Les auteurs remercient Adeline Dolly pour les photos de expiant sur la fibronectine, Eric Theveneau pour des discussions utiles et dans les animaleries de l&#39;Institut Curie. CM est un stage postdoctoral de la Région Ile de France (Domaine d&#39;Intérêt Majeur souches Pole), Université Paris Sud (Attaché Temporaire d&#39;Enseignement et de Recherche), et l&#39;Agence Nationale de la Recherche. Ce travail a été financé par l&#39;Université Paris Sud (Attractivité 2011), le Centre National de la Recherche Scientifique (Programme d&#39;Action Thématique et Incitative), Association pour la Recherche contre le cancer Grant SFI20101201882, Ligue contre le cancer, et l&#39;Agence Nationale de la Recherche (Agence Nationale de la Recherche Programme Blanc).

Les expériences sont conformes à la réglementation nationale et européenne sur la protection des animaux utilisés à des fins scientifiques et aux principes établis à l&#39;échelle internationale de remplacement, de réduction et de raffinement. Une. Préparation de plats fibronectine 14 <ol><li> Introduire à la pipette 500 ml de 10 mg / ml de fibronectine culture primaire dilué dans 1 x tampon phosphate salin (PBS) dans une boîte de Petri en plastique standard (Ø 40 x 11 mm). Incuber à 37 ° C pendant 1 heure. Remarque: si vous utilisez des plats en verre ou des lamelles de verre pour l&#39;imagerie ultérieure, utiliser un / ml solution fibronectine de 100 mg. </li><li> Rincer trois fois avec PBS 1x. </li><li> Remplacer PBS avec 500 ml de 1 x PBS contenant 0,1% albumine de sérum bovin (BSA). Incuber à 37 ° C pendant 30 min. </li><li> Rincer trois fois avec PBS 1x. </li><li> Remplissez avec PBS 1x et conserve à 4 ° C jusqu&#39;à utilisation (généralement pendant 24-48 h). </li></ol> 2. Dissection des explants NC &lt;ol&gt; <li> Préparer 3/4 normale amphibie moyen (NAM) (110 mM NaCl, 2 mM de KCl, 1 mM de Ca (NO 3) 2, 1 mM MgSO4, 0,1 mM d&#39;EDTA, 1 mM de NaHCO 3, 0,2 x PBS, 50 mg / ml gentamycine 10,15). Ne gardez pas plus de 1 semaine pour la dissection, conserver à 4 ° C. NAM plus ancien peut être utilisé pour la préparation de plats de dissection agarose revêtu (ci-dessous). </li><li> Préparer 60 mm boîtes de Pétri pour la dissection, revêtues avec 10 ml d&#39;agarose à 2% dans 3/4 NAM. </li><li> Récupérer des outils de dissection: pipettes Pasteur en plastique, une broche plus beaux insectes monté sur support de broche (voir le tableau des matériaux pour la référence), un &quot;couteau de sourcil&quot; (en poils de sourcils inclus dans la paraffine à l&#39;extrémité d&#39;une pipette Pasteur en verre 16), deux beaux pinces à dissection, P1000 pipette et des conseils, un stéréoscope binoculaire avec un grossissement 8-40X à 50, une source de lumière brillante avec des guides de fibres optiques. Tous les outils de dissection doivent être maintenus propres, après chaque expérience, les rincer deux fois avec de l&#39;eau distillée, une fois with 100% d&#39;éthanol et laisser sécher. Conserver à l&#39;abri de la poussière. </li><li> Remplissez le plat de dissection avec NAM frais presque au sommet. </li><li> Obtenir X. laevis embryons selon les procédures standard 10 et les vieillir jusqu&#39;à ce qu&#39;ils atteignent neurula étape 16 (selon Nieuwkoop et Faber tableau de développement 17). </li><li> Placez étape 16 embryons dans le plat de dissection à l&#39;aide de la pipette Pasteur en plastique. </li><li> Effectuer toutes les étapes ultérieures dans le cadre du champ binoculaire. Retirer la membrane vitelline avec deux paires de pinces. Veillez à ne pas endommager la partie dorsale de l&#39;embryon. </li><li> Attendez jusqu&#39;à ce que les embryons ont atteint le stade 17 pour commencer la dissection. Etape 17 est approprié depuis la crête neurale est clairement mis à part le tube neural, contrairement aux étapes précédentes. Après l&#39;étape 17, NC commence la migration et se mêlant à la mésenchyme environnant. </li><li> Creusez un petit trou dans l&#39;agarose avec des pinces pour faire un créneau pour les embryons afin de maintenireux au cours de la dissection. Placez un embryon dorsale vers le haut. Remarque: la pâte à modeler peut être utilisé pour maintenir les embryons de creuser un trou à la place dans l&#39;agarose. </li><li> Faire une incision dans la partie latérale du pli neural antérieur avec le couteau à sourcils et l&#39;axe d&#39;insecte. Veillez à ne pas aller trop loin dans l&#39;embryon: viser à couper à travers 2-3 couches de cellules (jamais atteindre le plus profond que le archenteron!). Pendant tout le processus de dissection, ne laissez jamais les tissus disséqués pour entrer en contact avec l&#39;interface air-liquide depuis X. tissus embryonnaires laevis sont lysées par la tension superficielle. </li><li> Faire un deuxième incision parallèle dans la partie la plus dorsale du pli de neurones en utilisant les mêmes instruments. </li><li> Si nécessaire, tourner le plat autour de 90 °. Faire une troisième incision avec le couteau perpendiculaire à sourcils et l&#39;axe d&#39;insecte, à la face postérieure des deux premières incisions. Le NC est une masse épaisse de cellules sous la couche externe pigmentée de l&#39;ectoderme, latéraldans le tube neural. </li><li> Utilisez la pointe du couteau de sourcil de détacher la couche pigmentée de l&#39;ectoderme NC sous-jacent, à partir du troisième bord de l&#39;incision. </li><li> Utiliser le côté du couteau de sourcil pour détacher le tissu NC partir de la troisième incision. Le tissu NC est plutôt grisâtre, tandis que le mésoderme céphalique sous-jacente est blanc. Séparez délicatement le NC du mésoderme. </li><li> Détachez le tissu NC antérieure de la vésicule optique, et couper à ce niveau pour libérer l&#39;expiant. Comme un contrôle de premières expériences, contrôle l&#39;expression de snail2 (un marqueur NC canonique) dans certains explants par hybridation in situ est recommandée. </li></ol> 3. Les explants de placage sur la fibronectine <ol><li> Remplissez le plat de fibronectine avec modification Danilchick moyen (MDM, NaCl 53 mM, Na2CO 3 5 mM, K gluconate de 4,5 mM, Na gluconate de 32 mM, MgSO 4 1 mM, CaCl2 1 mM, BSA 0,1%, ajuster le pH à 8,3 avec 1 M Bicine <sup&gt; 18). </li><li> Transférer les explants dans le plat de la fibronectine revêtu à l&#39;aide d&#39;une pipette P1000. Ne laissez jamais les explants de toucher l&#39;interface air-liquide. Tout d&#39;abord, la pipette du liquide dans la pointe, puis les explants, puis certains nouveau liquide. Ne pas permettre à l&#39;air dans la pointe tout en transférant les explants dans le plat de fibronectine. Une fois la pointe est à l&#39;intérieur du liquide de la boîte de fibronectine, laisser les explants vont lentement par gravité, ou pousser très doucement. Évitez d&#39;éjecter les explants trop vite. </li><li> Placez 10-30 explants dans le plat à l&#39;aide du couteau de sourcil. Éviter les parois de la capsule, qui ne sont pas adéquats pour l&#39;imagerie ultérieure. </li><li> Essayez de ne pas déplacer l&#39;antenne jusqu&#39;à ce que les explants ont attaché sur la fibronectine (cela prend environ 15-30 min). Si nécessaire, déplacez le plat très attentivement. </li><li> Placer le plat dans un incubateur réfrigéré (entre 15-20 ° C, pour le graphique de température voir 17). Lorsqu&#39;il est placé à 15 ° C, les cellules are la migration en général de manière efficace et de diffusion après 6 heures d&#39;incubation (ce calendrier peut varier entre les expériences). </li></ol> 4. Greffage explants dans des embryons d&#39;accueil <ol><li> Coupez une lamelle de verre bien en très petits morceaux à l&#39;aide de pinces grossières. La taille des pièces doit être d&#39;environ 1,5 mm 2. Plongez les morceaux dans une boîte de Pétri contenant 3/4 NAM ou PBS en utilisant une pince. </li><li> Préparation de l&#39;embryon hôte: disséquer l&#39;hôte NC à 3/4 NAM comme décrit dans 3) avec un soin particulier à ne pas endommager l&#39;embryon hôte tout en enlevant la membrane vitelline et la dissection du NC. La tension du tissu tend à augmenter la taille de l&#39;ablation: laisser l&#39;embryon hôte soigne partiellement tout en disséquant le donneur d&#39;embryon. </li><li> Dans un autre plat d&#39;agarose revêtues ou dans une autre partie du même plat, disséquer l&#39;expiant NC de l&#39;embryon donneur tel que décrit dans le protocole 3. Transférer la greffe NC dans le plat contenant l&#39;hôte avec soin, comme décrit dansProtocole 3. </li><li> Placer immédiatement l&#39;expiant NC sur la zone d&#39;ablation hôte en utilisant une pince. L&#39;explantation devrait fixer partiellement. Placer un morceau de lamelle de verre sur l&#39;embryon greffé à maintenir la greffe en place. Choisir un morceau de verre plus grand que l&#39;embryon pour éviter de l&#39;endommager. L&#39;embryon sera un peu aplatie. </li><li> Attendez au moins 15-20 minutes, puis retirez délicatement la lamelle. Que l&#39;embryon récupérer pendant 10 min et la pipette avec précaution dans un plat propre rempli de 3/4 NAM, en utilisant la pipette Pasteur en plastique. </li></ol>

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B., Hanken, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx2+is+essential+for+larval+hyobranchial+cartilage+formation+in+Xenopus+laevis.">Runx2 is essential for larval hyobranchial cartilage formation in Xenopus laevis.</a> Dev. Dyn. 236 (6), 1650-1662 (2007).</li><li>Yan, C. Y. I., Skourides, P., Chang, C., Samba Brivanlou, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=a+Xenopus+hnRNP+expressed+in+neural+and+neural+crest+tissues.">a Xenopus hnRNP expressed in neural and neural crest tissues.</a> Dev. Dyn. 238 (1), 204-209 (2009).</li><li>Acloque, H., Adams, M. S., Fishwick, K., Bronner-Fraser, M., Nieto, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epithelial-mesenchymal+transitions:+the+importance+of+changing+cell+state+in+development+and+disease.">Epithelial-mesenchymal transitions: the importance of changing cell state in development and disease.</a> J. Clin. Invest. 119 (6), 1438-1449 (2009).</li></ol>

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Ce protocole décrit une technique facile à explantation prémigratoire crânienne NC dans X. laevis embryons. Les embryons utilisés pour de telles expériences doivent être robustes et bien guérir. Jetez un lot d&#39;embryons malsaines. En outre, les embryons se développer à des températures différentes (de 12 à 18 à 20 ° C), afin d&#39;exciser la crête neurale, à l&#39;étape 17 et pas plus tard. Après l&#39;étape 17, la crête neurale peut être mélangé avec du mésoderme céphalique et ne p...

La crête neurale (NC) est une population de cellules embryonnaires transitoires qui se dégage du tube neural à la fin de la neurulation dans des embryons de vertébrés. Les événements de signalisation et génétiques qui contrôlent la spécification NC commencent dès la gastrulation. Le NC est spécifié à la frontière entre le neural et ectoderme non-neural par des signaux provenant de tissus dorsaux environnantes. A la fin de la neurulation, les cellules subissent une transition NC épithélium-mésenchyme à-(EMT) et migrent en détail dans la suite de l&#39;embryon routes stéréotypés en répondant à des signaux de guidage entourant. Une fois qu&#39;ils ont atteint leur destination finale, ils se différencient en une vaste gamme de produits dérivés, par exemple les neurones, les cellules gliales, les os, le cartilage et les cellules pigmentées 1-5. En raison de leur contribution à de nombreux types de cellules et de tissus embryonnaires, les défauts à toute étape du développement des cellules NC, de l&#39;induction de la différenciation finale, peut provoquer des syndromes congénitaux nommés neurocristopathies 6. Emanipulation Xperimental de la NC en développement à différents stades - Spécification, EMT, de migration et de différenciation - permettra d&#39;améliorer notre compréhension de neurocristopathies et permettre la conception de stratégies thérapeutiques potentielles. L&#39;embryon de Xenopus laevis est un modèle de choix pour étudier le développement NC. Un grand nombre d&#39;embryons sont faciles à obtenir, et la fécondation externe donne accès aux toutes premières étapes du développement. De nombreux outils sont disponibles pour manipuler expérimentalement X. laevis développement embryonnaire. Gain de fonction et knockdown gène sont faciles à effectuer par micro-injection de cellules individuelles de début blastulas. Tissus embryonnaires peuvent être coupés pour réassociation in vitro 7-11 ou tests de back-greffes 12,13. Dans ce protocole, nous décrivons comment disséquer prémigratoire crânienne NC dans X. laevis neurulas fin, avant la migration. Ces explants peuvent être cultivées sur fibronectine coateplaques d pour étudier la migration et la différenciation en contrôlées in vitro conditions expérimentales. NC explants peuvent également être greffés sur des embryons normaux de l&#39;hôte ou manipulées pour étudier leur migration et la différenciation in vivo.

<table><tbody><tr><td>Fibronectin from bovine plasma</td><td>Sigma</td><td>F4759-1MG</td><td></td></tr><tr><td>Bovine Serum Albumin, Fraction V</td><td>Euromedex</td><td>04-100-811-C</td><td>Lower quality grade may&amp;nbsp; be suitable for this application</td></tr><tr><td>Stainless Steel Insect Pins, Size 000</td><td>FST</td><td>26001</td><td></td></tr><tr><td>Dumont #5 forceps 0.05 mm x 0.02 mm</td><td>FST</td><td>11252-20</td><td></td></tr></tbody></table>

dissection of xenopus laevis neural crest for in vitro explant culture or in vivo transplantation

La crête neurale (NC) est une population de cellules du tube neural dorsal transitoire qui subit une transition épithélium-à-mésenchyme (EMT) à la fin de la neurulation, migre beaucoup vers divers organes, et se différencie en de nombreux types de produits dérivés (neurones, cellules gliales, cartilage et l&#39;os, les cellules pigmentées et endocriniens). Dans ce protocole, nous décrivons comment disséquer la prémigratoire crânienne NC de Xenopus laevis embryons, afin d&#39;étudier le développement NC in vivo et in vitro. Le modèle de grenouille offre de nombreux avantages pour étudier le développement précoce; lots abondantes sont disponibles, les embryons se développent rapidement, le gain in vivo et la perte de stratégies de fonctions permettent la manipulation de l&#39;expression des gènes avant NC dissection chez le donneur et / ou embryons hôtes. Les explants CN peuvent être plaqués sur la fibronectine et utilisés pour des études in vitro. Elles peuvent être mises en culture pendant plusieurs jours dans un milieu défini sans sérum. Nous décrivons également comment greffer NC explants de retourdans des embryons d&#39;accueil pour l&#39;étude de la migration et la différenciation NC in vivo.

Lorsque plaqué sur la fibronectine, les explants de la crête neurale attachent rapidement (15-30 min) et l&#39;expiant propage à plat dans 2 heures (figure 1A). Après 3-6 cellules h commencent à se disperser. À 24 heures à 15 ° C, de nombreuses cellules ont commencé à migrer loin de l&#39;explant (Figures 1B et 1C). Jaune rend les cellules très lumineux sous contraste de phase (figure 1B). protubérances cellulaires (filopodes et lamellipodes...

Watch this Scientific Journal Video about Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation at JoVE.com

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Ce protocole décrit comment disséquer la crête neurale crânienne prémigratoire (NC) de Xenopus laevis neurulas. Ces explants peuvent être étalées sur la fibronectine et cultivées in vitro, ou greffés retour dans des embryons hôtes. Cette technique permet d&#39;étudier les mécanismes de la NC transition épithélium-à-mésenchyme, la migration et la différenciation.

Dissection de<em&gt; Xenopus laevis</em&gt; Crête neurale pour<em&gt; In vitro</em&gt; Culture explantation ou<em&gt; In vivo</em&gt; Transplantation

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of ...

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Research

JoVE Journal

Biology

Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

13.2K vues.  Centre Universitaire.  Ce protocole décrit comment disséquer la crête neurale crânienne prémigratoire (NC) de Xenopus laevis neurulas. Ces explants peuvent être étalées sur la fibronectine et cultivées in vitro, ou greffés retour dans des embryons hôtes. Cette technique permet d'étudier les mécanismes de la NC transition épithélium-à-mésenchyme, la migration et la différenciation. 

Vidéo: Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

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, ...

Recherche

Biologie du développement

Neural Explant Cultures from Xenopus laevis

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets1. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system5-7 . In this method8, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Neural Explant Cultures from Xenopus laevis

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Des cultures d&#39;explants neuraux de<em&gt; Xenopus laevis</em

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During ...

Neurosciences

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

The process by which the anterior region of the neural plate gives rise to the vertebrate retina continues to be a major focus of both clinical and basic research. In addition to the obvious medical relevance for understanding and treating retinal disease, the development of the vertebrate retina continues to serve as an important and elegant model system for understanding neuronal cell type determination and differentiation1-16. The neural retina consists of six discrete cell types (ganglion, amacrine, horizontal, photoreceptors, bipolar cells, and M&uuml;ller glial cells) arranged in stereotypical layers, a pattern that is largely conserved among all vertebrates 12,14-18.
While studying the retina in the intact developing embryo is clearly required for understanding how this complex organ develops from a protrusion of the forebrain into a layered structure, there are many questions that benefit from employing approaches using primary cell culture of presumptive retinal cells 7,19-23. For example, analyzing cells from tissues removed and dissociated at different stages allows one to discern the state of specification of individual cells at different developmental stages, that is, the fate of the cells in the absence of interactions with neighboring tissues 8,19-22,24-33. Primary cell culture also allows the investigator to treat the culture with specific reagents and analyze the results on a single cell level 5,8,21,24,27-30,33-39. Xenopus laevis, a classic model system for the study of early neural development 19,27,29,31-32,40-42, serves as a particularly suitable system for retinal primary cell culture 10,38,43-45.
Presumptive retinal tissue is accessible from the earliest stages of development, immediately following neural induction 25,38,43. In addition, given that each cell in the embryo contains a supply of yolk, retinal cells can be cultured in a very simple defined media consisting of a buffered salt solution, thus removing the confounding effects of incubation or other sera-based products 10,24,44-45.
However, the isolation of the retinal tissue from surrounding tissues and the subsequent processing is challenging. Here, we present a method for the dissection and dissociation of retinal cells in Xenopus laevis that will be used to prepare primary cell cultures that will, in turn, be analyzed for calcium activity and gene expression at the resolution of single cells. While the topic presented in this paper is the analysis of spontaneous calcium transients, the technique is broadly applicable to a wide array of research questions and approaches (Figure 1).

Dissection, Culture, and Analysis of Xenopus laevis Embryonic Retinal Tissue

Xenopus laevis provides an ideal model system for studying cell fate specification and physiological function of individual retinal cells in primary cell culture. Here we present a technique for dissecting retinal tissues and generating primary cell cultures that are imaged for calcium activity and analyzed by in situ hybridization.

Dissection, de la culture, et de l&#39;analyse des<em&gt; Xenopus laevis</em&gt; Tissus embryonnaires rétine

The process by which  the anterior region of the neural plate gives rise to the vertebrate retina  continues to be a major focus of both clinical and ...

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and associated extracellular matrix. Because they provide a suitable organotypic environment, cortical slice explants are often used to investigate those interactions that control neuronal differentiation and development. Although beneficial, the slice explant model can suffer from drawbacks including aberrant cellular lamination and migration. Here we report a whole cerebral hemisphere explant system for studies of early cortical development that is easier to prepare than cortical slices and shows consistent organotypic migration and lamination. In this model system, early lamination and migration patterns proceed normally for a period of two days in vitro, including the period of preplate splitting, during which prospective cortical layer six forms. We then developed an ex utero electroporation (EUEP) approach that achieves ~80% success in targeting GFP expression to neurons developing in the dorsal medial cortex.
The whole hemisphere explant model makes early cortical development accessible for electroporation, pharmacological intervention and live imaging approaches. This method avoids the survival surgery required of in utero electroporation (IUEP) approaches while improving both transfection and areal targeting consistency. This method will facilitate experimental studies of neuronal proliferation, migration and differentiation.

Ex utero Electroporation and Whole Hemisphere Explants: A Simple Experimental Method for Studies of Early Cortical Development

This protocol describes an improved explant procedure that involves ex utero electroporation, dissection and culture of entire cerebral hemispheres from the embryonic mouse. The preparation facilitates pharmacological studies and assays of gene function during early cortical development.

Ex utero électroporation et explants hémisphère: Une méthode simple expérimental pour les études du développement cortical précoce

Cortical development involves complex interactions between neurons and non-neuronal elements including precursor cells, blood vessels, meninges and ...

Dissection, Culture and Analysis of Primary Cranial Neural Crest Cells from Mouse for the Study of Neural Crest Cell Delamination and Migration

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, the ability to study cell movements and differentiation in vivo is still very difficult. Neurocristopathies, or disorders of the neural crest lineage, are particularly challenging to study due to a lack of accessibility of key embryonic stages and the difficulties in separating out the neural crest mesenchyme from adjacent mesodermal mesenchyme. Here, we set out to establish a well-defined, routine protocol for the culture of primary cranial neural crest cells. In our approach we dissect out the mouse neural plate border during the initial neural crest induction stage. The neural plate border region is explanted and cultured. The neural crest cells form in an epithelial sheet surrounding the neural plate border, and by 24 h after explant, begin to delaminate, undergoing an epithelial-mesenchymal transition (EMT) to become fully motile neural crest cells. Due to our two-dimensional culturing approach, the distinct tissue populations (neural plate versus premigratory and migratory neural crest) can be readily distinguished. Using live imaging approaches, we can then identify changes in neural crest induction, EMT and migratory behaviors. The combination of this technique with genetic mutants will be a very powerful approach for understanding normal and pathological neural crest cell biology.

This protocol describes the dissection and culture of cranial neural crest cells from mouse models, primarily for the study of cell migration. We describe the live imaging techniques used and the analysis of speed and cell shape changes.

Dissection, culture et analyse des cellules neurales primaires de crête neurale de la souris pour l'étude de la délamination et de la migration neurales de cellules de crête

Over the past several decades there has been an increased availability of genetically modified mouse models used to mimic human pathologies. However, 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 ...

Preparation and Morphological Analysis of Chick Cranial Neural Crest Cell Cultures

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like the craniofacial skeleton and the peripheral nervous system. While it is critical to understand NCC migration in the context of a 3D embryo, isolating migratory cells in 2D culture facilitates visualization and functional characterization, complementing embryonic studies. The present protocol demonstrates a method for isolating chick cranial neural folds to generate primary NCC cultures. Migratory NCCs emerge from neural fold explants plated onto a fibronectin-coated substrate. This results in dispersed, adherent NCC populations that can be assessed by staining and quantitative morphological analyses. This simplified culture approach is highly adaptable and can be combined with other techniques. For example, NCC emigration and migratory behaviors can be evaluated by time-lapse imaging or functionally queried by including inhibitors or experimental manipulations of gene expression (e.g., DNA, morpholino, or CRISPR electroporation). Because of its versatility, this method provides a powerful system for investigating cranial NCC development.

This versatile protocol describes the isolation of premigratory neural crest cells (NCCs) through the excision of cranial neural folds from chick embryos. Upon plating and incubation, migratory NCCs emerge from neural fold explants, allowing for assessment of cell morphology and migration in a simplified 2D environment.

Préparation et analyse morphologique de cultures de cellules de crête neurale crânienne de poussin

During vertebrate development, neural crest cells (NCCs) migrate extensively and differentiate into various cell types that contribute to structures like ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

Un test pour évaluer le comportement des explants cellulaires du mésenchyme crânienne

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

Biologie

Facial Transplants in Xenopus laevis Embryos

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. To identify where signaling pathways and tissues act during patterning of the developing face, a 'face transplant' technique has been developed in embryos of the frog Xenopus laevis. A region of presumptive facial tissue (the "Extreme Anterior Domain" (EAD)) is removed from a donor embryo at tailbud stage, and transplanted to a host embryo of the same stage, from which the equivalent region has been removed. This can be used to generate a chimeric face where the host or donor tissue has a loss or gain of function in a gene, and/or includes a lineage label. After healing, the outcome of development is monitored, and indicates roles of the signaling pathway within the donor or surrounding host tissues. Xenopus is a valuable model for face development, as the facial region is large and readily accessible for micromanipulation. Many embryos can be assayed, over a short time period since development occurs rapidly. Findings in the frog are relevant to human development, since craniofacial processes appear conserved between Xenopus and mammals.

Facial Transplants in Xenopus laevis Embryos

A technique for transplanting "Extreme Anterior Domain" facial tissue between Xenopus laevis embryos has been developed. Tissue can be moved from one gene expression background into another, allowing the study of local requirements for craniofacial development and for signaling interactions between facial regions.

Transplantations faciales<em&gt; Xenopus laevis</em&gt; Embryons

Craniofacial birth defects occur in 1 out of every 700 live births, but etiology is rarely known due to limited understanding of craniofacial development. ...

Explant Culture of Neural Tissue

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and changes in cell morphology. Studying these processes is essential to our understanding of nervous system function as well as our ability to diagnose and treat disorders that result from abnormal development. However, neural tissues are relatively inaccessible for experimental manipulations, especially in embryonic mammals. As a result, many scientists take advantage of explant culture in order to study neurodevelopmental processes in an &#8220;organotypic&#8221; environment, meaning that the tissue is removed from the organism but its complex cellular architecture is maintained. Generally, explant cultures are created by careful dissection of neural tissue that is then submerged in carefully designed growth media and cultured in vitro.
This video will first provide a brief overview of neural explant culture, including its advantages over other in vitro methods and important considerations for maintaining healthy tissue. Next, a general protocol will be provided for setting up an explant culture from embryonic mouse brain, outlining the isolation of embryos from the mother and dissection of the brain. The presentation also includes an overview of slice culture, in which thin sections of nervous system tissue are generated for improved visual access to the developing cells. Lastly, a few applications of these techniques will be provided to demonstrate how they can be used to answer important questions in the neurodevelopmental field.

Culture d'explants de tissu neural

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and ...

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