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. High Throughput Morfolino Design Strategy <ol><li> Identificeer een gen (s) van belang (bijvoorbeeld kandidaat-gen benadering, cis-regulerende analyse RNAseq en / of proteomics differentiële schermen). </li><li> Gebruik genomische, transcriptoom, en genexpressie gegevens beschikbaar over regelmatig bijgewerkt websites (bv SpBase http://www.echinobase.org 20 en S. purpuratus Genome Search http: ///urchin.nidcr.nih.gov/blast/index .html) om vast te stellen dat de sp...

<ol>
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De hier gepresenteerde methodiek is een voorbeeld dat de kracht van het gebruik van embryo&#39;s met minder genomische en morfologische complexiteit dan gewervelde dieren aan de signalering-routes en GRNs betreffende fundamentele ontwikkelingsstoornissen mechanismen te begrijpen illustreert .. Veel labs worden met vergelijkbare testen tijdens de zee-egel vroege ontwikkeling van het ontleden signaalwegen betrokken bij andere lot van de cel specificatie evenementen (bijv Notch, Hedgehog, TGF-β, en FGF signalerin...

Gen regulerende netwerken (GRNs) en signaaltransductiewegen stellen de ruimtelijke en temporele expressie van genen tijdens de embryonale ontwikkeling die worden gebruikt om het volwassen dier lichaam kunnen samenstellen. Cel-tot-cel signaaloverdracht zijn essentiële componenten van deze regulerende netwerken die het mogelijk maakt waarbij cellen communiceren. Deze cellulaire interacties vast te stellen en verfijnen van de expressie van de regelgevende en differentiatie genen in en tussen de verschillende gebieden tijdens de embryogenese 1, 2. Interacties tussen uitgescheiden extracellulaire modulatoren (liganden, antagonisten), receptoren en co-receptoren controle over de activiteiten van signaaltransductieroutes. Een assortiment van intracellulaire moleculen transduceert deze ingangen resulteert in veranderde genexpressie, delen en / of vorm van een cel. Hoewel veel van de sleutelfactoren die de extracellulaire en intracellulaire niveaus in grote pathwaybekend is, is een onvolledige kennis grotendeels te danken aan de complexiteit van afzonderlijke signaalpaden. Bovendien hebben verschillende signaalwegen vaak met elkaar positief of negatief in de extracellulaire intracellulaire en transcriptionele niveau 3, 4, 5, 6. Belangrijk is dat de kerncomponenten van signaaltransductiewegen sterk geconserveerd in alle metazoan soort en, opmerkelijk meeste grote signaalwegen voeren vaak dezelfde ontwikkelingsfunctie in vele species bij vergelijking organismen van nauw verwante phyla name 7, 8, 9, 10, 11. De studie van signalering tijdens de ontwikkeling is een enorme taak in elk organisme, en erzijn verscheidene belangrijke uitdagingen voor het bestuderen signaalwegen meeste nieuwmondigen modellen (gewervelde, ongewervelde chordates, hemichordaten en stekelhuidigen): 1) In vertebraten er grote aantallen mogelijke ligand en receptor / co-modulator interacties intracellulaire transductie moleculen, alsmede mogelijke interacties tussen verschillende signaalwegen vanwege de complexiteit van het genoom 12, 13, 14; 2) Het complex morfologie en morfogenetische mutaties in gewervelden vaak moeilijker om functionele interacties in en tussen signaaltransductieroutes interpreteren; 3) Analyses in de meeste niet-echinoderm invertebrate nieuwmondigen model soorten worden beperkt door korte vensters van graviditeit met uitzondering van enkele soorten manteldier 15, 16. Dezeeëgel embryo enkele van de hierboven genoemde beperkingen en biedt een uitstekende eigenschappen voor het uitvoeren van een gedetailleerde analyse van signaaltransductie in vivo. Deze omvatten het volgende: 1) De relatieve eenvoud van de zee-egel genoom vermindert het aantal mogelijke ligand, receptor / coreceptor en intracellulaire transductie molecuul interacties 17; 2) De GRNs besturen van de specificatie en patroonvorming van de kiem lagen en grote embryonale assen zijn goed ingeburgerd in zeeëgel embryo, hulp bij het begrijpen van de regulerende context van de cel / gebied ontvangen van de signalen 18, 19; 3) Vele signaaltransductieroutes kunnen worden onderzocht tussen splitsing en vroege stadia, gastrula embryo uit één gelaagde epitheel waarvan de morfologie gemakkelijker te analyseren; 4) De moleculen omvattend in signaalwegen in de zee-egels zijn gemakkelijk te manipuleren; 5) Veel zee-egels zijn gravid gedurende 10 tot 11 maanden per jaar (bv Strongylocentrotus purpuratus en Lytechinus variegatus). Hier presenteren we een methode om systematisch en doeltreffend karakteriseren componenten van de signaalroutes die aangeven en patroon gebieden zeeegel&#39;s naar de voordelen die verschillende ongewervelde modelsystemen bieden in de studie van complexe moleculaire mechanismen illustreren.

<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.

In de zee-egel embryo hebben we aangetoond dat 3 verschillende Wnt signalering branches (Wnt / β-catenine, Wnt / JNK en Wnt / PKC) 4, 25 interactie vertonen een Wnt signalering netwerk anterior-posterior (AP) patronen regelt vormen. Een van de belangrijkste gevolgen van deze signalering gebeurtenissen is die de oorspronkelijke algemeen uitgedrukt anterior neuroectoderm (ANE) GRN wordt beperkt tot een klein gebied rond de oogbol ...

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

Deze video artikel beschrijft een eenvoudige in vivo methodiek die gebruikt kunnen worden om systematisch en efficiënt te karakteriseren componenten van complexe signaalwegen en regulatorische netwerken in vele ongewervelde embryo&#39;s.

De kracht van eenvoud: Sea Urchin Embryo&#39;s als<em&gt; In Vivo</em&gt; Developmental Modellen voor het bestuderen van complexe 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 ...

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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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