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. Rendimento Morpholino Estratégia de alto design <ol><li> Identificar um gene (s) de interesse (por exemplo, abordagem do gene candidato, análise de cis-regulatórias, RNA-Seq e ou telas diferenciais / proteômica). </li><li> Use genômica, transcriptomic, e dados de expressão de genes disponíveis em sites atualizados com freqüência (por exemplo SpBase http://www.echinobase.org 20 e S. purpuratus Genome Pesquisa http: ///urchin.nidcr.nih.gov/blast/index .html)...

<ol>
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A metodologia aqui apresentada é um exemplo que ilustra o poder de usar embriões com menos complexidade genômica e morfológica de vertebrados para compreender as vias de transdução de sinalização e GRNs regem mecanismos de desenvolvimento fundamentais .. Muitos laboratórios estão usando ensaios semelhantes durante o desenvolvimento do ouriço-do-mar cedo para dissecar o vias envolvidas em outros eventos de especificação destino de sinalização celular (por exemplo, Notch, Hedgehog, TGF-β, e FGF si...

redes de genes reguladores (GRNs) e vias de transdução de sinal estabelecer a expressão espacial e temporal dos genes durante o desenvolvimento embrionário que são usados ​​para construir o plano de adulto corpo do animal. Célula-a-célula vias de transdução de sinal são componentes essenciais destas redes reguladoras, que proporciona os meios pelos quais as células comunicam. Estas interacções celulares estabelecer e aperfeiçoar a expressão de genes reguladores e diferenciação dentro e entre os vários territórios durante a embriogênese 1, 2. Interações entre os moduladores secretadas extracelulares (ligantes, antagonistas), receptores e co-receptores controlar as atividades de vias de transdução de sinal. Uma variedade de moléculas intracelulares transduz estas entradas resultando na expressão alterada do gene, divisão, e / ou a forma de uma célula. Enquanto muitas das moléculas-chave utilizadas nos níveis extracelulares e intracelulares nas vias principais sãoconhecido, é um conhecimento incompleto, devido em grande parte à complexidade das vias de sinalização individuais. Além disso, diferentes vias de sinalização, muitas vezes interagem uns com os outros, positivamente ou negativamente na extracelular, intracelular, e os níveis de transcrição de 3, 4, 5, 6. É importante ressaltar que os principais componentes de vias de transdução de sinal são altamente conservadas em todas as espécies de metazoários, e, curiosamente, a maioria das principais vias de sinalização, muitas vezes executar funções de desenvolvimento semelhantes em muitas espécies quando se comparam os organismos de filos intimamente relacionados, em especial, 7, 8, 9, 10, 11. O estudo da sinalização durante o desenvolvimento é uma tarefa difícil em qualquer organismo, e hávários desafios significativos para estudar vias de sinalização na maioria dos modelos deuterostomia (vertebrados, cordados invertebrados, hemichordates, e equinodermes): 1) Em vertebrados há um grande número de possíveis ligandos e as interacções receptor / co-moduladores, moléculas de transdução intracelular, bem como potenciais interacções entre diferentes vias de sinalização, devido à complexidade do genoma de 12, 13, 14; 2) A morfologia complexa e movimentos morfogenéticos em vertebrados muitas vezes tornam mais difíceis de interpretar as interações funcionais em e entre vias de transdução de sinal; 3) As análises na maioria dos não-equinodermos espécie modelo de invertebrados deuterostomia são limitados por janelas curtas de gravidity com exceção de algumas espécies de tunicados 15, 16. oouriço-do-mar embrião tem algumas das limitações acima mencionadas e oferece muitas qualidades únicas para a realização de uma análise detalhada das vias de transdução de sinal in vivo. Estes incluem o seguinte: 1) A relativa simplicidade do genoma do ouriço-do-mar reduz significativamente o número de possíveis do ligando, do receptor / co-receptor e molécula de transdução intracelular interacções 17; 2) Os GRNs que controlam a memória descritiva e modelação das camadas germinativas e grandes eixos embrionários são bem estabelecida em embriões de ouriço-do-mar, auxiliando na compreensão do contexto regulamentar da célula / território receber os sinais 18, 19; 3) Muitas vias de transdução de sinal podem ser estudadas entre as fases de clivagem e de gástrula inicial quando embriões consistem de um único epitélio em camadas cuja morfologia é mais fácil de analisar; 4) As moléculas envolvemd nas vias de sinalização nas ouriços do mar são facilmente manipulados; 5) Muitas ouriços do mar estão grávidas de 10 a 11 meses por ano (por exemplo, Strongylocentrotus purpuratus e Lytechinus variegatus). Aqui, apresentamos um método para caracterizar de forma sistemática e eficiente componentes das vias de sinalização que especificam e territórios padrão em embriões de ouriço-do-mar para ilustrar as vantagens que vários sistemas modelo de invertebrados oferecem no estudo dos mecanismos moleculares complexas.

<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.

No embrião ouriço-do-mar temos demonstrado que 3 diferentes sinalização Wnt ramos (Wnt / β-catenina, Wnt / JNK, e Wnt / PKC) 4, 25 interagem de modo a formar uma rede de sinalização de Wnt que governa ântero-posterior padronização (AP). Uma das consequências mais importantes destes eventos de sinalização é que a GRN inicial amplamente expressa neuroectoderma anterior (ANE) torna-se restrita a um pequeno território ...

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

Este artigo de vídeo detalha uma simples em metodologia in vivo que pode ser utilizado para caracterizar de forma eficiente e sistematicamente componentes de vias de sinalização complexas e redes reguladoras em muitos embriões de invertebrados.

O Poder da Simplicidade: Sea Urchin embriões Na Vivo Modelos de desenvolvimento para estudar células de células-complexo de sinalização de rede Interações

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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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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