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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The authors have nothing to disclose.

Nous ici en détail une nouvelle méthode pour épuiser les facteurs maternels ou surexpression des facteurs exogènes avant la fécondation. Ce système exige deux micro-injections, mais au lieu saute l&#39;opération de grenouilles, utilisé dans le procédé de transfert de 7,17 hôte. Il est optimal pour supprimer l&#39;étape de la chirurgie de la grenouille en termes de soins aux animaux. En outre, nous ne avons pas à prendre en compte pour la qualité de l&#39;hôte femmes grenouilles pour les expériences de transfert, ce qui signifie que nous pouvons nous débarrasser d&#39;un facteur biologique qui affecte le succès d&#39;expériences. Par conséquent, dans ce système, la qualité des ovocytes obtenus à partir de grenouilles PMSG amorcées est un facteur biologique majeur qui est la clé pour les expériences réussies. La qualité des ovocytes peut être jugé après la collecte des defolliculation ovaire ou après. Si aucune anomalie ne est observée lors de ces étapes, il est optimal pour recueillir un autre ovaire d&#39;une nouvelle grenouille. Un autre point quand vous pouvez vérifier la qualité de l&#39;ovocyte est après la maturation des ovocytes. Si moins de 80% de la progestéroneovocytes e-traités montrent des signes de maturation, vous ne pouvez pas être en mesure d&#39;obtenir le nombre suffisant d&#39;embryons pour d&#39;autres analyses. L&#39;utilisation de defolliculation enzymatique lieu de defolliculation manuel est également un autre avantage de l&#39;utilisation de ce système afin de réduire le travail nécessaire pour defolliculation. Cependant, il est encore possible que defolliculation manuel peut donner un meilleur développement que le traitement enzymatique. Comme le montre la figure 3, près de 10% des ovocytes in vitro mûri peut atteindre le stade de têtard piscine. Ces données sont recueillies auprès de 13 expériences indépendantes, y compris ceux présentés dans huit et de nouvelles expériences d&#39;injection. méthode de transfert de l&#39;hôte a besoin de 75 à 150 ovocytes dans chaque traitement pour obtenir des données significatives depuis 30-60% des ovocytes transférés peut atteindre le stade neurula 17. Notre méthode commence normalement par 200 à 300 ovocytes dans chaque groupe expérimental depuis environ 40-60% des embryons clivés peut atteindre lestade de la réponse musculaire. Ces données suggèrent que les deux méthodes prennent en charge un taux de développement raisonnable. Nous avons jusqu&#39;ici obtenu 10 grenouilles vivant de contrôle ARNm injecté et le contrôle antisens ovocytes oligo-injecté à l&#39;aide de cette technique, indiquant que cette approche favorise le développement par la métamorphose. Notre méthode de manipulation des ovocytes-ICSI fournit l&#39;occasion de tester le rôle des facteurs maternels pendant le développement embryonnaire très tôt, dès après la fécondation. En outre, la surexpression de facteurs modificateurs de la chromatine avant la fécondation peut permettre de remodeler la chromatine maternelle avant la fécondation pour comprendre États chromatine maternelle nécessaire pour le développement. Cette stratégie peut aussi bien travailler avec les systèmes d&#39;édition de gènes actuels tels que la transcription activateur comme effecteur nucléase (TALEN) 18,19 CRISPR et / CAS9 20,21 puisque ceux-ci peuvent être exprimées avant même la fécondation. Par conséquent, notre nouvelle approche a un potential à être utilisé pour de nombreuses applications à l&#39;avenir.

Xenopus laevis est largement utilisé, puissant organisme modèle pour étudier le développement 1. Ce est parce que les œufs de Xenopus sont inhabituellement élevé (environ 1/2 à 1/4 mm, par rapport à leurs homologues de mammifère étant d&#39;environ 0,1 mm) et abondante. Les œufs contiennent des composants mère synthétisés et stockés, qui sont suffisants pour conduire le développement embryonnaire jusqu&#39;à la mi-blastula transition (MBT, commise au stade 8-8,5 avec 4,000-8,000 cellules). MBT est accompagné par l&#39;activation du génome zygotique, qui produit alors des produits de gènes embryonnaires qui dirigent le développement. De nombreuses études visant à identifier les facteurs maternels importants pour le développement. De nombreuses études se appuient sur ​​l&#39;injection d&#39;oligonucléotides antisens (oligos), y compris des oligonucléotides morpholino dans embryons fécondés, dans lesquels la dégradation des protéines maternels de cas peut être observé au stade de la gastrula 2-4. Alternativement, les ARNm sont injectés pour em fécondébryons de perturber les fonctions des gènes ou de retracer destins de protéines surexprimés. Cependant, l&#39;injection dans les embryons au stade une cellule-normalement ne affecte pas les niveaux de protéines d&#39;expression maternelle à des stades embryonnaires précoces très avant MBT. Heasman et Wylie établi la méthode de transfert de l&#39;hôte de surmonter ce problème 5. Dans leur méthode, défolliculés manuellement ovocytes sont injectés avec des oligos antisens et transférés dans des femelles hôtes 6. Les protéines sont régulés à la baisse avant la fécondation afin que les rôles de protéines réprimés dans le développement embryonnaire précoce peuvent être examinées. Cette méthode de déplétion maternelle a conduit à l&#39;identification de plusieurs rôles de développement uniques de protéines maternelle, tel que révisé à 7. Dans ce rapport, nous détaillons notre méthode développée récemment, dans lequel l&#39;épuisement maternel ou la surexpression de l&#39;ARNm avant la fécondation est réalisée sans defolliculation manuel et le transfert de l&#39;hôte8. Defolliculation manuel nécessite beaucoup de temps et le transfert de l&#39;hôte doit souvent techniques habiles et la licence spécifique pour la chirurgie des animaux, entravant ainsi l&#39;usage fréquent de la méthode de l&#39;épuisement maternel. Defolliculation et le transfert de l&#39;hôte sont substitués par defolliculation enzymatique 9,10 et injection intracytoplasmique de spermatozoïdes (ICSI) 11-13, respectivement. ICSI aux œufs a été utilisé pour produire des grenouilles transgéniques 12. Sperme et embryons noyaux ont également été transplantés dans des ovocytes in vitro amphibiens mûri 11,14,15. Ici, nous montrons notre méthode étape par étape pour injecter des spermatozoïdes in vitro ovocytes maturés qui ont été pré-injecté avec oligos antisens ou ARNm.

<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.

Le développement embryonnaire des embryons ICSI utilisant dans des ovocytes in vitro mûri a été examiné (figure 3A). taux d&#39;ovocytes GV à la phase de maturation MII sont variables et dépendent en grande partie sur la qualité de l&#39;ovocyte. Dans de bonnes expériences, presque 100% des ovocytes GV répondre à la progestérone et montrent des signes de maturation ovocytaire, devenant finalement des œufs au stade MII. Tous les ovocytes ont été soumis à mûri ICSI et environ 25% des œufs injectés clivé (figure 3A, n = 13 pour le contrôle brouillés ovocytes oligo-injectée et n = 7 pour aucun des ovocytes oligo-injectée). Environ 60% ou 80% des embryons clivés, produites à partir d&#39;ovocytes oligo-injecté contrôle ou ovocyte non-injecté, respectivement, ont atteint le stade blastula / gastrula. Parmi les embryons blastula / gastrula, environ la moitié des embryons dans des échantillons d&#39;oligo-injecté étaient de bonne qualité (presque aucun signe de division anormale et l&#39;apoptose), tandis que 82% des embryons blastula / gastrula étaient de bonne qualité nsur-injection des échantillons (figure 3A). Enfin, 41% et 11% des embryons clivés dans des échantillons d&#39;oligo-injecté a atteint la réponse musculaire natation et les étapes de têtards, respectivement, tandis que 60% ​​et 29% des embryons clivés dans des échantillons non injectée ont fait (figure 3A). Ces embryons ICSI sont le mélange d&#39;embryons normaux et anormaux (figure 3B). Certains d&#39;entre eux subissent une métamorphose et développer à mûrir grenouilles 8. Ces résultats suggèrent que l&#39;injection d&#39;oligonucléotides antisens dans des ovocytes de GV, suivis par l&#39;IVM et ICSI, permet le développement embryonnaire précoce efficace. Bien que l&#39;injection de antisens se oligos diminue développement embryonnaire, nous sommes toujours en mesure d&#39;obtenir suffisamment d&#39;embryons pour de nombreuses fins expérimentales telles que la vérification des taux de développement, RT-PCR, western blot et ainsi de suite. <img alt="Figure 1" src="/files/ftp_upload/52496/52496fig1highres.jpg" /> Figue<html> ure 1: Des exemples typiques de Xenopus laevis ovocytes de bonne qualité ou de mauvaise qualité pour des expériences de maturation in vitro. (A) Un exemple de mauvaises ovocytes de qualité. Par exemple, les ovocytes montrant la pigmentation inégale ne sont pas utilisés pour des expériences ultérieures. Chaque ovocyte de Xenopus laevis est d&#39;environ 1.2 à 1.4 mm de diamètre. (B) Une partie de l&#39;ovocyte défolliculés. La moitié droite de l&#39;ovocyte est recouverte par les cellules de follicules, ce qui peut être perçue que par la présence de vaisseaux sanguins. Une flèche indique une zone exempte de cellules folliculaires et dans lequel l&#39;aiguille d&#39;injection est injecté. (C) Un exemple de bonnes ovocytes de qualité, qui sont de même taille et montrent hémisphères animales uniformément pigmentés avec un contraste évident entre l&#39;hémisphère animal et de l&#39;hémisphère végétatif. 2496 / 52496fig2highres.jpg &quot;/&gt; Figure 2:. Xenopus laevis ovocytes avant et après la maturation Après la maturation des ovocytes, des taches blanches claires apparaissent en haut des hémisphères animales et les cellules folliculaires sont décollée. Chaque ovocyte de Xenopus laevis est d&#39;environ 1 mm de diamètre. <img alt="Figure 3" src="/files/ftp_upload/52496/52496fig3highres.jpg" /> Figure 3: Développement d&#39;embryons ICSI, produites à partir dans des ovocytes in vitro mûri. (A) le développement d&#39;embryons ICSI à chaque étape est résumée. Les ovocytes ont été injectés avec contrôle brouillé oligonucléotides antisens (injection d&#39;oligo de contrôle) ou sans injection (non injection), suivis par l&#39;IVM et ICSI. Le nombre correspondant d&#39;embryons à chaque étape est indiqué ci-dessus les barres. Moyenne ± SEM est indiquée. N = 4-13 expériences indépendantes / f différenteemales. (b) des exemples de survie des embryons ICSI. Ces embryons au stade de bourgeon caudal ont été produits dans une expérience, dans laquelle environ 200 ovocytes ont été utilisés en tant que matériau de départ.

Watch this Scientific Journal Video about Manipulation and In Vitro Maturation of Xenopus laevis Oocytes, Followed by Intracytoplasmic Sperm Injection, to Study Embryonic Development at JoVE.com

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 et<em&gt; In Vitro</em&gt; Maturation du<em&gt; Xenopus laevis</em&gt; ovocytes, Suivi par injection intracytoplasmique de spermatozoïdes, pour étudier le développement embryonnaire

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 vues.  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.

Vidéo: 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.

L&#39;expression de protéines fluorescentes en<em&gt; Branchiostoma lanceolatum</em&gt; Par injection dans l&#39;ARNm non fécondés ovocytes

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

Recherche

Biologie du développement

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.

Culture de souris embryonnaires cochléaires explants et Gene Transfer par électroporation

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.

Utilisation de l&#39;analyse confocale<em&gt; Xenopus laevis</em&gt; Chargé d&#39;enquêter sur les modulateurs de Wnt et Shh morphogène Dégradés

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.

Stem Cell-like<em&gt; Xenopus</em&gt; Embryonnaires explants à étudier les caractéristiques du développement précoce Neural<em&gt; In Vitro</em&gt; Et<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.

Technique à la cible microinjection du Developing<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.

Manipulation fonctionnelle du gène maternel Produits utilisant<em&gt; In vitro</em&gt; Ovocyte Maturation en Zebrafish

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.

Visualisation et analyse quantitative de l&#39;angiogenèse embryonnaire dans<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.

Méthode de Culture organotypique pour étudier le développement des tissus embryonnaires de poulet

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.

Microinjection d'ADN dans 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 ...

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.

Imagerie de calcium fluorescent et hybridation in situ subséquente pour la caractérisation neuronale de précurseur dans Les laevis de Xenopus

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 ...