Die Autoren danken für die freundliche Robert Kelsh teilen die Zebrafisch-Promotor sox10 Reagenz.

1. Embryo-Sammlung für Photokonversion <ol><li> Kaede transgenen Zebrafisch zwischen 5 und 18.00 Uhr am Abend: Bis mindestens zehn Paar sox10 ein. </li><li> Am nächsten Morgen, Trennwände ziehen und sammeln Eier gegen Mittag. Übertragen Sie sie auf Petrischalen und sie in 28,5 ° C Inkubator. </li><li> Mit rund 24 Stunden nach der Befruchtung, reinigen Sie die Petrischalen durch die Beseitigung von toten Embryonen. Überprüfen Sie das Entwicklungsstadium der Embryonen 13 mit Lichtfeld-Mikroskopie und jede Fluoreszenzmikroskop mit enhanced green fluorescent protein (EGFP)-Filter. Kaede wird durch diesen Filter sichtbar sein. </li><li> Wenn Embryonen erreichen 60 hpf Transfer fünf hell fluoreszierende Embryonen in einem separaten Petrischale und dechorionate sie gegebenenfalls. </li></ol> 2. Montage der Embryo für Photokonversion <ol><li> Montieren Sie den Embryo mit einem normalen Fluoreszenz-Mikroskop. Montage von Embryonen auf konfokalen Mikroskop ist sehr anspruchsvoll. </li><li> Verwenden Standard E3 Wasser oder frisch zubereiten E3 mit dem folgenden Rezept: Fügen Sie den folgenden pro einem Liter destilliertem Wasser dH 2 O, Folgendes zu 1x E3L machen: 0,292 g 5,0 mM NaCl 0,013 g KCl 0,17 mm 0,044 g CaCl 2 0,33 mm (Trockenmittel, 96%, ACS Reagens) 0,081 g 0,33 mM MgSO 4 (wasserfrei) Optional: mit 200 ul von 0,05% Methylenblau auf 1X E3 als Fungizid und umrühren. Medium hält bei Raumtemperatur für 1 Woche. </li><li> Verwenden Sie Standard-Tricaine oder bereiten Tricaine mit folgender Rezeptur: Für 5 L von 0,2% Tricaine die folgenden Zutaten mischen: 45 ml Tris-Cl (pH 9,5 1M) 5 LH 2 O 10 g Tricaine </li><li> Vorbereiten &quot;Film-Saft&quot; (50 ml E3, 0,015% Tricaine Lösung), durch Zugabe von 3,75 ml 0,2% auf 46,25 ml Tricaine E3. </li><li> Agarose vorbereiten, indem man 46,25 ml E3 Wasser mit 46,35 mg niedrigen Schmelzpunkt (LMP) Agarose in einem 50 ml Glaskolben und lösen Feststoffpartikel durch die Mikrowelle. Dann fügen3,73 ml 0,2% Tricaine an Agarose und legte Glaskolben mit Agarose in 50 ° C im Wasserbad bis zur Nutzung. </li></ol> 3. Vorbereitung und Montage der Embryo <ol><li> Anesthetize Embryonen mit E3/0.015% Tricaine Lösung in Schritt 1.1.2 und Transfer Embryo Kammer Bogen aus. </li><li> Genießen Sie alle überschüssige E3 mit Papiergewebe und der Put-Agarose auf Embryo. </li><li> Position Embryo perfekt dorsal mit Microloader Tipps. Stellen Sie sicher, die ganze Embryo nicht auf dem Agarose schweben, aber schieben Sie den ganzen Körper nach unten. Dann gießen E3/0.015% Tricaine Lösung über Agarose eingebettet Embryo bis Kammer ist vollständig mit Flüssigkeit gefüllt. </li></ol> 4. Anpassen der Software-Einstellungen auf Konfokalmikroskop <ol><li> Verwenden 20X Lufteint Ziel (numerische Apertur 0,75; Pinholegröße 20) zur Fokussierung des Embryos. Drücken Sie dann die L100 Licht-Taste am Mikroskop und wählen Sie Kanäle in der Software. Öffnen Sie die Icons sehen / Erwerb controls/A1settings und click die konfokale Setup-Taste. Nächstes klicken Sie auf &quot;Auto&quot; und wählen Sie folgende Kanäle vor dem Schließen des Fensters wieder: K1: DAPI-Kanal 403,4 nm (Wellenlängen können zwischen 350 bis 410 nm verwendet werden) Ch2: 488,0 nm Ch3: 561,9 nm </li></ol> 5. Photo <ol><li> Schalten Sie Kanal 1 und 3, und schalten Sie Ch2, bevor Sie auf den Entfernen-Taste Verriegelung und Scannen. </li><li> Konzentrieren Sie sich auf Zellen von Interesse für Photo, was ist am besten, indem Sie mit 1frame/sec und Hochspannung (HV) 200 und dann hinauf in den Frames / sec und die Senkung der HV entsprechend bis zur 1/32 Frames / s und HV rund getan 100-120, abhängig von dem Hintergrundrauschen. </li><li> Wenn der Embryo im Fokus ist, sehen Sie auf der rechten Bildschirmrand, greifen die grünen Rand und verkleinert so den Bereich von Interesse für Photo und klicken Sie rechts mit der Maus passt. Kleiner ist besser, weil die Photo ist irreversibel. </li><li> Drehen Sie Bilder / s auf 1 und HV 200 und schlugscannen. Ein Bild gezoomt in der Fläche der Photo ist jetzt sichtbar. </li><li> Photoconvert Zellen durch Drehen auf Kanal 1 (403,4 nm). Aussetzen des photoconverting Laser überall 5-60 Sekunden, abhängig von der Größe des Bereichs, bis die grüne kaede wie durch die GFP-Kanal gesehen ist fast nicht vorhanden. </li><li> Wenn das grüne Signal kaede fast abwesend, schalten K1 aus. Schalten Ch2 und 3 auf. Dann klicken Sie rechts auf die Fenster auf A1 Scanbereich Feld ein und klicken &quot;Rückkehr zur Originalgröße&quot;, um zu überprüfen, ob gewünschten Zellen wurden photokonvertiertem. </li></ol> 6. Einen Z-Stapel <ol><li> Finden Sie das Symbol &quot;Capture Z-Serie&quot; in der Menüleiste und stellen Sie Ober-und Untergrenzen der Z-Stapel, bevor Sie es. Stellen LUTs. </li></ol> 7. Software-Einstellungen Time-lapse <ol><li> Zum A1Simple GUI-Box und kreuzen Sie folgende Einstellungen vor: 1/32 Bilder / s Größe 1024 durchschnittlich 4 oder 8X abhängig von der Anzahl von Z-Stapeln, die in einer Schleife gezogen werden muss </li><li> Gehen Sie zu:view / Erwerb Kontrollen / ND Sequenzerfassung und Zeitrahmen festgelegt (8-30min), klicken Sie auf kontinuierliche und klicken Sie auf Z-Stapel. Legen Grenzen getestet, wie vor und Film starten. </li><li> Halten Sie das Hinzufügen E3/0.015% Tricaine Lösungskammer Folie den ganzen Tag. In der Nacht, füllen Sie Kammer vollständig. Das wird bis zum nächsten Morgen dauern. </li><li> Am nächsten Morgen füllen E3/0.015% Tricaine Lösung und prüfen, ob Embryo noch lebt. Verlust der Fluoreszenz zeigt Tod. Weiter Minen und prüft, den ganzen Tag. </li><li> Wenn die Struktur bilden abgeschlossen, oder der Embryo gestorben ist, beenden Sie den Film. </li></ol> 8. Post-Produktion Verarbeitung <ol><li> Klicken Sie auf &quot;Show Maximum Intensity Projection&quot; in der Werkzeugleiste, klicken Sie dann rechts auf das Bild und schaffen neues Dokument. Das Video ist jetzt in der bestmöglichen Qualität sichtbar. Gehen Sie auf Bilder, die nicht benötigt werden, zu löschen und stellen LUTs. Schließlich Speichern unter. Avi. Dieses Format wird gut für Windows handelt. Für Mac-Download-Software to konvertieren. mov oder. wmv. </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=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, 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=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., 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=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., 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=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., 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=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., 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=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., 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=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., 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=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., 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+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

Die Autoren erklären, dass sie keine finanziellen Interessen konkurrieren.

Hier wird ein neues Verfahren zur Darstellung von kraniofazialen Entwicklung im Zebrafischmodell dargestellt. Die sox10: kaede transgenen Zebrafischlinie wurde erfolgreich verwendet, um die Migrationsmuster der CNCC im Detail zu beschreiben ist als Modellorganismus 5 verwendet. Frühere Studien haben Brutto Sehenswürdigkeiten wie das Auge verwendet werden, um die Zielzellen und haben sich auf kaede mRNA Injektion, Photo Assays oder Käfig Fluorescein Dextran für Photo 10, 1...

Gesichtsspalten stellen die häufigsten kraniofazialen Fehlbildungen, mit 1/700-1, 000 betroffenen Lieferungen ein. Störung der frühen embryonalen kraniofazialen Entwicklung kann zur Bildung von Lippen-Kiefer-Gaumen-(CL / P) führen. Während Ursachen für syndromale Spalt weitgehend gezeigt wurde, müssen die genetischen und epigenetischen Grundlagen der nicht-syndromale Formen der orofazialen Spaltbildung noch aufgedeckt 2-4 werden. Um die Ätiologie und Pathogenese dieser Fehlbildungen zu verstehen, ist es notwendig, die Entwicklung der kraniofazialen Strukturen auf zellulärer Basis aufzuklären. In allen Wirbeltierarten Schädel Neuralleistenzellen (CNCC) wandern aus dem dorsalen Neuralrohr, die Kiemenbögen, die zur Bildung von orofazialen Strukturen beitragen füllen. Störung der frühen embryonalen Neuralleiste Entwicklung ist auf die Bildung von Gesichtsschädelfehlbildungen einschließlich CL / P 5-7 führen. In Ad-dition, strukturelle Ähnlichkeiten zwischen Zebrafisch-und Säugetier kraniofazialen Entwicklung (CNCCs befinden sich in homologen Regionen) wird das Gen regulatorische Netzwerk hoch konserviert. Es wurde auch gezeigt, dass CNCCs entwickeln in der gleichen Weise zwischen amniote Arten und Zebrafisch 8, so dass das Zebrafisch eine leistungsfähige Organismus für das Studium der Entwicklungs-und genetischen Grundlagen von CL / P. Es hat viele Vorteile, wie geringe Größe, schnelle und Ex-utero embryonalen Entwicklung und hohe Zuchtraten. Darüber hinaus ist der Embryo optisch transparent, so dass es für die Beobachtung von komplexen Entwicklungsschritte unter dem Mikroskop 9. Es ist ein ideales Tiermodell für die Untersuchung von Migration und Differenzierung von Hirn Neuralleistenzellen. Kaede transgenen Modell 5: Aufbauend auf zuvor veröffentlichte Arbeit 8, 10, 11, die Migrationsmuster der CNCC wurde ausführlich über die sox10 beschrieben. Kaede ist ein Foto-Cabrio-Protein, das turns von grün auf rot nach Foto-Aktivierung und macht es möglich, CNCCs genau verfolgen. Während dieser Umwandlung das Peptidrückgrat gespalten, was darauf hindeutet, dass die Umwandlung ist stabil, was bedeutet, können die Zellen an ihren endgültigen Bestimmungsort 12 verfolgt werden. Transgene Linien mit kaede unter der Transkriptionskontrolle sox10 beschriftet zeigte, dass die amniote Gaumen und das Siebbein Platte des Zebrafisch sind homolog durch Fusion von bilateralen Kiefer Protuberanzen (MXP) mit der Prominenz frontonasalen (FNP) gebildet wird und dass das Y-förmige Naht ist analog Fusion zwischen Arten. Unter anderen Anwendungen, die sox10: kaede transgenen Zebrafisch-Modell wurde verwendet, um Videos von Zebrafisch-Embryonen in verschiedenen Entwicklungsstadien zu erzeugen, um die Bildung von normalen und abnormalen kraniofazialen Strukturen zeigen. Photo bestimmter Gruppen von Zellen ist es möglich, ihre Entwicklung zu verfolgen. Mit dieser Methode ein Ansatz, um die Live-Darstellung der Entwicklung craniofac erstellenial Strukturen im Zebrafisch eingeführt wird, macht es einfach, dieses komplexe Entwicklungsprozess visuell demonstrieren. Kaede transgenen Zebrafisch als Beispiel: Dieses Protokoll wird auf die gemeinsame Nutzung der Erfahrungen der Erzeugung diese Videos mit der normalen Entwicklung des Siebbein Platte in Sox10 ab. Dieses Protokoll kann weiter zu machen Zeitraffer-Videos von jeder Struktur von Hirn Neuralleistenzellen in Zebrafisch-Regelung angewendet.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

Wirbel palatogenesis ist ein hoch choreographierte und komplexen Entwicklungsprozess, der Migration von Schädel Neuralleiste (CNC)-Zellen, Konvergenz und Erweiterung des Gesichts Protuberanzen und Reifung des kraniofazialen Skeletts beinhaltet. Kaede transgenen Zebrafischlinie generiert wurde: Um den Beitrag der Schädel Neuralleiste auf bestimmte Regionen des Zebrafisches Gaumen sox10 studieren. Sox10-Linie bietet Beschränkung der kaede Reporterprotein zu der Neuralleiste, wodurch die Zellmarkierung eine genauere Verfahren als traditionelle Farbstoff oder Reporter-mRNA-Injektion. Kaede ist ein Foto-Cabrio-Protein, das von grün auf rot schaltet sich nach Foto-Aktivierung und macht es möglich, Zellen genau zu folgen. Die sox10: kaede transgene Linie wurde verwendet, um Abstammung Analyse durchführen, um CNC-Zellpopulationen, die Anlass zu Oberkiefer-Unterkiefer gegenüber Elementen und veranschaulichen Homologie von Gesichts Protuberanzen zu amniotes abzugrenzen. Dieses Protokoll beschreibt den Schritts, eine Live-Zeitraffer-Video eines sox10 generieren: kaede Zebrafischembryo. Entwicklung des Siebbein Platte wird als praktisches Beispiel dienen. Dieses Protokoll kann zur Erstellung einer Zeitraffer-Aufnahme eines konfokalen kaede oder ähnliche photoconvertible Reporterprotein in transgenen Zebrafisch angewendet werden. Weiterhin kann es verwendet werden, um nicht nur normal, sondern auch abnorme Entwicklung der kraniofazialen Strukturen in den Zebrafisch-Mutanten zu erfassen.

In der sox10: kaede transgene Linie, Zug-und Postmigrations CNCCs fluoreszierend grün markiert. Die CNCC Zellen mit grün fluoreszierenden kaede beschriftet rekapitulieren sox10 endogene mRNA-Expression 5. Unter anderen Anwendungen wurde dieses Tiermodell verwendet, um die Entwicklung von CNCC abhängig kraniofazialen Strukturen besser sichtbar zu machen. Normale Entwicklung der spezifischen Strukturen und auch pathologische Entwicklung der kraniofazialen Fehlbildungen, insbesond...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Visualisierung von experimentellen Daten ist zu einem wichtigen Element in Präsentation der Ergebnisse für die wissenschaftliche Gemeinschaft. Generation von Live-Zeitraffer-Aufnahme von wachsenden Embryos trägt zu einer besseren Präsentation und Verständnis für komplexe Entwicklungsprozesse. Dieses Protokoll ist eine Schritt-für-Schritt-Anleitung zur Zellmarkierung über der Photo kaede Protein im Zebrafisch.

Visualisierung von Craniofacial Entwicklung in den sox10: kaede transgener Zebrafische Zeile mit Zeitraffer-konfokale Mikroskopie

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K Aufrufe.  Massachusetts General Hospital.  Visualisierung von experimentellen Daten ist zu einem wichtigen Element in Präsentation der Ergebnisse für die wissenschaftliche Gemeinschaft. Generation von Live-Zeitraffer-Aufnahme von wachsenden Embryos trägt zu einer besseren Präsentation und Verständnis für komplexe Entwicklungsprozesse. Dieses Protokoll ist eine Schritt-für-Schritt-Anleitung zur Zellmarkierung über der Photo kaede Protein im Zebrafisch. 

Serie: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Überwachung Aktin Demontage mit Zeitraffer-Mikroskopie

Forschung

Biologie

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

Zwei-Photonen-Axotomie und Zeitraffer-konfokale Bildgebung in lebenden Zebrafisch-Embryonen

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Live-Imaging der Zebrafisch embryonalen Gehirn durch konfokale Mikroskopie

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Zeitraffer-Imaging der Mitose Nach siRNA-Transfektion

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor Zeitraffer-Imaging transgener Zebrafische: Visualisierung von Retinal Stammzellen durch gezielte Nervenzellen Ablation Activated

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Zeitraffer-Mikroskopie der frühen Embryogenese in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Elektroporation von Craniofacial Mesenchym

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Quantitative Analyse der ungerichtete Migration von Zellen unter Verwendung Zeitraffer-Video-Mikroskopie

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

Langfristige, High-Resolution Konfokale Zeitraffer Bildgebung<em&gt; Arabidopsis Cotyledon</em&gt; Epidermis während der Keimung

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analyse Craniofacial Morphogenese in Zebrafisch Mit 4D konfokale Mikroskopie

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...