Name: the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions
Uploaded: 2017-02-16T14:00:00.000+00:00
Duration: 7 min 34 s
Description: 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

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><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 25 mg/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 ...

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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.  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. 

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

Microbead Implantation in the Zebrafish Embryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

The zebrafish is an excellent model system for genetic and developmental studies. Bead implantation is a valuable tissue manipulation technique that can be used to interrogate developmental mechanisms by introducing alterations in local cellular environments. This protocol describes how to perform microbead implantation in the zebrafish embryo.

Microbead Implantation im Zebrafischembryo

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic ...

Forschung

Entwicklungsbiologie

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Stammzell-ähnlichen<em&gt; Xenopus</em&gt; Embryonale Explantate bis Früh Neurale Entwicklungs- Eigenschaften Studieren<em&gt; In-vitro-</em&gt; Und<em&gt; In-vivo-</em

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance to man of conventional invertebrate model systems and the complexity of genomes in vertebrates are critical challenges to modeling human normal and pathological conditions. The chordate Ciona intestinalis is an invertebrate chordate that emerged from a common ancestor with the vertebrates and may represent features at the interface between invertebrates and vertebrates. A common body plan with much simpler cellular and genomic composition should unveil gene regulatory network (GRN) links and functional genomics readouts explaining phenomena in the vertebrate condition. The compact genome of Ciona, a fixed embryonic lineage with few divisions and large cells, combined with versatile community tools foster efficient gene functional analyses in this organism. Here, we present several crucial methods for this promising model organism, which belongs to the closest sister group to vertebrates. We present protocols for transient transgenesis by electroporation, along with microinjection-mediated gene knockdown, which together provide the means to study gene function and genomic regulatory elements. We extend our protocols to provide information on how community databases are utilized for in silico design of gene regulatory or gene functional experiments. An example study demonstrates how novel information can be gained on the interplay, and its quantification, of selected neural factors conserved between Ciona and man. Furthermore, we show examples of differential subcellular localization in embryonic cells, following DNA electroporation in Ciona zygotes. Finally, we discuss the potential of these protocols to be adapted for tissue specific gene interference with emerging gene editing methods. The in vivo approaches in Ciona overcome major shortcomings of classical model organisms in the quest of unraveling conserved mechanisms in the chordate developmental program, relevant to stem cell research, drug discovery, and subsequent clinical application.

Embryo Microinjection and Electroporation in the Chordate Ciona intestinalis

We present transient transgenesis and gene knockdown in Ciona intestinalis, a chordate sister group to vertebrates, using microinjection and electroporation techniques. Such methods facilitate functional genomics in this simple invertebrate that features rudimentary characteristics of vertebrates, including notochord and head sensory epithelia, and many orthologs of human disease associated genes.

Embryo Mikroinjektions und Elektroporation in der bei Chor<em&gt; Schlauchseescheide</em

Simple model organisms are instrumental for in vivo studies of developmental and cellular differentiation processes. Currently, the evolutionary distance ...

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily accessible at all stages of development. Moreover, their optical clarity allows high resolution imaging of cell and molecular dynamics in the natural environment of the intact embryo. We are using a live imaging approach to analyze cell behaviors during neural crest cell migration and the outgrowth and guidance of neuronal axons.
Live imaging is particularly useful for understanding mechanisms that regulate cell motility processes. To visualize details of cell motility, such as protrusive activity and molecular dynamics, it is advantageous to label individual cells. In zebrafish, plasmid DNA injection yields a transient mosaic expression pattern and offers distinct benefits over other cell labeling methods. For example, transgenic lines often label entire cell populations and thus may obscure visualization of the fine protrusions (or changes in molecular distribution) in a single cell. In addition, injection of DNA at the one-cell stage is less invasive and more precise than dye injections at later stages.
Here we describe a method for labeling individual developing neurons or neural crest cells and imaging their behavior in vivo. We inject plasmid DNA into 1-cell stage embryos, which results in mosaic transgene expression. The vectors contain cell-specific promoters that drive expression of a gene of interest in a subset of sensory neurons or neural crest cells. We provide examples of cells labeled with membrane targeted GFP or with a biosensor probe that allows visualization of F-actin in living cells1.
Erica Andersen, Namrata Asuri, and Matthew Clay contributed equally to this work.

This protocol describes imaging of individual neurons or neural crest cells in living zebrafish embryos. This method is used to examine cellular behaviors and actin localization using fluorescence confocal time-lapse microscopy.

Live-Imaging von Zellbewegung und Aktin-Zytoskelett von einzelnen Neuronen und Zellen der Neuralleiste in Zebrafisch-Embryos

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

Biologie

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

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

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

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

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

Microinjection of Medaka Embryos for use as a Model Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1, as in zebrafish and the mouse. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions.
To take advantage of medaka fish whose embryos are transparent and develop externally, microinjection is an essential technique to inject cell-tracers for labeling cells, mRNAs or anti-sense oligonucleotides for over-expressing and knocking-down genes of interest, and DNAs for making transgenic lines.

Medaka and zebrafish are complementary for genetic dissection of vertebrate genome functions. This protocol highlights the key points for successful microinjection into medaka embryos, an important technique for embryological and genetic analysis using medaka and zebrafish in a laboratory.

Mikroinjektion von Medaka Embryonen für die Verwendung als Modell Genetic Organism

In this video, we demonstrate the technique of microinjection into one-cell stage medaka embryos. Medaka is a small egg-laying freshwater fish that allows ...

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing (Frog) Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design efficient remedies for diseases. Technologies enabling accurate identification and quantification of diverse types and large numbers of proteins would provide still missing information on molecular mechanisms orchestrating tissue and organism development in space and time. Here, we present a mass spectrometry-based protocol that enables the measurement of thousands of proteins in identified cell lineages in Xenopus laevis (frog) embryos. The approach builds on reproducible cell-fate maps and established methods to identify, fluorescently label, track, and sample cells and their progeny (clones) from this model of vertebrate development. After collecting cellular contents using microsampling or isolating cells by dissection or fluorescence-activated cell sorting, proteins are extracted and processed for bottom-up proteomic analysis. Liquid chromatography and capillary electrophoresis are used to provide scalable separation for protein detection and quantification with high-resolution mass spectrometry (HRMS). Representative examples are provided for the proteomic characterization of neural-tissue fated cells. Cell-lineage-guided HRMS proteomics is adaptable to different tissues and organisms. It is sufficiently sensitive, specific, and quantitative to peer into the spatio-temporal dynamics of the proteome during vertebrate development.

Cell-Lineage Guided Mass Spectrometry Proteomics in the Developing Frog Embryo

Here we describe a mass spectrometry-based proteomic characterization of cell lineages with known tissue fates in the vertebrate Xenopus laevis embryo.

Zellliniengesteuerte Massenspektrometrie-Proteomik im sich entwickelnden (Frosch-)Embryo

Characterization of molecular events as cells give rise to tissues and organs raises a potential to better understand normal development and design ...

Chemie

Generation of Na&#239;ve Blastoderm Explants from Zebrafish Embryos

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. However, because of the complexity and redundancy of embryonic signals, it can be challenging to discern the complete role of any single signal during early embryogenesis. By explanting the animal region of the zebrafish blastoderm, relatively na&#239;ve clusters of embryonic cells are generated that can be easily cultured and manipulated ex vivo. By introducing a gene of interest by RNA injection before explantation, one can assess the effect of this molecule on gene expression, cell behaviors, and other developmental processes in relative isolation. Furthermore, cells from embryos of different genotypes or conditions can be combined in a single chimeric explant to examine cell/tissue interactions and tissue-specific gene functions. This article provides instructions for generating zebrafish blastoderm explants and demonstrates that a single signaling molecule - a Nodal ligand - is sufficient to induce germ layer formation and extension morphogenesis in otherwise na&#239;ve embryonic tissues. Due to their ability to recapitulate embryonic cell behaviors, morphogen gradients, and gene expression patterns in a simplified ex vivo system, these explants are anticipated to be of great utility to many zebrafish researchers.

Zebrafish blastoderm explants are generated by isolating embryonic cells from endogenous signaling centers within the early embryo, producing relatively na&#239;ve cell clusters easily manipulated and cultured ex vivo. This article provides instructions for making such explants and demonstrates their utility by interrogating roles for Nodal signaling during gastrulation.

Erzeugung naiver Blastoderm-Expflanzen aus Zebrafisch-Embryonen

Due to their optical clarity and rapid development, zebrafish embryos are an excellent system for examining cell behaviors and developmental processes. ...

Assessing Signaling Properties of Ectodermal Epithelia During Craniofacial Development

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue interactions that regulate patterning and morphogenesis of vertebrates (e.g., 1, 2, 3, 4). Here, we illustrate a method that exploits this accessibility to test the signaling and patterning properties of ectodermal tissues during facial development. In these experiments, we create quail-chick 5 or mouse-chick 6 chimeras by transplanting the surface cephalic ectoderm that covers the upper jaw from quail or mouse onto either the same region or an ectopic region of chick embryos. The use of quail as donor tissue for transplantation into chicks was developed to take advantage of a nucleolar marker present in quail but not chick cells, thus allowing investigators to distinguish host and donor tissues 7. Similarly, a repetitive element is present in the mouse genome and is expressed ubiquitously, which allows us to distinguish host and donor tissues in mouse-chick chimeras 8. The use of mouse ectoderm as donor tissue will greatly extend our understanding of these tissue interactions, because this will allow us to test the signaling properties of ectoderm derived from various mutant embryos.

This article describes a tissue transplantation technique that was designed to test the signaling and patterning properties of surface cephalic ectoderm during craniofacial development.

Die Beurteilung Signaling Eigenschaften von ektodermale Epithel Während Craniofacial Entwicklung

The accessibility of avian embryos has helped experimental embryologists understand the fates of cells during development and the role of tissue ...

High Throughput Microinjections of Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of treatment solution is loaded into a microinjection needle that is used to physically inject individual immobilized cells or embryos. Despite the need for initial training to perform this procedure for high-throughput delivery, microinjection offers maximum efficiency and reproducible delivery of a wide variety of treatment solutions (including complex mixtures of samples) into cells, eggs or embryos. Applications to microinjections include delivery of DNA constructs, mRNAs, recombinant proteins, gain of function, and loss of function reagents. Fluorescent or colorimetric dye is added to the injected solution to enable instant visualization of efficient delivery as well as a tool for reliable normalization of the amount of the delivered solution. The described method enables microinjection of 100-400 sea urchin zygotes within 10-15 min.

Microinjection is a common technique used to deliver DNA constructs, mRNAs, morpholino antisense oligonucleotides or other treatments into eggs, embryos, and cells of various species.

Hochdurchsatz-Mikroinjektionen von Sea Urchin Zygotes

Microinjection into cells and embryos is a common technique that is used to study a wide range of biological processes. In this method a small amount of ...

Die Kraft der Einfachheit: Seeigel Embry als

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