wir eine organotypischen Kultivierung Protokoll zur embryonalen Organe wachsen in-vitro-. Mit dieser Methode kann die Entwicklung der embryonalen Gewebe untersucht werden und gleichzeitig ein hohes Maß an Kontrolle über die Kultur Umwelt.

Serie: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair cells are aligned in one row of inner hair cells and three rows of outer hair cells that extend along the basal to apical axis of the cochlea. The explant culture technique described here provides an efficient method to isolate and maintain cochlear explants from the embryonic mouse inner ear. Also, the morphology and molecular characteristics of sensory hair cells and nonsensory supporting cells within the cochlear explant cultures resemble those observed in vivo and can be studied within its intrinsic cellular environment. The cochlear explants can serve as important experimental tools for the identification and characterization of molecular and genetic pathways that are involved in cellular specification and patterning. Although transgenic mouse models provide an effective approach for gene expression studies, a considerable number of mouse mutants die during embryonic development thereby hindering the analysis and interpretation of developmental phenotypes. The organ of Corti from mutant mice that die before birth can be cultured so that their in vitro development and responses to different factors can be analyzed. Additionally, we describe a technique for electroporating embryonic cochlear explants ex vivo which can be used to downregulate or overexpress specific gene(s) and analyze their potential endogenous function and test whether specific gene product is necessary or sufficient in a given context to influence mammalian cochlear development1-8.

We present a method that describes isolation and culture of cochlear explants from embryonic mouse inner ear. We also demonstrate a method for gene transfer into cochlear explants via square-wave electroporation. The in vitro explant culture coupled with gene transfer technique enables researchers to study the effects of altering gene expression during development.

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

Auditory hair cells located within the mouse organ of Corti detect and transmit sound information to the central nervous system. The mechanosensory hair ...

Forschung

Entwicklungsbiologie

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during oogenesis. In order to study roles of such maternal factors in early embryonic development, it is desirable to manipulate their functions from the very beginning of embryonic development. Conventional ways of gene interference are achieved by injection of antisense oligonucleotides (oligos) or mRNA into fertilized eggs, enabling under- or over-expression of specific proteins, respectively. However, these methods normally require more than several hours until protein expression is affected, and, hence, the interference of gene functions is not effective during early embryonic stages. Here, we introduce an experimental system in which expression levels of maternal proteins can be altered before fertilization. Xenopus laevis oocytes obtained from ovaries are defolliculated by incubating with enzymes. Antisense oligos or mRNAs are injected into defolliculated oocytes at the germinal vesicle (GV) stage. These oocytes are in vitro matured to eggs at the metaphase II (MII) stage, followed by intracytoplasmic sperm injection (ICSI). By this way, up to 10% of ICSI embryos can reach the swimming tadpole stage, thus allowing functional tests of specific gene knockdown or overexpression. This approach can be a useful way to study roles of maternally stored factors in early embryonic development.

Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development

We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

Manipulation und<em&gt; In-vitro-</em&gt; Ausbau der<em&gt; Xenopus laevis</em&gt; Eizellen, von Intrazytoplasmatische Spermieninjektion gefolgt, um embryonale Entwicklungsstudie

Amphibian eggs have been widely used to study embryonic development. Early embryonic development is driven by maternally stored factors accumulated during ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transkriptom Profilieren<em&gt; In-Vivo</em&gt; Produziert Bovine Präimplantationsembryonen Mit Zwei-Farben-Microarray-Plattform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Epigenetische Regulation der Herz Differenzierung von embryonalen Stammzellen und Gewebe

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

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 ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Kultur von murinen embryonalen Mittelfuß: ein physiologisches Modell der enchondralen Verknöcherung

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissektion und Kultur der Maus Embryonale Niere

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Isolierung der embryonalen Gewebe und die Bildung von Wachteln-Huhn Chimären Organe Thymus am Beispiel

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.

Organotypischen Kultur-Methode, die Entwicklung der embryonalen Gewebe zu studieren

Zusammenfassung

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Ein Roman

Kultur von murinen embryonalen Mittelfuß: ein physiologisches Modell der enchondralen Verknöcherung

Dissektion und Kultur der Maus Embryonale Niere

Computergestützte Analyse der Keimbahn Caenorhabditis Elegans , die Verteilung der Kerne, Proteine und das Zytoskelett zu studieren

Isolierung der embryonalen Gewebe und die Bildung von Wachteln-Huhn Chimären Organe Thymus am Beispiel

Ein Instrument zur Analyse der C. elegans Embryonal development