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. Strategia High Throughput Morpholino design <ol><li> Identificare un gene (s) di interesse (ad esempio, candidato approccio del gene, l&#39;analisi cis-normativo, RNA-Seq e / o schermi differenziali proteomica). </li><li> Utilizzare genomica, trascrittomica, e dati di espressione genica disponibili sui siti web aggiornati di frequente (ad es SpBase http://www.echinobase.org 20 e S. purpuratus Genome Ricerca http: ///urchin.nidcr.nih.gov/blast/index .html) per deter...

<ol>
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La metodologia qui presentata è un esempio che illustra il potere di usare embrioni con una minore complessità genomica e morfologica di vertebrati per capire le vie di segnalazione di trasduzione e GRN regolano meccanismi di sviluppo fondamentali .. Molti laboratori stanno utilizzando dosaggi simili durante lo sviluppo precoce riccio di mare a sezionare il coinvolti in percorsi di altri eventi di specifica destino della cellula di segnalazione (ad esempio Notch, Riccio, TGF-β, e la segnalazione FGF) <sup cl...

reti gene regolatore (GRN) e vie di trasduzione del segnale stabiliscono l&#39;espressione spaziale e temporale dei geni durante lo sviluppo embrionale, che vengono utilizzati per costruire il piano di adulti corpo animale. Cell-a-cella trasduzione del segnale sono componenti essenziali di queste reti di regolazione, fornendo i mezzi con cui le cellule comunicano. Queste interazioni cellulari stabilire e perfezionare l&#39;espressione di geni regolatori e differenziazione nei e tra i diversi territori durante l&#39;embriogenesi 1, 2. Le interazioni tra i modulatori secreti extracellulari (ligandi, antagonisti), recettori, e co-recettori controllano le attività delle vie di trasduzione del segnale. Un assortimento di molecole intracellulari transduces questi ingressi conseguente alterata espressione genica, divisione, e / o la forma di una cellula. Mentre molte delle molecole chiave utilizzati ai livelli extracellulari e intracellulari nelle principali vie sononoto, è una conoscenza incompleta dovuto in gran parte alla complessità delle vie di segnalazione individuali. Inoltre, diverse vie di segnalazione spesso interagiscono tra loro positivamente o negativamente al extracellulare, intracellulare e livelli trascrizionali 3, 4, 5, 6. È importante sottolineare che i componenti di base delle vie di trasduzione del segnale sono altamente conservati in tutte le specie metazoi, e, straordinariamente, la maggior parte delle vie di segnalazione spesso svolgono funzioni di sviluppo simili in molte specie quando si confrontano gli organismi da phyla strettamente correlati, in particolare 7, 8, 9, 10, 11. Lo studio di segnalazione durante lo sviluppo è un compito arduo in qualsiasi organismo, e cisono diverse sfide significative per studiare percorsi di segnalazione nella maggior parte dei modelli deuterostomi (vertebrati, cordati invertebrati, hemichordata, ed echinodermi): 1) Nei vertebrati ci sono un gran numero di possibili ligando e recettore / co-modulatore, molecole di trasduzione intracellulare, così come potenziali interazioni tra diverse vie di segnalazione a causa della complessità del genoma 12, 13, 14; 2) La morfologia complessa e movimenti morfogenetici nei vertebrati spesso rendono più difficile interpretare le interazioni funzionali e tra le vie di trasduzione del segnale; 3) Analisi in maggior parte delle specie modello invertebrato deuterostomi non echinoderma sono limitate da brevi finestre gravidity con l&#39;eccezione di alcune specie tunicate 15, 16. Ilriccio di mare embrione ha alcune delle limitazioni di cui sopra ed offre molte qualità uniche per l&#39;esecuzione di un&#39;analisi dettagliata delle vie di trasduzione del segnale in vivo. Questi sono i seguenti: 1) La relativa semplicità del genoma riccio riduce significativamente il numero di possibili ligando, recettore / co-recettore e molecola di trasduzione intracellulare interazioni 17; 2) I GRN che controllano la specifica e patterning degli strati germinali e le principali assi embrionali sono ben stabiliti in embrioni di riccio di mare, aiutando la comprensione del contesto normativo della cella / territorio, che riceve i segnali 18, 19; 3) Molti vie di trasduzione del segnale possono essere studiati tra gli stadi scissione e gastrula quando gli embrioni sono costituiti da un singolo strato dell&#39;epitelio la cui morfologia è più facile da analizzare; 4) Le molecole comportanod in vie di segnalazione nei ricci di mare sono facilmente manipolati; 5) Molti ricci di mare sono gravide per 10 a 11 mesi l&#39;anno (ad esempio Strongylocentrotus purpuratus e Lytechinus variegatus). Qui, vi presentiamo un metodo per caratterizzare in modo sistematico ed efficiente componenti delle vie di segnalazione che specificano e territori del modello negli embrioni di riccio di mare per illustrare i vantaggi che diversi sistemi modello invertebrati offrono nello studio dei meccanismi molecolari complessi.

<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.

Nell&#39;embrione riccio di mare abbiamo dimostrato che 3 diverse di segnalazione Wnt rami (Wnt / β-catenina, Wnt / JNK, e Wnt / PKC) 4, 25 interagiscono per formare una rete di segnalazione Wnt che governa antero-posteriore (AP) patterning. Una delle più importanti conseguenze di questi eventi di segnalazione è che il ampiamente espresso anteriore neuroectoderma (ANE) GRN iniziale diventa limitato ad un piccolo territorio att...

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

Questo articolo descrive il video di una semplice metodologia vivo che può essere utilizzata per caratterizzare in modo sistematico ed efficiente componenti di vie di segnalazione complessi e reti di regolazione in molti embrioni invertebrati.

La forza della semplicità: Sea Urchin embrioni<em&gt; In Vivo</em&gt; Modelli di sviluppo per lo studio delle cellule cellula-Complex segnalazione di rete Interazioni

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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