Name: the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions
Uploaded: 2017-02-16T14:00:00
Description: Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

We would like to thank Dr. Robert Angerer for his careful reading and editing of the manuscript. NIH R15HD088272-01 as well as the Office of Research and Development, and Department of Biological Sciences at Mississippi State University provided support for this project to RCR.

1. Stratégie de conception morpholino Throughput haut <ol><li> Identifier un gène (s) d&#39;intérêt (par exemple , l&#39; approche de gène candidat, l&#39; analyse des cis-régulatrices, et RNA-seq ou écrans différentiels / protéomique). </li><li> Utilisez génomique, transcriptomique, et des données d&#39;expression génique disponibles sur les sites Web fréquemment mis à jour (par exemple SpBase http://www.echinobase.org 20 et S. purpuratus Genome Recherc...

<ol>
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La méthodologie présentée ici est un exemple qui illustre le pouvoir d&#39;utiliser des embryons avec une complexité moins génomique et morphologique que les vertébrés à comprendre les voies de signalisation de la transduction et GRNS régissant les mécanismes de développement fondamentaux .. De nombreux laboratoires utilisent des tests similaires au début du développement de l&#39;oursin à disséquer le voies impliquées dans d&#39; autres événements de spécification du destin cellulaire de signalisatio...

réseaux de gènes de régulation (GRNS) et voies de signalisation établissent l&#39;expression spatiale et temporelle des gènes au cours du développement embryonnaire qui sont utilisés pour construire le plan corporel de l&#39;animal adulte. Cell-à-cellule des voies de transduction du signal sont des composants essentiels de ces réseaux de régulation, en fournissant les moyens par lesquels les cellules communiquent. Ces interactions cellulaires établir et affiner l&#39;expression des gènes de régulation et de différenciation dans et entre les différents territoires pendant l&#39; embryogenèse 1, 2. Les interactions entre les modulateurs sécrétées extracellulaires (ligands, des antagonistes), des récepteurs et co-récepteurs contrôlent les activités des voies de transduction du signal. Un assortiment de molécules intracellulaires transduction ces entrées résultant de l&#39;expression altérée du gène, la division et / ou la forme d&#39;une cellule. Bien que plusieurs des molécules clés utilisés aux niveaux extracellulaires et intracellulaires dans les principales voies sontconnu, il est une connaissance incomplète due en grande partie à la complexité des voies de signalisation individuelles. En outre, les différentes voies de signalisation interagissent souvent entre eux de façon positive ou négative au extracellulaire, intracellulaire, et les niveaux de transcription 3, 4, 5, 6. Fait important, les composants de base de voies de signalisation sont hautement conservées dans toutes les espèces de métazoaires, et, fait remarquable, la plupart des grandes voies de signalisation exercent souvent des fonctions de développement similaires dans de nombreuses espèces lorsque l&#39;on compare les organismes de phyla étroitement liés , en particulier 7, 8, 9, 10, 11. L&#39;étude de la signalisation au cours du développement est une tâche ardue dans tout organisme, et il yPlusieurs défis importants pour l&#39; étude des voies de signalisation dans la plupart des modèles deutérostomes (vertébrés, chordés invertébrés, hémichordés et échinodermes): 1) Chez les vertébrés , il y a un grand nombre de possibles ligand et les interactions récepteur / co-modulateurs, des molécules de transduction intracellulaire, ainsi que les interactions possibles entre les différentes voies de signalisation en raison de la complexité du génome 12, 13, 14; 2) La morphologie complexe et mouvements morphogénétiques chez les vertébrés font qu&#39;il est souvent plus difficile d&#39;interpréter les interactions fonctionnelles et entre les voies de transduction du signal; 3) Les analyses de la plupart des espèces modèles invertébré deutérostomien non échinodermes sont limitées par de courtes fenêtres de gravidity à l&#39;exception de certaines espèces de tuniciers 15, 16. leoursin embryon a quelques - unes des limitations mentionnées ci-dessus et offre de nombreuses qualités uniques pour effectuer une analyse détaillée des voies de transduction du signal in vivo. Ceux - ci sont les suivantes: 1) La relative simplicité du génome de l&#39; oursin réduit de manière significative le nombre de possible ligand, un récepteur / co-récepteur et la molécule de transduction intracellulaire interactions 17; 2) Les GRNS contrôlant la spécification et la structuration des couches germinales et les grands axes embryonnaires sont bien établis dans les embryons d&#39;oursins, d&#39; aider à la compréhension du contexte réglementaire de la cellule / territoire recevant les signaux 18, 19; 3) De nombreuses voies de transduction du signal peuvent être étudiés entre les étapes de clivage et gastrula lorsque les embryons sont constitués d&#39;un seul épithélium stratifié dont la morphologie est plus facile à analyser; 4) Les molécules comportentd dans les voies dans les oursins sont facilement manipulables de signalisation; 5) De nombreux oursins sont gravides pendant 10 à 11 mois par an (par exemple Strongylocentrotus purpuratus et Lytechinus variegatus). Ici, nous présentons une méthode pour caractériser systématiquement et efficacement les composants des voies de signalisation qui précisent et territoires de modèle dans les embryons d&#39;oursins pour illustrer les avantages que plusieurs systèmes de modèles d&#39;invertébrés offrent dans l&#39;étude des mécanismes moléculaires complexes.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Translational-blocking morpholino and/or splice-blocking morpholino</td>
			<td>Gene Tools LLC</td>
			<td>Customized</td>
			<td>More information at www.gene-tools.com</td>
		</tr>
		<tr>
			<td>Glycerol</td>
			<td>Invitrogen</td>
			<td>15514-011</td>
			<td></td>
		</tr>
		<tr>
			<td>FITC (dextran fluorescein isothiocyanate)</td>
			<td>Invitrogen, Life Technologies</td>
			<td>D1821</td>
			<td>Make 25mg/mL stock solution</td>
		</tr>
		<tr>
			<td>Paraformaldehyde 16% solution EM Grade</td>
			<td>Electron Microscopy Sciences</td>
			<td>15710</td>
			<td></td>
		</tr>
		<tr>
			<td>MOPS</td>
			<td>Sigma Aldrich</td>
			<td>M1254-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Sigma Aldrich</td>
			<td>23336-0010</td>
			<td></td>
		</tr>
		<tr>
			<td>Formamide</td>
			<td>Sigma Aldrich</td>
			<td>47671-1L-F</td>
			<td></td>
		</tr>
		<tr>
			<td>Yeast tRNA</td>
			<td>Invitrogen</td>
			<td>15401-029</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Goat Serum</td>
			<td>Sigma Aldrich</td>
			<td>G9023-10mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Alkaline Phosphatase-conjugated anti-digoxigenin antibody</td>
			<td>Roche</td>
			<td>11 093 274 910</td>
			<td></td>
		</tr>
		<tr>
			<td>Tetramisole hydrochloride (levamisole)</td>
			<td>Sigma Aldrich</td>
			<td>L9756-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris Base UltraPure</td>
			<td>Research Products Internationall Corp</td>
			<td>56-40-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Chloride</td>
			<td>Fisher Scientific</td>
			<td>BP358-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>Sigma Aldrich</td>
			<td>7786-30-3</td>
			<td></td>
		</tr>
		<tr>
			<td>BCIP (5-Bromo-4-Chloro-3-indolyl-phosphate</td>
			<td>Roche</td>
			<td>11 383 221 001</td>
			<td></td>
		</tr>
		<tr>
			<td>4 Nitro blue tetrazolium chloride (NBT)</td>
			<td>Roche</td>
			<td>11 383 213 001</td>
			<td></td>
		</tr>
		<tr>
			<td>Dimethyl Formamide</td>
			<td>Sigma Aldrich</td>
			<td>D4551-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>P9541-5KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Bicarbonate</td>
			<td>Sigma Aldrich</td>
			<td>S5761-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium Sulfate</td>
			<td>Sigma Aldrich</td>
			<td>M7506-2KG</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Chloride</td>
			<td>Sigma Aldrich</td>
			<td>C1016-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

the power of simplicity: sea urchin embryos as in vivo developmental models for studying complex cell-to-cell signaling network interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and tissues in the adult body form. Yet, each year more components of individual signaling pathways are discovered, and studies indicate that depending on the context there is significant cross-talk among most of these pathways. This complexity makes studying cell-to-cell signaling in any in vivo developmental model system a difficult task. In addition, efficient functional analyses are required to characterize molecules associated with signaling pathways identified from the large data sets generated by next generation differential screens. Here, we illustrate a straightforward method to efficiently identify components of signal transduction pathways governing cell fate and axis specification in sea urchin embryos. The genomic and morphological simplicity of embryos similar to those of the sea urchin make them powerful in vivo developmental models for understanding complex signaling interactions. The methodology described here can be used as a template for identifying novel signal transduction molecules in individual pathways as well as the interactions among the molecules in the various pathways in many other organisms.

Dans l&#39;embryon d&#39;oursin , nous avons montré que 3 différents de signalisation Wnt branches (Wnt / β-caténine, Wnt / JNK et Wnt / PKC) 4, 25 interagissent pour former un réseau de signalisation Wnt qui régit antéro-postérieur (AP) patterning. Une des conséquences les plus importantes de ces événements de signalisation est que la large exprimé neurectoderme antérieure (ANE) GRN initial devient restreint à un p...

Watch this Scientific Journal Video about The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions at JoVE.com

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

Cet article vidéo détaille un simple dans la méthode in vivo qui peut être utilisé pour caractériser systématiquement et efficacement les composants de voies de signalisation complexes et les réseaux de régulation dans de nombreux embryons d&#39; invertébrés.

La puissance de la simplicité: Sea Urchin Embryons comme<em&gt; In Vivo</em&gt; Modèles de développement pour l&#39;étude de la cellule cellule-à-Complex de signalisation réseau Interactions

Remarkably few cell-to-cell signal transduction pathways are necessary during embryonic development to generate the large variety of cell types and ...

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

Unit 1

Principles of Cell Signaling

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