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. Rendimiento estrategia de diseño de alta morfolino <ol><li> Identificar un gen (s) de interés (por ejemplo, enfoque de genes candidatos, el análisis de cis-regulador, RNAseq y pantallas diferenciales / o proteómica). </li><li> Utilice genómica, transcriptómica, y los datos de expresión génica disponibles en los sitios web se actualizan con frecuencia (por ejemplo SpBase http://www.echinobase.org 20 y S. purpuratus genoma Búsqueda http: ///urchin.nidcr.nih....

<ol>
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La metodología que aquí se presenta es un ejemplo que ilustra el poder de utilizar embriones con menos complejidad genómica y morfológica de los vertebrados para entender las vías de transducción de señales y GRNs que rigen los mecanismos fundamentales del desarrollo .. Muchos laboratorios están usando ensayos similares durante el desarrollo de erizo de mar temprano para diseccionar el vías que participan en otros eventos de especificación del destino celular de señalización (por ejemplo, Notch, Hed...

las redes de genes reguladores (GRNs) y las vías de transducción de señales establecen la expresión espacial y temporal de los genes durante el desarrollo embrionario que se utilizan para construir el plan de adultos cuerpo del animal. Célula a célula de las vías de transducción de señales son componentes esenciales de estas redes reguladoras, que proporcionan el medio por el cual las células se comunican. Estas interacciones celulares establecer y perfeccionar la expresión de genes reguladores y diferenciación en y entre los diversos territorios durante la embriogénesis 1, 2. Las interacciones entre los moduladores secretadas extracelulares (ligandos, antagonistas), receptores y co-receptores controlan las actividades de las vías de transducción de señales. Un surtido de moléculas intracelulares transduce estas entradas que resulta en la expresión alterada de genes, la división y / o la forma de una célula. Si bien muchas de las moléculas clave que se utilizan en los niveles extracelulares e intracelulares en las principales vías sonconocido, es un conocimiento incompleto debido en gran parte a la complejidad de las vías de señalización individuales. Además, las diferentes vías de señalización a menudo interactúan uno con el otro, ya sea positiva o negativamente en el extracelular, intracelular y los niveles de la transcripción 3, 4, 5, 6. Es importante destacar que los componentes centrales de las vías de transducción de señales están altamente conservadas en todas las especies de metazoos, y, notablemente, la mayoría de las principales vías de señalización menudo realizan funciones de desarrollo similares en muchas especies cuando se comparan los organismos de filos estrechamente relacionados, en particular, 7, 8, 9, 10, 11. El estudio de la señalización durante el desarrollo es una tarea de enormes proporciones en cualquier organismo, y hayson varios retos importantes para el estudio de las vías de señalización en la mayoría de los modelos deuterostome (vertebrados, los cordados invertebrados, hemicordados y equinodermos): 1) En los vertebrados hay un gran número de posibles ligando y las interacciones de receptor / co-moduladores, moléculas de transducción intracelular, así como potenciales interacciones entre diferentes vías de señalización debido a la complejidad del genoma de 12, 13, 14; 2) La morfología compleja y movimientos morfogenéticos en los vertebrados a menudo hacen que sea más difícil la interpretación de las interacciones funcionales en y entre las vías de transducción de señales; 3) Análisis de la mayoría de especies modelo de invertebrados deuterostome no equinodermo están limitados por las ventanas cortas de gravidez con la excepción de algunas especies de tunicados 15, 16. losembrión de erizo de mar tiene algunas de las limitaciones mencionadas anteriormente y ofrece muchas cualidades únicas para la realización de un análisis detallado de las vías de transducción de señales in vivo. Estos incluyen los siguientes: 1) La relativa simplicidad del genoma del erizo de mar reduce significativamente el número de posibles ligando, receptor / co-receptor y la molécula de transducción intracelular las inter- 17; 2) Los GRNs que controlan la memoria y en los patrones de las capas germinales embrionarias y los principales ejes están bien establecidos en embriones de erizo de mar, ayudar en la comprensión del contexto normativo de la célula / territorio que recibe las señales 18, 19; 3) Muchas vías de transducción de señal pueden ser estudiados entre las etapas de escisión y gástrula primeros cuando los embriones se componen de una sola epitelio estratificado cuya morfología es más fácil de analizar; 4) Las moléculas implicand en las vías de los erizos de mar son fáciles de manipular señalización; 5) Muchos erizos de mar están grávidas de 10 a 11 meses al año (por ejemplo, Strongylocentrotus purpuratus y variegatus Lytechinus). A continuación, presentamos un método para caracterizar de manera sistemática y eficientemente los componentes de las vías de señalización que especifican y territorios de patrón en embriones de erizo de mar para ilustrar las ventajas que varios sistemas modelo de invertebrados ofrecen en el estudio de los mecanismos moleculares complejas.

<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.

En el embrión de erizo de mar hemos demostrado que 3 de señalización Wnt diferentes ramas (Wnt / β-catenina, Wnt / JNK, y Wnt / PKC) 4, 25 interactúan para formar una red de señalización Wnt que gobierna anterior-posterior patrón (AP). Una de las consecuencias más importantes de estos eventos de señalización es que el expresado en términos generales neuroectodermo anterior (ANE) GRN inicial se restringe a un pequeño ...

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 artículo de vídeo detalla una sencilla metodología vivo que se puede utilizar para caracterizar de manera sistemática y eficiente los componentes de las vías de señalización complejos y las redes de regulación en muchos embriones de invertebrados en.

El poder de la simplicidad: Erizo de mar como embriones<em&gt; En Vivo</em&gt; Modelos de desarrollo para el estudio celular de la célula-a-complejo de señalización de red Interacciones

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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Cell Biology

Developmental Biology

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

SCIENTISTS IN ACTION

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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 ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

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Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues ...