We are grateful to Gurdon laboratory members for useful discussion. K.M. is a Research Fellow at Wolfson College and is supported by the Herchel Smith Postdoctoral Fellowship and the Great Britain Sasakawa Foundation. Gurdon laboratory is supported by grants from the Wellcome Trust (RG69899) and MRC to J.B.G.

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P., 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=Mammalian+nuclear+transplantation+to+Germinal+Vesicle+stage+Xenopus+oocytes+-+a+method+for+quantitative+transcriptional+reprogramming.">Mammalian nuclear transplantation to Germinal Vesicle stage Xenopus oocytes - a method for quantitative transcriptional reprogramming.</a> Methods. 51 (1), 56-65 (2010).</li><li>Astrand, C., Belikov, S., Wrange, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histone+acetylation+characterizes+chromatin+presetting+by+NF1+and+Oct1+and+enhances+glucocorticoid+receptor+binding+to+the+MMTV+promoter.">Histone acetylation characterizes chromatin presetting by NF1 and Oct1 and enhances glucocorticoid receptor binding to the MMTV promoter.</a> Experimental cell research. 315 (15), 2604-2615 (2009).</li><li>Amaya, E., Kroll, K. 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The authors have nothing to disclose.

Wir hier ausführlich eine neue Methode, um mütterliche Faktoren zum Abbau oder exogene Faktoren vor der Befruchtung überexprimieren. Dieses System erfordert zwei Mikroinjektionen, sondern überspringt die Operation von Fröschen, in der Host-Übertragungsmethode 7,17 verwendet. Es ist optimal, um den Frosch Operation Schritt im Hinblick auf die Pflege der Tiere zu entfernen. Darüber hinaus haben wir nicht zu berücksichtigen, für die Qualität der Gast weiblichen Frösche für die Transferexperimente zu nehmen, was bedeutet, dass wir von einem biologischen Faktor, der den Erfolg der Experimente betrifft loszuwerden. Daher in diesem System die Qualität der Eizellen von PMSG grundiert Frösche erhalten ist ein wichtiger Faktor, der biologischen Schlüssel für erfolgreiche Experimente ist. Die Qualität der Eizellen nach der Entnahme der Eierstöcke oder nach defolliculation beurteilt werden. Wenn irgendeine Abnormalität in diesen Phasen beobachtet wird, ist es optimal, einen weiteren Ovar von neuen Frosches sammeln. Ein weiterer Punkt, wenn man die Qualität der Oozyten zu überprüfen, ist nach Eizellreifung. Wenn weniger als 80% der progesteronE-behandelten Oozyten zeigen Anzeichen von Reife, können Sie nicht in der Lage, die genug Anzahl von Embryonen für weitere Analysen zu erhalten. Die Verwendung von enzymatischen defolliculation statt manuell defolliculation ist auch ein weiterer Vorteil der Verwendung dieses Systems, um die Arbeit für defolliculation reduzieren. Jedoch ist es immer noch möglich, dass eine manuelle defolliculation kann eine besser als der enzymatischen Behandlung. Wie in 3 gezeigt, fast 10% der in vitro gereiften Eizellen können die Schwimm Kaulquappenstadium erreichen. Diese Daten werden aus 13 unabhängigen Experimenten einschließlich der in 8 und neue Injektion Versuche berichtet gesammelt. Host-Übertragungsmethode benötigt 75-150 Oozyten in jeder Behandlung zur Gewinnung von aussagekräftigen Daten, da 30-60% der übertragenen Oozyten können die Neurulastadium 17 zu erreichen. Unsere Methode beginnt normalerweise mit 200-300 Oozyten in jeder Versuchsgruppe seit etwa 40-60% der gespalten Embryonen können die zu erreichenMuskelreaktion Bühne. Diese Daten legen nahe, dass beide Methoden unterstützt eine angemessene Entwicklungsrate. Bisher haben wir erhalten 10 Leben Frösche von Steuer mRNA injiziert und Kontrollantisense-Oligo-injizierten Oozyten mit dieser Technik, was darauf hinweist, dass dieser Ansatz unterstützt die Entwicklung durch Metamorphose. Unsere Oozyte manipulations ICSI Verfahren bietet die Möglichkeit, die Rolle der mütterliche Faktoren in sehr frühen embryonalen Entwicklung zu testen, kurz nach der Befruchtung. Darüber hinaus kann die Überexpression von Chromatin modifizierenden Faktoren vor der Befruchtung ermöglichen es, mütterlichen Chromatin vor der Befruchtung für das Verständnis für die Entwicklung notwendige mütterliche Chromatinzustände umzugestalten. Diese Strategie kann auch gut mit aktuellen Gen-Editing-Systeme wie TALENs (TALEN) 18,19 und CRISPR / CAS9 20,21 da diese schon vor der Befruchtung ausgedrückt werden zu arbeiten. Daher hat unser neuer Ansatz eine potential, für viele Anwendungen in der Zukunft verwendet werden.

Xenopus laevis ist eine weit verbreitete, leistungsfähige Modellorganismus, um die Entwicklung 1 zu untersuchen. Dies liegt daran, Xenopus Eiern ungewöhnlich groß sind (ungefähr 1,2-1,4 mm, im Vergleich zu Säugetier-Gegenstücke etwa 0,1 mm) und reichlich vorhanden. Eier enthalten mütterlich synthetisiert und gespeichert Komponenten, die genug, um die embryonale Entwicklung bis Mitte Blastula Übergang fahren sind (MBT, in der Phase 8-8,5 auftretenden mit 4.000-8.000 Zellen). MBT wird durch zygotischen Genom-Aktivierung, die dann produziert embryonalen Genprodukten Weiterentwicklung direkt verbunden. Zahlreiche Studien zielte darauf ab, mütterliche Faktoren wichtig für die Entwicklung zu identifizieren. Viele Untersuchungen beruhen auf der Injektion von Antisense-Oligonukleotide (Oligos) mit Morpholino-Oligonukleotide in befruchtete Embryonen, wobei Abbau der mütterlichen Proteine ​​können im Gastrula-Stadium 2-4 einzuhalten. Alternativ werden mRNAs befruchteten em injiziertbryonen um Genfunktionen zu stören oder zu Schicksale überexprimiert Proteine ​​zu verfolgen. Jedoch ist die Injektion in die Ein-Zell Embryonen normalerweise nicht beeinflusst Expressionsniveaus von mütterlichen Proteine ​​bei den sehr frühen embryonalen Stadien vor MBT. Heasman und Wylie gründete die Host-Übertragungsverfahren, um dieses Problem zu überwinden, 5. In ihrer Methode werden manuell defollikuliert Oozyten, die mit Antisense-Oligos injiziert und in Wirts Weibchen 6 übertragen. Proteine ​​werden vor der Befruchtung herunterreguliert, so dass die Rollen von regulierte Proteine ​​in frühen embryonalen Entwicklung untersucht werden können. Das mütterliche Abreicherungsverfahren führte zur Identifizierung von mehreren einzigartigen Entwicklungsrollen mütterlicher Proteine, wie in 7 überprüft. In diesem Bericht werden wir ausführlich unsere neu entwickelte Methode, bei der mütterliche Erschöpfung oder Überexpression von mRNA vor der Befruchtung wird ohne manuelle defolliculation und Host-Übertragungs erreicht8. Hand defolliculation erfordert viel Zeit und Host-Übertragungs braucht oft geschickte Techniken und die spezifische Lizenz für Tierchirurgie, wodurch die häufige Verwendung von mütterlichem Erschöpfung Verfahren behindern. Defolliculation und Host-Übertragungs durch enzymatische defolliculation 9,10 und intrazytoplasmatische Spermieninjektion (ICSI) von 11 bis 13 ersetzt sind. ICSI, Eier wurde ursprünglich zur transgenen Fröschen 12 zu produzieren. Sperma und embryonalen Kerne wurden in in vitro gereiften Eizellen Amphibien 11,14,15 transplantiert. Hier zeigen wir unsere Schritt-für-Schritt-Methode, um Spermien in vitro gereiften Eizellen, die waren zu injizieren mit Antisense-Oligos oder pre-mRNA injiziert.

<table border="0" cellpadding="0" cellspacing="0" width="589">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:87px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:167px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Pregnant Mare Serum Gonadotropin (PMSG)&#160;</td>
			<td style="width:87px;">MSD Animal Health</td>
			<td style="width:119px;">Vm 01708/4309</td>
			<td>PMSG-Intervet 5000iu powder and solvent for solution for injection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethyl 3-aminobenzoate methanesulfonate salt (MS222)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">A5040</td>
			<td>For anesthesia</td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Liberase TM Research Grade</td>
			<td style="width:87px;">Roche</td>
			<td style="width:119px;">05 401 127 001</td>
			<td>For oocyte defolliculation. Store at -20oC</td>
		</tr>
		<tr height="72">
			<td height="72" style="height:72px;width:216px;">Drummond Nanoject</td>
			<td style="width:87px;">Drummond Scientific Company</td>
			<td style="width:119px;">3-000-205/206</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">glass capillary&#160;</td>
			<td style="width:87px;">Alpha laboratories</td>
			<td style="width:119px;">7&#8221; Drummond #3-000-203-G/XL</td>
			<td>For microinjection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Micropipette Puller&#160;</td>
			<td style="width:87px;">SUTTER Instrument</td>
			<td style="width:119px;">Model D-97</td>
			<td>For microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">UltraPure Agarose</td>
			<td style="width:87px;">Invitrogen</td>
			<td style="width:119px;">16500-500</td>
			<td>For invitro maturation and ICSI</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">14 ml ultra-clear centrifuge tube&#160;</td>
			<td style="width:87px;">Beckman Coulter United Kingdom Ltd</td>
			<td style="width:119px;">344060</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">OptiPrep Density Gradient Medium (Iodixanol)</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D1556</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Digitonin</td>
			<td style="width:87px;">Sigma</td>
			<td style="width:119px;">D141</td>
			<td>Cell permeabilization reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Shaker</td>
			<td style="width:87px;">Hybaid&#160;</td>
			<td style="width:119px;">HB-SHK-1</td>
			<td>For oocyte defolliculation.</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dissecting microscope (Stereo zoom microscope)</td>
			<td style="width:87px;">ZEISS</td>
			<td style="width:119px;">Semi SV6</td>
			<td>For oocyte collection and microinjection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">50 &#956;m pore filter</td>
			<td style="width:87px;">CellTrics</td>
			<td style="width:119px;">04-0042-2317</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Ultracentrifuge</td>
			<td style="width:87px;">Beckman Coulter</td>
			<td style="width:119px;">Optima L-100XP</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">50 ml centrifuge tube</td>
			<td style="width:87px;">Cellstar Greiner Bio-One</td>
			<td style="width:119px;">227261 or 210261</td>
			<td>Oocyte collection and defolliculation reaction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tube</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">352097</td>
			<td>For sperm purification</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">90 mm Petri dish&#160;</td>
			<td style="width:87px;">Thermo Scientific</td>
			<td style="width:119px;">101VR20/C</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 60x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">353004</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Easy-Grip Cell Culture Dish, 35x15 mm</td>
			<td style="width:87px;">FALCON</td>
			<td style="width:119px;">351008</td>
			<td></td>
		</tr>
	</tbody>
</table>

manipulation and in vitro maturation of xenopus laevis oocytes, followed by intracytoplasmic sperm injection, to study embryonic development

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

Embryonalentwicklung ICSI-Embryonen unter Verwendung von in vitro gereiften Eizellen wurde (3A) untersucht. Reifungsgeschwindigkeiten von GV-Oozyten, um den MII Stufe sind variabel und hängt weitgehend von der Qualität der Oozyten. In guten Experimente fast 100% der GV-Oozyten reagieren auf Progesteron und zeigen Anzeichen von Eizellreifung, zuletzt als Eier am MII Stadium. Alle gereiften Eizellen wurden ICSI (7 ohne Oligo-injizierten Oozyten 3A, n = 13 für die Steuerung verwürfelten oligo-injizierten Oocyten und n =) unterzogen und etwa 25% der injizierten Eier abgespalten. Etwa 60% oder 80% des gespaltenen Embryonen aus Steuer oligo-injizierten Oozyten oder nicht injizierten Oozyten erzeugt jeweils erreichte die Blastula / Gastrulastadium. Unter den Blastula / Gastrula Embryonen waren von guter Qualität (fast keine Anzeichen von anomaler Spaltung und Apoptose) etwa die Hälfte der Embryonen in Oligo-Proben injiziert, während 82% der Blastula / Gastrula Embryonen waren von guter Qualität in non-injizierten Proben (3A). Schließlich, 41% und 11% der gespaltenen Embryonen in Oligo-injizierten Proben erreichten die Muskelreaktionen und die Schwimm Kaulquappe Stufen eingesetzt werden, wobei 60% und 29% der gespaltenen Embryonen nicht injizierten Proben war (3A). Diese ICSI-Embryonen werden die Mischung aus normalen und abnormalen Embryonen (3B). Einige von ihnen Metamorphose und zu entwickeln, um Frösche 8 reifen. Diese Ergebnisse legen nahe, dass die Injektion von Antisense-Oligonukleotide in GV-Oozyten, gefolgt von IVM und ICSI, ermöglicht eine effiziente frühen Embryonalentwicklung. Obwohl die Injektion von Antisense-Oligos selbst abnimmt Embryonalentwicklung, sind wir immer noch in der Lage, genug Embryonen für viele experimentelle Zwecke wie etwa die Überprüfung Entwicklungsraten zu erhalten, RT-PCR, Western-Blot und so weiter. <img alt="Figur 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Feige<html> ure 1: Typische Beispiele für Xenopus laevis Oozyten von guter Qualität oder schlechte Qualität für in-vitro-Reifung Experimente. (A) Ein Beispiel für eine schlechte Qualität Oozyten. Beispielsweise werden Oozyten zeigt fleckige Pigmentierung nicht für nachfolgende Experimente verwendet. Jede Xenopus laevis etwa 1,2-1,4 mm im Durchmesser. (B) Ein teilweise defollikuliert Eizelle. Die rechte Hälfte der Eizelle durch Follikelzellen, die durch das Vorhandensein der Blutgefäße erkennen kann abgedeckt. Ein Pfeil zeigt einen Bereich frei von Follikel-Zellen und in der die Injektionsnadel injiziert wird. (C) Ein Beispiel für gute Qualität Oozyten, die gleich große sind und zeigen, gleichmäßig pigmentierte Tierhalbkugeln mit klaren Kontrast zwischen dem Tier Hemisphäre und der pflanzlichen Hemisphäre. 2496 / 52496fig2highres.jpg &quot;/&gt; Abbildung 2:. Xenopus laevis Oozyten vor und nach der Reifung Nach Eizellreifung, an der Spitze der Tierhälften und Follikelzellen erscheinen klare weiße Flecken sind abgezogen. Jede Xenopus laevis Oozyten ist etwa 1 mm im Durchmesser. <img alt="Figur 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Abbildung 3: Entwicklung der ICSI-Embryonen aus in vitro gereiften Eizellen produziert. (A) Entwicklung von ICSI-Embryonen auf jeder Stufe zusammengefasst. Oocyten injiziert mit Steuer verwürfelten Antisense-Oligonukleotide (Oligo Steuereinspritzung) oder ohne Injektion (non-Injektion), gefolgt von IVM und ICSI. Die entsprechende Anzahl von Embryonen in jeder Stufe oberhalb der Balken dargestellt. Mittelwert ± SEM dargestellt. N = 4-13 unabhängigen Experimenten / verschiedene females. (B) Beispiele für überlebende ICSI-Embryonen. Diese Schwanzknospen-Embryonen wurden in einem Experiment, bei dem etwa 200 Oozyten wurden als Ausgangsmaterial verwendet wird produziert.

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

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

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

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

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Research

JoVE Journal

Developmental Biology

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

22.1K Aufrufe.  University of Cambridge. We describe methods of manipulating Xenopus laevis immature oocytes, in vitro maturation of oocytes to eggs, and intracytoplasmic sperm injection. This protocol allows degradation of some maternal proteins and overexpression of genes of interest at fertilization, and hence is valuable to study roles of specific factors in early embryonic development.

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

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the cephalochordate amphioxus, Branchiostoma lanceolatum. We use constructs for membrane and nuclear targeted mCherry and eGFP that have been modified to accommodate amphioxus codon usage and Kozak consensus sequences. We describe the type of injection needles to be used, the immobilization protocol for the unfertilized oocytes, and the overall injection set-up. This technique generates fluorescently labeled embryos, in which the dynamics of cell behaviors during early development can be analyzed using the latest in vivo imaging strategies. The development of a microinjection technique in this amphioxus species will allow live imaging analyses of cell behaviors in the embryo as well as gene-specific manipulations, including gene overexpression and knockdown. Altogether, this protocol will further consolidate the basal chordate amphioxus as an animal model for addressing questions related to the mechanisms of embryonic development and, more importantly, to their evolution.

Expression of Fluorescent Proteins in Branchiostoma lanceolatum by mRNA Injection into Unfertilized Oocytes

We report here the robust and efficient expression of fluorescent proteins after mRNA injection into unfertilized oocytes of Branchiostoma lanceolatum. The development of the microinjection technique in this basal chordate will pave the way for far-reaching technical innovations in this emerging model system, including in vivo imaging and gene-specific manipulations.

Expression von fluoreszierenden Proteinen in<em&gt; Branchiostoma lanceolatum</em&gt; Durch mRNA Injektion in ungedüngte Eizellen

We report here a robust and efficient protocol for the expression of fluorescent proteins after mRNA injection into unfertilized oocytes of the ...

Forschung

Entwicklungsbiologie

Culture of Embryonic Mouse Cochlear Explants and Gene Transfer by Electroporation

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

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

Kultur von embryonalen Maus Cochlear Explantate und Gentransfer durch Elektroporation

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

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

Using Confocal Analysis of Xenopus laevis to Investigate Modulators of Wnt and Shh Morphogen Gradients

The manuscript here provides a simple set of methods for analysing the secretion and diffusion of fluorescently tagged ligands in Xenopus. This provides a context for testing the ability of other proteins to modify ligand distribution and allowing experiments that may give insight into mechanisms regulating morphogen gradients.

Mittels konfokaler Analyse<em&gt; Xenopus laevis</em&gt; Um Modulatoren der Wnt und Shh Morphogen Gradienten Untersuchen

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the ...

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, ...

Technique to Target Microinjection to the Developing Xenopus Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus embryos are large, develop externally, and can be easily manipulated by microinjection or surgical procedures. In addition, fate maps have been established for early Xenopus embryos. Targeted microinjection into the individual blastomere that will eventually give rise to an organ or tissue of interest can be used to selectively overexpress or knock down gene expression within this restricted region, decreasing secondary effects in the rest of the developing embryo. In this protocol, we describe how to utilize established Xenopus fate maps to target the developing Xenopus kidney (the pronephros), through microinjection into specific blastomere of 4- and 8-cell embryos. Injection of lineage tracers allows verification of the specific targeting of the injection. After embryos have developed to stage 38 - 40, whole-mount immunostaining is used to visualize pronephric development, and the contribution by targeted cells to the pronephros can be assessed. The same technique can be adapted to target other tissue types in addition to the pronephros.

Technique to Target Microinjection to the Developing Xenopus Kidney

Here, we present a protocol to use fate maps and lineage tracers to target injections into individual blastomeres that give rise to the kidney of Xenopus laevis embryos.

Technik auf Zielmikroinjektions an die Entwicklungs<em&gt; Xenopus</em&gt; Kidney

The embryonic kidney of Xenopus&#160;laevis (frog), the pronephros, consists of a single nephron, and can be used as a model for kidney disease. Xenopus ...

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into the developing oocyte. Because these events rely on maternal products which typically act very soon after fertilization-that preexist inside the egg, standard approaches for expression and functional reduction involving the injection of reagents into the fertilized egg are typically ineffective. Instead, such manipulations must be performed during oogenesis, prior to or during the accumulation of maternal products. This article describes in detail a protocol for the in vitro maturation of immature zebrafish oocytes and their subsequent in vitro fertilization, yielding viable embryos that survive to adulthood. This method allows the functional manipulation of maternal products during oogenesis, such as the expression of products for phenotypic rescue and tagged construct visualization, as well as the reduction of gene function through reverse-genetics agents.

Functional Manipulation of Maternal Gene Products Using In Vitro Oocyte Maturation in Zebrafish

An optimized protocol for the in vitro maturation of zebrafish oocytes used for the manipulation of maternal gene products is presented here.

Funktionelle Manipulation der mütterlichen Gene Produkte verwenden<em&gt; In Vitro</em&gt; Oozytenreifung in Zebrabärbling

Cellular events that take place during the earliest stages of animal embryonic development are driven by maternally derived gene products deposited into ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualisierung und quantitative Analyse der embryonalen Angiogenese in<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

The embryonic chicken is commonly used as a reliable model organism for vertebrate development. Its accessibility and short incubation period makes it ideal for experimentation. Currently, the study of these developmental pathways in the chicken embryo is conducted by applying inhibitors and drugs at localized sites and at low concentrations using a variety of methods. In vitro tissue&#160;culturing is a technique that enables the study of tissues separated from the host organism, while simultaneously bypassing many of the physical limitations present when working with whole embryos, such as the susceptibility of embryos to high doses of potentially lethal chemicals. Here, we present an organotypic culturing protocol for culturing the embryonic chicken half head in vitro, which presents new opportunities for the examination of developmental processes beyond the currently established methods.

Here, we present an organotypic culturing protocol to grow embryonic chicken organs in vitro. Using this method, the development of embryonic chicken tissue can be studied, while maintaining a high degree of control over the culture environment.

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

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

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Mikroinjektion von DNA in Augenknospen in Xenopus laevis Embryonen und Bildgebung von GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological processes. In particular, appropriate regulation of calcium activity patterns during embryogenesis is necessary for many aspects of vertebrate neural development, including proper neural tube closure, synaptogenesis, and neurotransmitter phenotype specification. While the observation that calcium activity patterns can differ in both frequency and amplitude suggests a compelling mechanism by which these fluxes might transmit encoded signals to downstream effectors and regulate gene expression, existing population-level approaches have lacked the precision necessary to further explore this possibility. Furthermore, these approaches limit studies of the role of cell-cell interactions by precluding the ability to assay the state of neuronal determination in the absence of cell-cell contact. Therefore, we have established an experimental workflow that pairs time-lapse calcium imaging of dissociated neuronal explants with a fluorescence in situ hybridization assay, allowing the unambiguous correlation of calcium activity pattern with molecular phenotype on a single-cell level. We were successfully able to use this approach to distinguish and characterize specific calcium activity patterns associated with differentiating neural cells and neural progenitor cells, respectively; beyond this, however, the experimental framework described in this article could be readily adapted to investigate correlations between any time-series activity profile and expression of a gene or genes of interest.

Fluorescent Calcium Imaging and Subsequent In Situ Hybridization for Neuronal Precursor Characterization in Xenopus laevis

We present a two-part protocol that combines fluorescent calcium imaging with in situ hybridization, allowing the experimenter to correlate patterns of calcium activity with gene expression profiles on a single-cell level.

Fluoreszierende Calcium-Bildgebung und anschließende In-Situ-Hybridisierung zur neuronalen Vorläufercharakterisierung bei Xenopus laevis

Spontaneous intracellular calcium activity can be observed in a variety of cell types and is proposed to play critical roles in a variety of physiological ...