We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. Hoher Durchsatz Morpholino Design Strategy <ol><li> Identifizieren eines Gens (e) von Interesse (zB Kandidatengen - Ansatz, cis-regulatorische Analyse, RNA-Seq und / oder Proteom - Differential - Bildschirme). </li><li> Verwenden Sie genomischen, Transkriptom und Genexpressionsdaten auf häufig aktualisierte Webseiten (zB SpBase http://www.echinobase.org 20 und S. purpuratus Genom suchen http: ///urchin.nidcr.nih.gov/blast/index .html), um festzustellen, dass der Raum-Zeit-Expressionsprofil überlappt sich mit dem Entwicklungsmechanismus in Frage. Wenn kein Ausdruckdaten verfügbar ist, dann erzeugen qPCR Primer und / oder ein Antisense - in - situ - Sonde , die die Genexpressionsmuster zu beurteilen. </li><li> Nach der räumlichen und zeitlichen Expressionsmuster zu bestimmen, erhalten von den genomischen Websites die DNA-Sequenz. <ol><li> Zur Erzeugung von Morpholino-Oligonukleotide Translation-blocking, erhalten Sequenz of die 5 &#39;un-translatierte Region (5&#39; - UTR) direkt stromaufwärts vom Initiationscodon von exprimierten Sequenz - Tag (EST) der verfügbaren Datenbanken auf SpBase oder S. purpuratus Genom Search Websites. Wenn diese Informationen für das Gen von Interesse nicht verfügbar ist, führen Sie dann 5 &#39;RACE. </li><li> So entwerfen Sie einen Spleiß-Blocking - Morpholino, suchen Sie nach der genomischen Sequenz einschließlich der Exons und Introns mit dem Gerüst blastn- Werkzeug in SpBase oder S. purpuratus Genom Search Tool. </li></ol></li><li> Verwenden Sie das Oligonukleotid-Design Website http://oligodesign.gene-tools.com, um die gewünschte 25-Basenpaar-Morpholino-Sequenz zu entwerfen. <ol><li> Für eine translatorische blockierungs Morpholino, verwenden, um die mRNA-Sequenz von 5 &#39;zu 3&#39; als Quelle der Translationszielsequenz. Fügen Sie die UTR-Sequenz 5 &#39;(ca. 70 Nukleotide stromaufwärts von der Transkriptionsstartstelle) plus 25 Basen der kodierenden Region (hinter der Startstelle) mit dem Start-Codon markierten with Klammer (ATG). </li><li> Für eine Spleiß-blocking Morpholino, wählen Intron-Exon oder Exon-Intron-Grenze-Sequenz und umfassen 50 Basen (25 Basen von Exon-Sequenz und 25 Basen von Intron-Sequenz) um die Grenzbereiche. Scannen des Genoms mit dem Morpholino-Sequenz zu überprüfen, ob die Sequenz einzigartig ist. </li></ol></li></ol> 2. Mikroinjektion von Morpholino Oligonukleotiden <ol><li> Bereiten Sie 3 mM Stammlösungen der Morpholino-Oligonukleotide durch Zugabe von 100 ul Nuklease-freies Wasser in die 300 nmol Morpholino Fläschchen. HINWEIS: Verwenden Sie nicht DEPC behandeltem Wasser für die Wieder Suspension verwenden, da Diethylpyrocarbonat die Morpholino beschädigen können. </li><li> Zum ersten Rekonstitution Spin-Down das Fläschchen mit dem Lager-Oligonukleotid-Lösung für 30 s bei voller Geschwindigkeit, die (14.000 - 16.000 · g), kurz Wirbel, Wärme bei 65 ° C für 5 bis 10 min, kurz Wirbel, und halten Sie die Morpholino Lager bei Raum Temperatur für mindestens 1 h. Morpholino-Stammlösung<html> s werden von -20 ° C bis + 4 ° C gelagert. </li><li> Bereiten Injektionslösung morpholino Oligonukleotiden in der gewünschten Konzentration enthält. Diese Lösung enthält in der Regel 20% Glycerin oder 125 mM KCl als Trägerstoff und 15% FITC-Dextran (Fluorescein-Isothiocyanat-Dextran 10.000 MW 2,5 mg / &amp; mgr; l Vorratslösung). FITC-Dextran und anderen fluoreszierenden Dextran-Konjugate werden routinemäßig zur Identifizierung injizierten Embryonen durch Epifluoreszenzmikroskopie verwendet. Store Injektionslösungen bei -20 ° C. </li><li> Erhitzen Sie das Morpholino-Lösung bei 65 ° C in einem Heizblock oder Wasserbad für mindestens 2 - 5 min. </li><li> Kurz Spin für 30 s bei voller Geschwindigkeit die Morpholino-Lösung (14.000 - 16.000 xg), Wirbel für 1 min und Zentrifuge bei voller Geschwindigkeit (14.000 - 16.000 · g) für mindestens 10 min. </li><li> Laden Sie die Injektionsnadeln mit der Morpholino-Lösung. Für eine detaillierte Mikroinjektionsprotokoll finden Sie unter Stepicheva und Song, 2014 21 und Prost und Ettensohn 2004&quot;&gt; 22. </li></ol> 3. Fixierung und In - Situ - Protokoll bei 24 h nach der Befruchtung (HPF) in S. purpuratus Embry Hinweis: Dieses Protokoll von Arenas-Mena et al modifiziert wird. 2000 23 und Sethi et al. 2014 24. <ol><li> Fixierung <ol><li> Fügen Sie einige Tropfen Fixierungsmittel (siehe unten) zu Vertiefungen, die Embryonen vorsichtig mischen durch Pipettieren, und es ihnen ermöglichen, zu begleichen. Entfernen Sie die Fixierungslösung, und dann Embryonen mischen mit zwei zusätzlichen 180 ul Waschungen von Fixiermittel. Hinweis: Während der Wäschen durch sanftes Auf- und Abpipettieren mehrmals Es ist wichtig, gründlich die Embryonen mischen. Geschieht dies nicht , kann das Signal - Rausch - Verhältnis zu senken. </li><li> Lassen Sie die Embryonen in der zweiten Fixierungsmittel waschen zu fixieren für 50 min bis 1 h bei Raumtemperatur (RT) in 4% Elektronenmikroskopie Klasse Paraformaldehyd, bestehend aus 10 mM MOPS pH 7,0, 0,1% Tween-20 und künstliche seawater (ASW). Machen Sie diese Lösung jedes Mal für die besten Ergebnisse frisch. Zusätzlich zur einfachen und Praktikabilität Embryonen werden in Platten mit 96 Vertiefungen fixiert. <ol><li> Für 20 ml Fixiermittel Verwendung 5 ml 16% Paraformaldehyd, 15 ml ASW, 200 &amp; mgr; l 1 M MOPS pH 7,0, und 20 &amp; mgr; l Tween-20. </li></ol></li><li> Waschen 5mal bei RT mit 180 &amp; mgr; l MOPS-Waschpuffer von 0,1 M MOPS pH 7,0, bestehend, 0,5 M NaCl und 0,1% Tween-20 für mindestens 5 min oder bis Embryonen bis zum Boden des Bohrlochs fallen. Auch hier ist es wichtig, dass die Embryonen in die Waschpuffer durch vorsichtiges Auf- und Abpipettieren mehrmals gründlich zu mischen. MOPS Waschpuffer kann für 2 Tage verwendet werden, wenn bei 4 gespeichert ° C. <ol><li> Für 40 ml waschen MOPS - Puffer Gebrauch 4 ml 1 M MOPS pH 7,0, 4 ml 5 M NaCl, 32 ml dH 2 O und 40 ul Tween-20. </li><li> Fest Embryonen für 2 Tage bei 4ºC gelagert werden. HINWEIS: Wenn fixierte Embryonen länger speichern, dann mit 0,2% Natriumazid in MOPS-Puffer waschen Bakterienwachstum zu verhindern.</li></ol></li></ol></li><li> Vorhybridisierung <ol><li> Absaugen MOPS Waschpuffer und 180 ul einer 2 hinzuzufügen: 1-Verhältnis von MOPS waschen, um Hybridisierungspuffer Puffer, mischen Embryonen in die Lösung durch vorsichtiges Pipettieren mehrmals, und Inkubation mindestens 20 min bei RT. Hybridisierungspuffer besteht aus 70% Formamid, 0,1 M MOPS pH 7,0, 0,5 M NaCl, 1 mg / ml BSA und 0,1% Tween-20. <ol><li> Für 40 ml Hybridisierungspuffer Verwendung 4 ml 1 M MOPS pH 7,0, 4 ml 5 M NaCl, 4 ml dH 2 O, 0,04 g BSA und 40 ul Tween-20. Gut mischen durch Vortexen, fügen Sie 28 ml Formamid und Wirbel wieder. </li></ol></li><li> Entfernen Sie die 2: 1-Verhältnis von MOPS waschen und Hybridisierungspuffer und fügen 180 ul einer 1: 2-Verhältnis von MOPS-Puffer waschen, um Hybridisierungspuffer, mischen Embryonen in die Lösung und Inkubation für mindestens 20 min bei RT. </li><li> Vor sanft Sondenhybridisierung Embryonen mischen in 100-150 ul Hybridisierungspuffer. Inkubieren Embryonen bei 50 ° C für mindestens 1h. HINWEIS: Inkubieren Embryonen über Nacht akzeptabel ist. Vor der Inkubation dichten die Vertiefungen mit einer Klebefolie um ein Verdampfen zu verhindern. </li></ol></li><li> Hybridisation <ol><li> 0,3 ng / &amp; mgr; l-Sonde und 500 ug / ml Hefe-tRNA in Hybridisierungspuffer, dann Wirbel sanft Sonde Lösung zu schaffen - in einem separaten Röhrchen 0,1 hinzuzufügen. Hefe-tRNA wird an die Sondenlösung hinzugefügt des anti-sense Sondenbindungs ​​unspezifische zu verringern. Vorzuwärmen, diese Lösung auf 50 ° C und absaugen Hybridisierungspuffer. </li><li> Mischungs vorhybridisiert Embryonen in 100 &amp; mgr; l der Sondenlösung, Abdichten mit einer Klebefolie und hybridisieren bei 50 ° C für 2 bis 7 Tage , je nach der Sonde (die foxq2 Sonde kann für 2 Tage inkubiert werden). HINWEIS: Die Inkubationszeiten variiert die Sonde basiert. Einige Gene auf einem niedrigen Niveau exprimiert benötigen bis zu einer 7-tägigen Inkubation. 96-Well-Platten können in einer feuchten Box als Versicherung gegen Verdunstung, wenn die Klebs platziert werdenive Blatt nicht richtig abdichten. </li><li> Waschen 5 mal bei 50 ° C mit 180 &amp; mgr; l frisch zubereiteter MOPS-Waschpuffer für insgesamt 3 h. Dann wird 3x waschen (15 min) mit 180 &amp; mgr; l MOPS-Waschpuffer bei RT. Denken Sie daran, Embryonen in die Wäsche zu mischen jedes Mal Puffer durch mehrmals vorsichtig pipettieren. </li></ol></li><li> Antikörperinkubation <ol><li> Absaugen MOPS Waschpuffer und mischen Embryonen in 180 &amp; mgr; l der Blockierung über Nacht Puffer, bestehend aus 10% normalem Schafserum und 5 mg / ml BSA in MOPS Waschpuffer und Inkubation für mindestens 45 min bei RT oder 4 ° C. </li><li> Entfernen Blocking-Puffer und dann Embryonen Mischung in einen Blockierungspuffer, enthaltend 1: 1.500-Verdünnung von mit alkalischer Phosphatase konjugierten anti-Digoxigenin-Antikörper, verdünnt in Blockierungspuffer. Inkubieren über Nacht bei RT in einem verschlossenen Platte Verdunstung zu vermeiden. HINWEIS: Lassen Sie keine Antikörper auf mehr als über Nacht. </li><li> Waschen Embryonen 6-mal (5 min oder bis Embryonen fallen) in MOPS-Waschpuffer bei RT. Embryonenwerden über Nacht bei 4 ° C gelagert. </li></ol></li><li> In - situ - Entwicklung <ol><li> Waschen Embryonen 3 mal (10 min) bei RT mit frisch pH 9,5 - Puffer, bestehend aus 0,1 M Tris pH 9,5, 100 mM NaCl, 50 mM MgCl 2, 1 mM Levamisol und 0,1% Tween-20. <ol><li> 20 ml pH 9,5 - Puffer verwendet 2 ml 1 M Tris pH 9,5, 400 &amp; mgr; l 5M NaCl, 1 ml 1 M MgCl 2, 20 &amp; mgr; l Tween-20, 16,6 ml dH 2 O und 0,0048 g Levamisol. </li></ol></li><li> Inkubieren Embryonen bei 37 ° C in Färbepuffers vor Licht geschützt. Färbepuffer besteht aus 10% Dimethylformamid, 4,5 &amp; mgr; l / ml 4-Nitroblautetrazoliumchlorid (NBT) und 3,5 ul / ml 5-Brom-4-chlor-3-indolyl-phosphat (BCIP) in frisch pH 9,5-Puffer, . <ol><li> Für 1 ml Färbepuffers verwenden 100 ul Dimethylformamid, 4,5 ul NBT und 3,5 ul BCIP. </li></ol></li><li> Stoppen Sie die alkalische Phosphatase-Reaktion von 3 bis 5 Mal in MOPS Waschen Waschpuffer. Die Reaktion with die foxq2 Sonde dauert in der Regel 30 min bis 1 h. Einige Sonden benötigen Inkubation über Nacht bei entweder RT oder bei 4 ° C. </li><li> Mischen Sie Embryonen in eine Lösung von 70% MOPS waschen und 30% Glycerin. Das Glycerin stellt einen Brechungsindex, die für die Mikroskopie. Embryos können in dieser Lösung für mehrere Wochen gelagert werden. Verschließen Sie die Platten mit Kunststoff-Paraffin Verdunstung zu verhindern. </li></ol></li><li> Slide Vorbereitung und Bilderfassung <ol><li> Vorbereitung einer Folie durch drei kleine Streifen des doppelseitigen Klebebandes in ein Dreieck mit kleinen Lücken zwischen den Streifen auf einem Objektträger angeordnet werden. </li><li> Übertragen Sie die Embryonen in der 70% MOPS waschen und 30% Glycerin-Lösung in die Mitte des Dreiecks und die Abdeckung mit einem Deckglas. </li><li> Deckglasränder durch eine Schicht von Auftragen von Nagellack um die Ränder des Deckglases. </li><li> Zum Aufzeichnen von Bildern eine Verbindung Lichtmikroskop mit einem 20x-Objektiv mit und einer angeschlossenen Kamera. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, 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=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, 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=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. R. <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+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. 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=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

The authors have nothing to disclose.

Die Methodik präsentiert hier ist ein Beispiel, das die Macht der Verwendung von Embryonen mit weniger genomischen und morphologische Komplexität als Wirbeltiere zeigt die Signalübertragungswege und KNS Regierungs grundlegenden Entwicklungsmechanismen .. Viele Labors verwenden ähnliche Tests während der frühen Seeigel Entwicklung zu verstehen, die zu sezieren Signalisierung in anderen Zellschicksal Spezifikation Ereignisse beteiligt Wege (zB Notch, Igel, TGF - β und FGF - Signalisierung) <sup class="xref...

Gene regulatorische Netzwerke (KNS) und Signaltransduktionswege schaffen die räumliche und zeitliche Expression von Genen während der embryonalen Entwicklung, die verwendet werden, um die erwachsenen tierischen Körper Plan zu bauen. Zell-Zell-Signalwege sind wesentliche Bestandteile dieser regulatorischen Netzwerke, die Bereitstellung der Mittel, durch die Zellen in Verbindung stehen. Diese zellulären Interaktionen etablieren und die Expression von regulatorischen und Differenzierung Gene in und zwischen den verschiedenen Gebieten während der Embryonalentwicklung 1, 2 zu verfeinern. Wechselwirkungen zwischen sezerniert extrazellulären Modulatoren (Liganden, Antagonisten), Rezeptoren und Co-Rezeptoren steuern die Aktivitäten von Signaltransduktionswegen. Eine Auswahl von intrazellulären Molekülen wandelt diese Eingänge in veränderter Genexpression, Abteilung führt, und / oder die Form einer Zelle. Während viele der Schlüsselmoleküle bei den extrazellulären und intrazellulären Spiegel in den Hauptwegen verwendet werden,bekannt ist, ist es eine unvollständige Kenntnis der Komplexität der einzelnen Signalwege zu einem großen Teil. Zusätzlich interagieren verschiedene Signalwege oft miteinander entweder positiv oder negativ auf den extrazellulären, intrazellulären und Transkriptionsebenen 3, 4, 5, 6. Wichtig ist , dass die Kernkomponenten von Signaltransduktionswegen hoch in allen metazoan Spezies konserviert, und bemerkenswert, führen die meisten der wichtigsten Signalwege oft ähnliche Entwicklungsfunktionen in vielen Arten , wenn Organismen von eng verwandten Stämmen insbesondere 7, 8, 9 zu vergleichen, 10, 11. Die Studie der während der Entwicklung Signalgebung ist eine schwierige Aufgabe in jedem Organismus, undmehrere bedeutende Herausforderungen zu studieren Signalwege in den meisten Deuterostomia Modelle (Vertebraten, Invertebraten Chordaten , Kiemenlochtiere und Echinoderme): 1) In Vertebraten gibt es eine große Anzahl von möglichen Liganden und Rezeptor / Co-Modulator - Wechselwirkungen, die intrazelluläre Transduktion Moleküle sowie mögliche Wechselwirkungen zwischen verschiedenen Signalwegen aufgrund der Komplexität des Genoms 12, 13, 14; 2) Die komplexe Morphologie und morphogenetische Bewegungen bei Wirbeltieren oft erschweren funktionelle Interaktionen in und unter Signaltransduktionswege zu interpretieren; 3) Die Analysen in den meisten Nicht-echinoderm invertebrate Deuterostomia Modellarten werden durch kurze Fenster von gravidity mit Ausnahme einiger tunicate Arten 15, 16 begrenzt. DasSeeigel embryo hat einige der oben erwähnten Beschränkungen und bietet viele einzigartige Eigenschaften für eine detaillierte Analyse von Signaltransduktionswegen in vivo durchgeführt wird . Dazu gehören die folgenden: 1) Die relative Einfachheit des Genoms Seeigel reduziert signifikant die Anzahl der möglichen Ligand, Rezeptor / Co-Rezeptor und intrazellulären Transduktion Molekül - Wechselwirkungen 17; 2) Die KNS die Spezifikation und die Strukturierung der Keimblätter und die wichtigsten embryonalen Achsen steuern gut in Seeigel - Embryonen etabliert, in dem Verständnis der regulatorischen Kontext der Zelle / Gebiet Beihilfe Empfangen der Signale 18, 19; 3) Viele Signaltransduktionswege können zwischen frühen Spaltung und Gastrula Stadien untersucht werden , wenn Embryonen von einem einschichtigen Epithel , deren Morphologie bestehen ist einfacher zu analysieren; 4) Die Moleküle beinhaltend in Bahnen in den Seeigeln Signalisierung leicht manipuliert werden; 5) Viele Seeigel sind für 10 bis 11 Monate im Jahr (zB Strongylocentrotus purpuratus und Lytechinus variegatus trächtig). Hier präsentieren wir eine Methode, um systematisch und effizient Komponenten der Signalwege zu charakterisieren, die angeben, und das Muster Gebiete in Seeigel-Embryonen, die Vorteile zu verdeutlichen, dass mehrere wirbellosen Modellsysteme in der Studie von komplexen molekularen Mechanismen bieten.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

Im Seeigel Embryo wir haben gezeigt , dass drei verschiedene Wnt - Signalzweige (Wnt / β-Catenin, Wnt / JNK und Wnt / PKC) 4, 25 interact ein Wnt - Signalnetzwerk zu bilden , die von vorne nach hinten (AP) Strukturierung regelt. Eine der wichtigsten Folgen dieser Signalisierungsereignisse besteht darin , dass die anfängliche breit ausgedrückt anterioren Neuroektoderm (ANE) GRN auf ein kleines Gebiet um den vorderen Pol durch d...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Dieses Video Artikel beschreibt eine einfache In - vivo - Methodik , die verwendet werden können , um systematisch und effizient Komponenten komplexer Signalwege und regulatorische Netzwerke in vielen wirbellosen Embryonen zu charakterisieren.

Die Kraft der Einfachheit: Seeigel Embry als<em&gt; In Vivo</em&gt; Entwicklungsmodelle für komplexe Zell-zu-Zell-Signalnetzwerk Wechselwirkungen Studieren

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Unit 1

Principles of Cell Signaling

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7.8K Aufrufe.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

Serie: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protokolle für die Beschaffung Zygotic und somatischen Embryonen zur Untersuchung der Regulation der frühen Embryo-Entwicklung in der Modell-Hülsenfrucht<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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Entwicklungsbiologie

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Charakterisierung von Thymic Einschwingzeit Vorläufern in der Mouse Embryo Verwendung<em&gt; In Vivo</em&gt; Und<em&gt; In-vitro-</em&gt; Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

Studieren Wnt Signaling Während Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Induzierende komplette Polyp Regeneration von aboraler Physa des Starlet Sea Anemone<em&gt; Nematostella vectensis</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

Medikamentöse Behandlung und<em&gt; In Vivo</em&gt; Imaging von Osteoblasten-Osteoklasten-Wechselwirkungen in Medaka-Fisch Osteoporose Modell

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

In - vivo - Bildgebung von Expression Transgene Gene in einzelnen Retinal Vorläufern in Chimäre Zebrabärblingembryonen Studie Zellnichtautonome Einflüsse

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Live Imaging der Maus Sekundärer Gaumen Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualisierung der Knoten und Notochordal Platte In Gastrulating Maus-Embryonen mittels Rasterelektronenmikroskopie und ganze Mount Immunfluoreszenz

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Bewertung von oxidativen Schäden in den primären Maus-Augen-Oberflächenzellen/Stammzellen als Reaktion auf Uv-C-Schäden (UV-C)

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Eine geschichtete Montagemethode für die verlängerte Zeitraffer-Konfokalmikroskopie ganzer Zebrafisch-Embryonen

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...