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. Gennemløb Morpholino Design Strategy High <ol><li> Identificer et gen (er) af interesse (f.eks kandidat gen tilgang, cis-regulatoriske analyse, RNAseq og / eller proteom differential skærme). </li><li> Brug genomisk, transkriptom, og genekspression data om hyppigt opdaterede hjemmesider (fx SpBase http://www.echinobase.org 20 og S. purpuratus Genome Søg http: ///urchin.nidcr.nih.gov/blast/index .html) for at fastslå, at Spatiotemporal udtryk profil overlapper d...

<ol>
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Metoden præsenteres her er et eksempel, der illustrerer styrken i at bruge embryoner med mindre genomisk og morfologisk kompleksitet end hvirveldyr at forstå signalering transduktionsveje og GRNs for grundlæggende udviklingsmæssige mekanismer .. Mange laboratorier anvender lignende analyser i den tidlige søpindsvin udvikling at dissekere signalveje, der er involveret i andre celleskæbne specifikation hændelser (f.eks Notch, Hedgehog, TGF β, og FGF-signalering) 27, <sup...

Gen regulatoriske netværk (GRNs) og signaltransduktionsveje etablere den rumlige og tidsmæssige ekspression af gener under fosterudviklingen, der anvendes til at bygge det voksne dyr krop plan. Celle-til-celle signaltransduktionsveje er væsentlige komponenter af disse regulatoriske netværk, som giver de midler, hvormed celler kommunikerer. Disse cellulære interaktioner etablere og forfine udtryk for regulatoriske og differentiering gener i og mellem de forskellige områder under embryogenese 1, 2. Interaktioner blandt udskilles ekstracellulære modulatorer (ligander, antagonister), receptorer, og co-receptorer styrer aktiviteterne i signaltransduktionsveje. Et udvalg af intracellulære molekyler transducerer disse input resulterer i ændret genekspression, division, og / eller formen af ​​en celle. Mens mange af de vigtigste molekyler, der anvendes ved de ekstracellulære og intracellulære niveauer i de store veje erkendt, er det et ufuldstændigt kendskab i vid udstrækning skyldes kompleksiteten af ​​individuelle signalveje. Desuden forskellige signalveje ofte interagere med hinanden enten positivt eller negativt på den ekstracellulære, intracellulære, og transkriptionelle niveau 3, 4, 5, 6. Vigtigere er de centrale komponenter i signaltransduktionsveje stærkt konserveret i alle metazoiske art og, bemærkelsesværdigt, fleste af de store signalveje udfører ofte lignende udviklingsmæssige funktioner i mange arter, når man sammenligner organismer fra nært beslægtede phyla navnlig 7, 8, 9, 10, 11. Studiet af signalering under udvikling er en skræmmende opgave i enhver organisme, og derer flere væsentlige udfordringer for at studere signalveje i de fleste deuterostom modeller (hvirveldyr, hvirvelløse chordater, hemichordates og pighuder): 1) I hvirveldyr der er et stort antal mulige ligand og receptor / co-modulator interaktioner, intracellulære transduktion molekyler, samt potentielle interaktioner mellem forskellige signalveje grund af kompleksiteten af genomet 12, 13, 14; 2) Den komplekse morfologi og morfogenetiske bevægelser hos hvirveldyr ofte gøre det vanskeligere at fortolke funktionelle interaktioner i og blandt signaltransduktionsveje; 3) Analyser i de fleste ikke-pighuder hvirvelløse deuterostom modelarter er begrænset af korte vinduer af gravidity med undtagelse af nogle tunicate arter 15, 16. Detsøpindsvin embryo har nogle af de ovennævnte begrænsninger og tilbyder mange enestående kvaliteter til at udføre en detaljeret analyse af signaltransduktionsveje in vivo. Disse omfatter følgende: 1) Den relative enkelhed af søpindsvin genomet reducerer antallet af mulige ligand, receptor / co-receptor og intracellulære transduktion molekyle væsentligt interaktioner 17; 2) GRNs styrer specifikation og mønster af kim lag og større embryonale akser er veletablerede i søpindsvin embryoner, medvirken i forståelsen af de lovgivningsmæssige sammenhæng med celle / område modtager signalerne 18, 19; 3) Mange signaltransduktionsveje kan studeres mellem spaltnings- og gastrula stadier tidlige når embryoner består af et enkelt lag epitel hvis morfologi er lettere at analysere; 4) Molekylerne omfatterd i signalveje i søpindsvin let manipuleret; 5) Mange søpindsvin er den drægtige i 10 til 11 måneder om året (f.eks Strongylocentrotus purpuratus og Lytechinus variegatus). Her præsenteres en metode til systematisk og effektivt karakterisere komponenter af signalveje, der angiver og mønster territorier i søpindsvin embryoner at illustrere de fordele, som en række hvirvelløse modelsystemer tilbyder i studiet af komplekse molekylære mekanismer.

<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.

I havet urchin embryo har vi vist, at 3 forskellige Wnt signalering grene (Wnt / β-catenin, Wnt / JNK, og Wnt / PKC) 4, 25 samvirker til dannelse af et Wnt signalering netværk, der styrer anterior-posterior (AP) mønsterdannelse. En af de vigtigste konsekvenser af disse signaleringsbegivenheder er, at den oprindelige udtrykkes bredt anterior neuroectoderm (ANE) GRN bliver begrænset til et lille område omkring den forreste pol...

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

Denne video artikel beskriver en enkel in vivo metode, der kan anvendes til systematisk og effektivt karakterisere bestanddele af komplekse signalveje og regulerende net i mange hvirvelløse embryoer.

The Power of Simplicity: Søpindsvin embryoner som<em&gt; I Vivo</em&gt; Developmental modeller til undersøgelser Complex celle-til-cellesignalering netværksinteraktioner

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

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Principles of Cell Signaling

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