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. hög genomströmning morfolino Design Strategy <ol><li> Identifiera en gen (er) av intresse (t.ex. kandidatgen tillvägagångssätt, cis-regulatoriska analys RNAseq och / eller proteomik differential skärmar). </li><li> Använd iskt, transcriptomic och genuttryck data tillgängliga på ofta uppdaterade webbplatser (t.ex. SpBase http://www.echinobase.org 20 och S. purpuratus Genome Sök http: ///urchin.nidcr.nih.gov/blast/index .html) för att fastställa att Spati...

<ol>
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Den metod som presenteras här är ett exempel som illustrerar kraften i att använda embryon med mindre iska och morfologiska komplexitet än ryggradsdjur att förstå signalering överföringsvägar och GRNs styr grundläggande mekanismer utvecklings .. Många labb använder liknande analyser under tidigt sjöborre utveckling dissekera signalvägar som är involverade i andra cell öde specifikation händelser (t.ex. Notch, Hedgehog, TGF β, och FGF signalering) 27, <sup cla...

Genreglerande nät (GRNs) och signaltransduktionsvägar fastställa den rumsliga och tidsmässiga uttryck av gener under embryonal utveckling som används för att bygga det vuxna djuret kropp plan. Cell till cell signaltransduktionsvägar är viktiga komponenter i dessa regulatoriska nätverk, vilket ger det sätt på vilket celler kommunicerar. Dessa cellulära interaktioner etablera och förbättra uttrycket av regulatoriska och differentierings gener i och mellan de olika områdena under embryogenes 1, 2. Interaktioner mellan utsöndrade extracellulära modulatorer (ligander, antagonister), receptorer och co-receptorer kontrollera verksamheten i signaltransduktionsvägar. Ett sortiment av intracellulära molekyler omvandlar dessa ingångar leder till förändrad genexpression, division, och / eller formen av en cell. Medan många av de viktiga molekyler som används vid de extracellulära och intracellulära nivåer i de stora vägar ärkänd, är det en ofullständig kunskap beror till stor del på komplexiteten i enskilda signaleringsvägar. Dessutom har olika signalvägar samverkar ofta med varandra, antingen positivt eller negativt på den extracellulära, intracellulära och transkriptions nivåer 3, 4, 5, 6. Viktigt är de centrala delarna av signaltransduktionsvägar högkonserverade i alla flercelliga arter, och anmärkningsvärt, de flesta av de stora signalvägar utför ofta likartade utvecklingsfunktioner i många arter när man jämför organismer från närbesläktade provinsar i synnerhet 7, 8, 9, 10, 11. Studien av signalering under utveckling är en svår uppgift i en organism, och därfinns flera stora utmaningar för att studera signalvägar i de flesta deuterostomer modeller (ryggradsdjur, ryggradslösa chordates, svalgsträngsdjur och tagghudingar): 1) I ryggradsdjur finns det ett stort antal möjliga ligand och receptor / co-modulator interaktioner, intracellulära överföringsmolekyler, samt potentiella interaktioner mellan olika signalvägar på grund av komplexiteten av genomet 12, 13, 14; 2) Den komplexa morfologi och morfogenetiska rörelser hos ryggradsdjur ofta göra det svårare att tolka funktionella interaktioner i och bland signaltransduktionsvägar; 3) Analyser i de flesta icke-Echinoderm ryggradslös deuterostomer modellarter begränsas av korta fönster gravidity med undantag för vissa manteldjursarter 15, 16. Desjöborre embryot har få av de ovannämnda begränsningarna och erbjuder många unika egenskaper för att utföra en detaljerad analys av signaltransduktionsvägar in vivo. Dessa innefattar följande: 1) Den relativa enkelhet sjöborre genomet minskar signifikant antalet möjliga ligand, receptor / co-receptor och intracellulära transduktionen molekyl interaktioner 17; 2) De GRNs styr specifikation och mönstring av grodden lager och stora embryonala axlar är väl etablerade i sjöborre embryon, medhjälp i förståelsen av regelverket ramen för cell / område som tar emot signalerna 18, 19; 3) Många signaltransduktionsvägar kan studeras mellan tidiga klyvnings och gastrula steg när embryona består av ett enda lager epitel vars morfologi är lättare att analysera; 4) Molekylerna involverard i signalvägar i sjöborrar lätt manipuleras; 5) Många sjöborrar är gravid i 10 till 11 månader om året (t.ex. Strongylocentrotus purpuratus och lytechinus variegatus). Här presenterar vi en metod för att systematiskt och effektivt karaktärisera komponenter av signalvägar som anger och mönster områden i sjöborre embryon för att illustrera de fördelar som flera ryggradslösa modellsystem erbjuder i studiet av komplexa molekylära 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 sjöborre embryot har vi visat att 3 olika Wnt signalering grenar (Wnt / β-catenin, Wnt / JNK, och Wnt / PKC) 4, 25 samverkar för att bilda ett Wnt signaleringsnätet som styr anterior-posterior (AP) mönstring. En av de viktigaste konsekvenserna av dessa signaleringshändelser är att den ursprungliga brett uttryckt främre neuroectoderm (ANE) GRN blir begränsad till ett litet område runt den främre stolpen i början av g...

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

Denna video artikeln beskriver en enkel in vivo metod som kan användas för att systematiskt och effektivt karaktärisera komponenter i komplexa signaleringsvägar och regulatoriska nätverk i många ryggradslösa embryon.

The Power of Enkelhet: Sea Urchin embryon som<em&gt; In vivo</em&gt; Utvecklings modeller för att studera komplexa cell till cellsignalering 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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Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

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Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka ...

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In vivo avbildning av Transgena genuttryck i Individuell Retinal stamceller i chimär Zebrafish embryon att studera Cell Nonautonomous Influenser

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The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...

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Bedömning av oxidativ skada i de primära musokulära ytcellerna/stamcellerna som svar på ultraviolett-C (UV-C) Skada

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

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