Les auteurs voudrais remercier Gregory Haller (Mount Saint Vincent University) pour son travail préliminaire à l’élaboration du protocole. Les auteurs tiens également à remercier Nicholas Jones (Mount Saint Vincent University) pour son expertise technique et d’assistance avec le tournage et la production de la portion audio/visuel du manuscrit. Daniel Andrews a été appuyée par le financement de l’Université Mount Saint Vincent et les Sciences naturelles et génie conseil recherche du Canada (CRSNG) le via une bourse de recherche de premier cycle. Tamara A. Franz-Odendaal est soutenue par une subvention à la découverte du CRSNG.

Remarque : Pour les stades de l’embryon, utiliser le Hamburger et Hamilton12 (HH) mise en scène de table.
1. embryon Incubation
<ol><li>Incuber les œufs de poule fécondés dans un incubateur stérile, contrôle de la température à 37 ° C ± 1 ° C et environ 40 % d’humidité.</li>
	<li>Tournez les oeufs 1 x par jour et laisser incuber à HH34 (fécondation après 8 jours).</li>
</ol>2. préparation et stérilisation du matériel
<ol><li>Pour 12 embryons, autoclave 2 L d’eau distillée, 1 L de solution saline à poussin 0,85 %, 12 pipettes en verre, 1 boîte de mouchoir en papier, 24 2,5 cm x 2,5 cm carrés de papier-filtre semi poreux et carrés de 24 2,5 cm x 2,5 cm de fil d’acier de maille avec les bords courbés vers le bas. Pour stériliser le matériel, autoclave à 121 ° C pendant au moins 15 min. Remarque : L’autoclavage des matériaux peut se faire à l’avance.</li>
	<li>Stériliser l’incubateur avec l’éthanol à 70 % en l’essuyant les côtés et étagères porte. Placer 2 récipients en plastique contenant autoclavés distillée à l’intérieur de l’incubateur à conserver l’humidité. Préchauffer l’incubateur de culture à 37 ° C ± 1 ° C et environ 40 % d’humidité. S’assurer que l’incubateur est complètement noir ; Si la porte est transparente, couvrez-la de papier d’aluminium.</li>
	<li>Réchauffer au préalable les 100 mL de milieu nutritif et 1 mL de pénicilline en les plaçant dans l’incubateur de culture.</li>
	<li>Couvrir l’espace de travail avec du papier essuie-tout et stériliser le banc en vaporisant avec l’éthanol à 70 %. Stériliser 2 paires de pinces, ciseaux de dissection, une lame de rasoir et une cuillère en plastique avec l’éthanol à 70 % de la même manière.</li>
	<li>Plats de Pétri stérile 100 mm lieu 12 et 24 boîtes de Pétri stériles 35 mm sur le banc pour utilisation. S’assurer que les plats restent dans un sachet stérile jusqu'à utilisation.</li>
</ol>3. préparation de l’embryon
NOTE : Partir de là, porter un masque de protection poussière pour éviter de contaminer les cultures. Une infection bactérienne ou fongique va ruiner la culture, et il y a un risque de s’étendre rapidement à toutes les cultures dans l’incubateur.
<ol><li>Crack ouvrir le œuf et transférer l’embryon à un 100 mm boîte de Pétri contenant du sérum physiologique poussin à 0,85 %. Stade de l’embryon selon les caractéristiques anatomiques décrites dans le Hamburger et Hamilton, mise en scène de directives12 et confirmer l’embryon est à HH34.</li>
	<li>Couper le cou et puis coupent la tête de l’embryon à la ligne médiane à l’aide d’une lame de rasoir stérilisée. À l’aide d’une pince stérile, enlever le cerveau. Le bec laisse intacte.</li>
</ol>4. culture Setup
<ol><li>2 place de la Pétri de 35 mm à l’intérieur d’une boîte de Pétri 100 mm.</li>
	<li>Placer un treillis en acier à l’intérieur de chaque boîte de Pétri 35 mm.</li>
	<li>À l’aide de pinces, transférer 1 des têtes coupées à un morceau de papier filtre semi poreux de 2,5 cm x 2,5 cm avec le œil vers le haut. S’assurer que le tissu est sécurisé et ne glisse pas éteint en l’inclinant légèrement.</li>
	<li>À l’aide de pinces, placer soigneusement les tissus de le œil et le filtre en papier sur le dessus de la maille de fil d’acier en 1 des boîtes de Pétri le 35 mm, créant une scène surélevée avec les tissus de le œil sur le dessus.</li>
	<li>Répétez les étapes 4.3 et 4.4 avec l’autre moitié de tête.</li>
	<li>À l’aide d’une pipette de verre stérile, ajouter avec précaution le milieu nutritif directement dans chaque boîte de Pétri 35 mm jusqu'à ce qu’il atteigne le niveau du papier-filtre. Ne pas immerger la moitié de la tête dans le milieu nutritif.</li>
	<li>Ajouter 50 µL de 10 000 U/mL de pénicilline-streptomycine dans le milieu nutritif dans chaque boîte de Pétri.</li>
	<li>Plier un morceau de papier de soie dans un petit carré et le placer à l’intérieur de la boîte de Pétri de 100 mm. Humidifier avec de l’eau distillée autoclavé.</li>
	<li>Placez la boîte de Pétri dans l’incubateur stérile, sombre à 37 ° C ± 1 ° C et environ 40 % d’humidité pendant 4 jours.</li>
	<li>Répétez les étapes 3 et 4 pour chaque embryon. Remarque : Le nombre d’embryons nécessaire dépendra de l’expérience spécifique ; Nous décrivons ici le protocole pour 12 embryons.</li>
</ol>5. culture entretien
<ol><li>Une fois par jour, remplir les contenants de plastique dans l’incubateur avec l’eau distillée froide, stérilisés à l’autoclave.</li>
	<li>Tous les jours, vérifier toutes les cultures pour des infections bactériennes ou fongiques. Cultures qui se sont désintégrées ou dans lequel le milieu a changé de couleur sont infectés. Jeter toutes les cultures qui sont infectés.</li>
</ol>6. fixation
<ol><li>Après mise en culture, sortir tous les plats de l’incubateur.</li>
	<li>À l’aide d’une paire de pinces, retirez délicatement le œil du papier filtre, prenant soin de ne pas pour déchirer le tissu.</li>
	<li>Fixer le tissu oculaire dans 4 % de paraformaldéhyde dans 1 x tampon phosphate salin pendant la nuit à 4 ° C ou 10 % de formol tamponné neutre durant la nuit à température ambiante.</li>
	<li>Stocker les tissus à 4 ° C en 1 x solution saline tamponnée au phosphate.</li>
</ol>

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Les auteurs n’ont rien à divulguer.

Ce protocole utilise des tissus établie des techniques de culture pour atteindre la croissance d’un oeil de poulet embryonnaire jour 8 (HH34) in vitro pendant 4 jours. Cette méthode de mise en réseau-culture a été initialement décrite par Trowell15. Nous avons optimisé un protocole de Pinto et Hall 1991 étude16 utilisant une membrane semi poreuse avec le réseau pour étudier les signaux inductifs entre les couches de tissus séparés des yeux embryonnair...

L’embryonnaire poulet (Gallus gallus) est un organisme d’excellent modèle couramment utilisé dans le domaine de la biologie. Sa période d’incubation est d’environ 21 jours et beaucoup d’oeufs peut être incubés simultanément, rendant les expérimentation rapide et efficace. Peut-être plus important encore, l’embryon est également facilement manipulé, permettant l’étude approfondie des principaux processus de développement et des gènes et des protéines qui animent ces processus.
L’oeil de l’embryon de poulet est un organe complexe qui se développe par l’interaction d’un certain nombre de différents tissus semblables à beaucoup d’autres systèmes de corps. Cette méthode permet l’étude de l’évolution de ces tissus, en particulier à un stade avancé de développement. Par exemple, la rétine multicouche peut être particulièrement intéressante pour ceux qui étudient le développement du système nerveux. Des méthodes alternatives qui permettent l’étude des autres tissus de l’oeil comme la cornée, le corps vitréennes, la lentille, la sclérotique et les paupières sont bénéfiques pour les chercheurs. L’oeil embryonnaire de poulet contient également une série d’os plats, les osselets sclérales, qui peuvent servir de modèle pour l’étude des os intramembranaires induction et l’ossification en vertébrés1.
Actuellement, il existe un certain nombre de méthodes utilisées pour étudier le développement embryonnaire. Microinjections d’anticorps inhibiteurs ou autres molécules inhibitrices de la2,3, implanté chirurgicalement des microbilles imbibé d’inhibiteur4et électroporation5 sont toutes des méthodes pouvant servir à des gènes régulent ou protéines d’intérêt dans un embryon. Des méthodes similaires sont utilisés pour réguler positivement les protéines. Ces méthodes ne sont pas sans leurs limites. Par exemple, lors de l’utilisation de produits chimiques pour modifier le développement embryonnaire, l’effet létal sur l’embryon doit être évalué, et cela limite l’utilisation des méthodes susmentionnées pour les sites localisés d’application à des doses suffisamment bas pour assurer la survie de la embryon.
Culture de tissus in vitro a été utilisée dans un large éventail d’organismes pour étudier le développement et peut être utilisé pour contourner les limitations susmentionnées. Par exemple, les fémurs6et plume bourgeons7,8branches9 du poulet ont tous étudiés à l’aide de tissus cultivant des méthodes, comme ont les testicules des souris10 et les racines et les tiges des plantes11. Ces méthodes accordent aux scientifiques un degré élevé de contrôle sur le développement des tissus, comme la possibilité de varier la température et de modifier la disponibilité des nutriments. L’isolement des tissus de l’embryon entier rend également beaucoup moins sensibles à l’effet létal des produits chimiques, permettant ainsi à des études de manipulation à l’échelle mondiale à des concentrations plus élevées. Un autre avantage notable de la mise en culture in vitro est la préservation de l’environnement cellulaire du tissu ; l’arrangement des tissus reste relativement inchangée, ce qui permet d’étudier les interactions entre les différents tissus types9. Ainsi, in vitro culture ouvre portes supplémentaires expérimentale approches non disponible dans des modèles in vivo ou in ovo .
Actuellement, il est particulièrement difficile d’étudier l’évolution de le œil de poulet embryonnaire à l’aide de produits chimiques. Un certain nombre de membranes extra-embryonnaires couvre l’embryon, rendant difficile l’application de microbilles ou produits chimiques ; l’embryon est également très active dans l’oeuf comme elle vieillit, ce qui complique une méthode déjà difficile. Ce protocole permet un accès facile à le œil et des tissus entourant, en éliminant ces obstacles, tout en offrant de nouvelles possibilités d’examiner les processus de développement intérieur de le œil. Ce protocole a été créé pour étudier l’induction des osselets sclérales intérieur de l’oeil embryonnaire.

<table border="0" cellpadding="0" cellspacing="0" style="width:1081px;" width="1082">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">35 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353001</td>
			<td style="width:676px;">easy grip tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">100 mm cell culture petri dishes</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">353003</td>
			<td>tissue culture dish, polystyrene, non-pyrogenic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper tissue</td>
			<td style="width:71px;">Kimtech</td>
			<td style="width:119px;">34155</td>
			<td>Kimtech Science Brand Task Wipers, 280 per carton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">wire mesh</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>stainless steel wire mesh (grid size 0.7 mm)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable glass pipettes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">14673-010</td>
			<td>Borosilicate glass disposable 5 3/4&#34;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">nutrient medium</td>
			<td style="width:71px;">Gibco</td>
			<td style="width:119px;">12591-038</td>
			<td>Fitton-Jackson Modification, [+] L-glutamine with phenol red (BGJB)</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">penicillin-streptomycin</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P4458</td>
			<td>10000 units/mL penicillin streptomycin solution stabilised</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">filter paper</td>
			<td style="width:71px;">Whatman</td>
			<td style="width:119px;">1454 090</td>
			<td>semi-porous filter paper 90mm</td>
		</tr>
		<tr height="147">
			<td height="147" style="height:147px;width:216px;">fertilized chicken eggs</td>
			<td style="width:71px;">Dalhousie University Agricultural College</td>
			<td style="width:119px;">n/a</td>
			<td>can be obtained from local farms</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">sodium chloride (NaCl)</td>
			<td style="width:71px;">EMD</td>
			<td style="width:119px;">SX0420-3</td>
			<td>sodium chloride crystals, reagent grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">1 L glass bottle</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">89000-240</td>
			<td>1 L pyrex autoclavable glass bottle</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">ethanol</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td>BP82011</td>
			<td>70% molecular biology grade</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tupperware containers</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">disposable razor blades</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td>single edge industrial razor blades (surgical carbon steel)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">plastic spoons</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought and sterilized with EtOH</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">dust mask</td>
			<td style="width:71px;">3M</td>
			<td style="width:119px;">n/a</td>
			<td>3M 8500 Comfort Mask</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">paraformaldehyde</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">P6148</td>
			<td>paraformaldehyde, reagent grade, crystalline</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">neutral-buffered formalin</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">72210</td>
			<td>10% neutral buffered formalin</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">phosphate buffered saline (PBS)</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>10X phosphate buffered saline pH 7.4 (137mM NaCl, 2.5mM KCl, 4.3mM Na2HPO4, 1.4mM KH2PO4)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml falcon tubes</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">21008-216</td>
			<td>presterilized centrifuge tubes</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">forceps</td>
			<td style="width:71px;">FST</td>
			<td style="width:119px;">n/a</td>
			<td>fine forceps</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">chick saline&#160;</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a&#160;</td>
			<td>0.85% NaCl</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">tinfoil</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">paper towel</td>
			<td style="width:71px;">n/a</td>
			<td style="width:119px;">n/a</td>
			<td>store-bought</td>
		</tr>
	</tbody>
</table>

organotypic culture method to study the development of embryonic chicken tissues

Le poulet embryonnaire est couramment utilisé comme un organisme modèle fiable pour le développement des vertébrés. Son accessibilité et son incubation courte période le rend idéal pour l’expérimentation. Actuellement, l’étude de ces voies de développement de l’embryon de poulet est réalisée en appliquant des inhibiteurs et des médicaments à des sites localisés et à de faibles concentrations en utilisant une variété de méthodes. Culture de tissus in vitro est une technique qui permet l’étude des tissus séparés de l’organisme hôte, tout en contournant en même temps beaucoup de limitations physiques présentes lorsque vous travaillez avec des embryons entiers, tels que la susceptibilité des embryons doses élevées de produits chimiques potentiellement mortels. Nous présentons ici un organotypique cultivant protocole pour cultiver le poulet embryonnaire demi tête in vitro, qui présente de nouvelles opportunités pour l’examen des processus de développement au-delà des méthodes actuellement établies.

En utilisant cette méthode, un oeil embryonnaire de poulet peut être cultivé de jour 8 de son développement (HH34) in vitro pendant 4 jours. Quatre jours de in ovo développement correspond à HH38.
Cette méthode de culture soutient le développement des bourgeons de plumes autour de le œil et des paupières (Figure 1 b). Ces bourgeons de plumes ne sont pas présents dans...

Watch this Scientific Journal Video about Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues at JoVE.com

Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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

Nous présentons ici un organotypique cultivant protocole pour cultiver des organes embryonnaires de poulet in vitro. En utilisant cette méthode, le développement des tissus embryonnaires de poulet peut être étudié, tout en conservant un haut degré de contrôle sur le milieu de culture.

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

organotypic-culture-method-to-study-development-embryonic-chicken

Research

JoVE Journal

Developmental Biology

5.9K vues.  Mount Saint Vincent University. Nous présentons ici un organotypique cultivant protocole pour cultiver des organes embryonnaires de poulet in vitro. En utilisant cette méthode, le développement des tissus embryonnaires de poulet peut être étudié, tout en conservant un haut degré de contrôle sur le milieu de culture.

Vidéo: Organotypic Culture Method to Study the Development Of Embryonic Chicken Tissues

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 ...

Recherche

Biologie du développement

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.

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 ...

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo development due to their similar developmental process. Although genetic factors are known to affect early embryonic development, the discovery of such factors has been a serious challenge. Microarray technology allows quantitative measurement and gene expression profiling of transcript levels on a genome-wide basis. One of the main decisions that have to be made when planning a microarray experiment is whether to use a one- or two-color approach. Two-color design increases technical replication, minimizes variability, improves sensitivity and accuracy as well as allows having loop designs, defining the common reference samples. Although microarray is a powerful biological tool, there are potential pitfalls that can attenuate its power. Hence, in this technical paper we demonstrate an optimized protocol for RNA extraction, amplification, labeling, hybridization of the labeled amplified RNA to the array, array scanning and data analysis using the two-color analysis strategy.

Transcriptome Profiling of In-Vivo Produced Bovine Pre-implantation Embryos Using Two-color Microarray Platform

Microarray technology allows quantitative measurement and gene expression profiling of transcripts on a genome-wide basis. Therefore, this protocol provides an optimized technical procedure in a two-color custom made bovine array using Day 7 bovine embryos to demonstrate the feasibility of using low amount of total RNA.

Transcriptome profilage des<em&gt; In-Vivo</em&gt; embryons de bovins pré-implantation à l&#39;aide de deux couleurs Microarray Plate-forme

Early embryonic loss is a large contributor to infertility in cattle. Moreover, bovine becomes an interesting model to study human preimplantation embryo ...

Epigenetic Regulation of Cardiac Differentiation of Embryonic Stem Cells and Tissues

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated by transcription factors which bind genomic regulatory regions including enhancers and promoters of cardiac constitutive genes. DNA is wrapped around histones that are subjected to chemical modifications. Modifications of histones further lead to repressed, activated or poised gene transcription, thus bringing another level of fine tuning regulation of gene transcription. Embryonic Stem cells (ES cells) recapitulate within embryoid bodies (i.e., cell aggregates) or in 2D culture the early steps of cardiac development. They provide in principle enough material for chromatin immunoprecipitation (ChIP), a technology broadly used to identify gene regulatory regions. Furthermore, human ES cells represent a human cell model of cardiogenesis. At later stages of development, mouse embryonic tissues allow for investigating specific epigenetic landscapes required for determination of cell identity. Herein, we describe protocols of ChIP, sequential ChIP followed by PCR or ChIP-sequencing using ES cells, embryoid bodies and cardiac specific embryonic regions. These protocols allow to investigating the epigenetic regulation of cardiac gene transcription.

A fine tuning regulation of gene transcription underlies embryonic cell fate decision. Herein, we describe chromatin immunoprecipitation assays used to investigate epigenetic regulation of both cardiac differentiation of stem cells and cardiac development of mouse embryos.

Régulation épigénétique de Cardiac Différenciation des cellules et tissus souches embryonnaires

Specific gene transcription is a key biological process that underlies cell fate decision during embryonic development. The biological process is mediated ...

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and molecular level. To alter intracardiac hemodynamics, fertilized chicken eggs are incubated in a humidified chamber to obtain embryos of the desired stage (HH17). Once this developmental stage is achieved, the embryo is maintained ex ovo and hemodynamics in the embryonic heart are altered by partially constricting the outflow tract (OFT) with a surgical suture at the junction of the OFT and ventricle (OVJ). Control embryos are also cultured ex ovo but are not subjected to the surgical intervention. Banded and control embryos are then incubated in a humidified incubator for the desired period of time, after which 2D ultrasound is employed to analyze the change in blood flow velocity at the OVJ as a result of OFT banding. Once embryos are maintained ex ovo, it is important to ensure adequate hydration in the incubation chamber so as to prevent drying and eventually embryo death. Using this new banded model, it is now possible to perform analyses of changes in the expression of key players involved in valve development and to understand the role of hemodynamics on cellular responses in vivo, which could not be achieved previously.

A Novel Ex Ovo Banding Technique to Alter Intracardiac Hemodynamics in an Embryonic Chicken System

For the first time we present here a reproducible banding procedure to alter hemodynamics in the developing heart ex ovo. This is achieved by partially constricting the outflow tract (OFT).

Un roman<em&gt; Ovo Ex</em&gt; Banding Technique d&#39;Alter intracardiaque hémodynamique dans un système embryonnaire de poulet

The new model presented here can be used to understand the influence of hemodynamics on specific cardiac developmental processes, at the cellular and ...

Culture of Murine Embryonic Metatarsals: A Physiological Model of Endochondral Ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or longitudinal bone growth. As such, it is imperative that we further our understanding of the underpinning molecular mechanisms involved in such disorders so as to provide advances towards human and animal patient benefit. The mouse metatarsal organ explant culture is a highly physiological ex vivo model for studying endochondral ossification and bone growth as the growth rate of the bones in culture mimic that observed in vivo. Uniquely, the metatarsal organ culture allows the examination of chondrocytes in different phases of chondrogenesis and maintains cell-cell and cell-matrix interactions, therefore providing conditions closer to the in vivo situation than cells in monolayer or 3D culture. This protocol describes in detail the intricate dissection of embryonic metatarsals from the hind limb of E15 murine embryos and the subsequent analyses that can be performed in order to examine endochondral ossification and longitudinal bone growth.

We present a protocol to dissect and culture embryonic day 15 (E15) murine metatarsal bones. This highly physiological ex vivo model of endochondral ossification provides conditions closer to the in vivo situation than cells in monolayer or 3D culture and is a vital tool for investigating bone growth and development.

Culture de murines embryonnaires Métatarsiens: un modèle physiologique de endochondrale ossification

The fundamental process of endochondral ossification is under tight regulation in the healthy individual so as to prevent disturbed development and/or ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Dissection et culture du rein embryonnaire de la souris

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

Analyse de calcul de la lignée germinale de Caenorhabditis elegans pour étudier la Distribution des noyaux, les protéines et le cytosquelette

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

Isolation of Embryonic Tissues and Formation of Quail-Chicken Chimeric Organs Using The Thymus Example

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed contributions to unveiling key processes in developmental biology.
Herein is described an optimized method to isolate embryonic tissues from quail and chickens by microsurgery and enzymatic digestion while preserving its biological properties. After isolation, tissues from both species are associated in an in vitro organotypic assay for 48 h. Quail and chicken tissues can be discriminated by distinct nuclear features and molecular markers allowing the study of the cellular cross-talk between heterospecific association of tissues. This approach is, therefore, a useful tool for studying complex tissue interactions in developmental processes with highly dynamic spatial modifications, such as those occurring during pharyngeal morphogenesis and the formation of the foregut endoderm-derived organs. This experimental approach was first developed to study the epithelial-mesenchymal interactions during early-stages of thymus formation. In this, the endoderm-derived prospective thymic rudiment and mesoderm-derived mesenchyme, were isolated from quail and chicken embryos, respectively.
The capacity of the associated tissues to generate organs can be further tested by grafting them onto the chorioallantoic membrane (CAM) of a chicken embryo. The CAM provides nutrients and allows gas exchanges to the explanted tissues. After 10 days of in ovo development, the chimeric organs can be analyzed in the harvested explants by conventional morphological methods. This procedure also allows studying tissue-specific contributions during organ formation, from its initial development (in vitro development) to the final stages of organogenesis (in ovo development).
Finally, the improved isolation method also provides three-dimensionally (3D) preserved embryonic tissues, that can also be used for high-resolution topographical analysis of tissue-specific gene-expression patterns.

This article provides a method to isolate pure embryonic tissues from quail and chicken embryos that can be combined to form ex vivo chimeric organs.

Isolement des tissus embryonnaires et la Formation des organes chimériques caille-poulet à l’aide de l’exemple du Thymus

The capacity to isolate embryonic tissues was an essential step for establishing the quail-chicken chimera system, which in turn has provided undisputed ...

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

C. elegans is the premier system for the systematic analysis of cell fate specification and morphogenetic events during embryonic development. One challenge is that embryogenesis dynamically unfolds over a period of about 13 h; this half day-long timescale has constrained the scope of experiments by limiting the number of embryos that can be imaged. Here, we describe a semi-high-throughput protocol that allows for the simultaneous 3D time-lapse imaging of development in 80&#8211;100 embryos at moderate time resolution, from up to 14 different conditions, in a single overnight run. The protocol is straightforward and can be implemented by any laboratory with access to a microscope with point visiting capacity. The utility of this protocol is demonstrated by using it to image two custom-built strains expressing fluorescent markers optimized to visualize key aspects of germ-layer specification and morphogenesis. To analyze the data, a custom program that crops individual embryos out of a broader field of view in all channels, z-steps, and timepoints and saves the sequences for each embryo into a separate tiff stack was built. The program, which includes a user-friendly graphical user interface (GUI), streamlines data processing by isolating, pre-processing, and uniformly orienting individual embryos in preparation for visualization or automated analysis. Also supplied is an ImageJ macro that compiles individual embryo data into a multi-panel file that displays maximum intensity fluorescence projection and brightfield images for each embryo at each time point. The protocols and tools described herein were validated by using them to characterize embryonic development following knock-down of 40 previously described developmental genes; this analysis visualized previously annotated developmental phenotypes and revealed new ones. In summary, this work details a semi-high-throughput imaging method coupled with a cropping program and ImageJ visualization tool that, when combined with strains expressing informative fluorescent markers, greatly accelerates experiments to analyze embryonic development.

A Semi-high-throughput Imaging Method and Data Visualization Toolkit to Analyze C. elegans Embryonic Development

This work describes a semi-high-throughput protocol that allows simultaneous 3D time-lapse imaging of embryogenesis in 80&#8211;100 C. elegans embryos in a single overnight run. Additionally, image processing and visualization tools are included to streamline data analysis. The combination of these methods with custom reporter strains enables detailed monitoring of embryogenesis.