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.gov/blast/index .html) para determinar que el perfil de expresión espacio-temporal se solapa con el mecanismo de desarrollo en cuestión. Si no hay datos de expresión está disponible, a continuación, generar cebadores qPCR y / o un antisentido en situ sonda para evaluar el patrón de expresión génica. </li><li> Después de determinar el patrón de expresión espacial y temporal, obtener la secuencia de ADN de los sitios web genómicos. <ol><li> Para la generación de oligonucleótidos de morfolino traducción de bloqueo, obtener la secuencia of la &#39;región traducida por la ONU (5&#39; UTR 5) directamente aguas arriba del codón de iniciación de bases de datos de etiqueta de secuencia expresada (EST) disponibles en SpBase o S. purpuratus Genoma sitios web de búsqueda. Si esta información no está disponible para el gen de interés, a continuación, realizar 5 &#39;RACE. </li><li> Para diseñar un empalme morfolino-bloqueo, la búsqueda de la secuencia genómica que incluye los exones e intrones utilizando la herramienta de andamio BLASTN en SpBase o S. purpuratus genoma herramienta de búsqueda. </li></ol></li><li> Utilice el diseño de página web oligonucleótido http://oligodesign.gene-tools.com con el fin de diseñar la secuencia de pares de bases deseada morfolino 25. <ol><li> Para un morfolino de traslación de bloqueo, utilizar la secuencia de ARNm de 5 &#39;a 3&#39; como la fuente de la secuencia diana de la traducción. Incluir la secuencia 5 &#39;UTR (aproximadamente 70 nucleótidos aguas arriba del sitio de inicio de transcripción), además de 25 bases de la región codificadora (aguas abajo del sitio de inicio) con el codón de inicio marcada wiTH paréntesis (ATG). </li><li> Para un empalme morfolino-bloqueo, seleccione el exón-intrón o secuencia límites exón-intrón e incluyen 50 bases (25 bases de la secuencia exón y 25 bases de la secuencia del intrón) en torno a las regiones limítrofes. Analiza el genoma con la secuencia de morfolino para verificar que la secuencia es única. </li></ol></li></ol> 2. La microinyección de morfolino oligonucleótidos <ol><li> Preparar 3 mM de soluciones madre de los oligonucleótidos de morfolino mediante la adición de 100 l de agua libre de nucleasa en el vial morfolino 300 nmol. NOTA: No utilice agua tratada con DEPC para la resuspensión debido pirocarbonato de dietilo puede dañar el morfolino. </li><li> Por primera vuelta de la reconstitución por el vial que contiene la solución de oligonucleótido durante 30 s a la velocidad máxima (14.000 - 16.000 xg) y agitar brevemente, el calor a 65 ° C durante 5 a 10 minutos, vórtice brevemente y mantener a la población de morfolino en la sala temperatura durante al menos 1 h. solución de morfolino<html> s se almacenan de -20 ° C a + 4 ° C. </li><li> Preparar la solución de inyección que contiene oligonucleótidos de morfolino a la concentración deseada. Esta solución por lo general contiene 20% de glicerol o KCl 125 mM como vehículo y 15% de FITC-dextrano (isotiocianato de fluoresceína dextrano 10.000 MW 2,5 mg solución madre / l). FITC-dextrano y otros conjugados de dextrano fluorescentes se utilizan habitualmente para identificar embriones inyectados por microscopía de epifluorescencia. soluciones de inyección almacenar a -20 ° C. </li><li> Calentar la solución morfolino a 65 ° C en un bloque de calor o baño de agua durante por lo menos 2 - 5 min. </li><li> Brevemente girar la solución morfolino durante 30 s a la velocidad máxima (14.000 - 16.000 xg) y agitar durante 1 min y centrifugar a toda velocidad (14.000 - 16.000 xg) durante al menos 10 min. </li><li> Cargar las agujas de inyección con la solución morfolino. Para un protocolo detallado microinyección consulte Stepicheva y Song, 2014 21 y Saludos y Ettensohn de 2004&quot;&gt; 22. </li></ol> 3. Fijación y en el Protocolo Situ a las 24 h después de la fecundación (HPF) en S. purpuratus embriones NOTA: Este protocolo se modificó a partir de Arenas-Mena et al. , 2000 23 y Sethi et al. 2014 24. <ol><li> Fijación <ol><li> Añadir unas gotas de fijador (véase más adelante) a los pocillos que contenían embriones, mezclar suavemente con la pipeta, y dejar que se asienten. Retire la solución de fijación, y luego mezclar los embriones con dos lavados de 180 mu l adicionales de fijador. NOTA: Es importante mezclar bien los embriones durante los lavados por pipeteo suave hacia arriba y abajo varias veces. El no hacerlo puede disminuir la relación señal a ruido. </li><li> Dejar los embriones para fijar en el segundo lavado fijador durante 50 min a 1 h a temperatura ambiente (RT) en paraformaldehído al grado microscopía electrónica de 4% que consiste en MOPS 10 mM pH 7,0, 0,1% de Tween-20, y artificial seawater (ASW). Hacer esta solución fresca cada vez para obtener mejores resultados. Además, para la facilidad y practicidad embriones se fijan en placas de 96 pocillos. <ol><li> Para 20 ml uso fijador 5 ml 16% de paraformaldehído, 15 ml ASW, 200 l 1 M MOPS pH 7.0, y 20 l de Tween-20. </li></ol></li><li> Lavar 5 veces a temperatura ambiente con 180 l de MOPS tampón de lavado que consiste en 0,1 M MOPS pH 7,0, 0,5 M NaCl y 0,1% de Tween-20 durante al menos 5 min o hasta que los embriones de caer al fondo del pozo. Una vez más, es importante para mezclar completamente los embriones en el tampón de lavado pipeteando suavemente arriba y abajo varias veces. tampón de lavado MOPS se puede utilizar durante 2 días si se almacenan a 4 DO. <ol><li> Por 40 ml de MOPS lavan uso de búferes de 4 ml 1 M MOPS pH 7,0, 4 ml de NaCl 5 M, 32 ml dH2O y 40 l de Tween-20. </li><li> Fija los embriones pueden ser almacenadas a 4 ° C durante 2 días. NOTA: Si el almacenamiento de los embriones ya fijos, a continuación, añadir azida de sodio al 0,2% en MOPS tampón de lavado para evitar el crecimiento bacteriano.</li></ol></li></ol></li><li> Pre-hibridación <ol><li> Aspirar el MOPS tampón de lavado y añadir 180 l de una proporción de 2: 1 de MOPS tampón de lavado en tampón de hibridación, la mezcla embriones en la solución mediante pipeteado suave varias veces, y se incuba durante al menos 20 minutos a temperatura ambiente. El tampón de hibridación consiste en 70% de formamida, 0,1 M MOPS pH 7,0, 0,5 M NaCl, 1 mg / ml de BSA y 0,1% de Tween-20. <ol><li> Para uso 40 ml de tampón de hibridación 4 ml 1 M MOPS pH 7,0, 4 ml de NaCl 5 M, 4 ml dH2O, 0,04 g de BSA, y 40 l de Tween-20. Mezclar bien por agitación, añadir 28 ml de formamida, y agitar de nuevo. </li></ol></li><li> Retire la proporción 2: 1 de MOPS de lavado y tampones de hibridación y añadir 180 l de una proporción de 1: 2 de MOPS tampón de lavado en tampón de hibridación, la mezcla embriones en la solución, y se incuba durante al menos 20 minutos a temperatura ambiente. </li><li> Antes de la hibridación de la sonda mezclar suavemente los embriones en 100 - 150 ml de tampón de hibridación. Incubar embriones a 50 ° C durante al menos 1marido. NOTA: La incubación de los embriones durante la noche es aceptable. Antes de la incubación, sellar los pocillos con una lámina adhesiva con el fin de evitar la evaporación. </li></ol></li><li> Hibridación <ol><li> En un tubo separado añadir 0,1 - 0,3 ng / l de sonda y 500 mg / ml de ARNt de levadura en un tampón de hibridación, a continuación, vórtice suavemente para crear solución de la sonda. tRNA de levadura se añade a la solución de la sonda para disminuir la unión no específica de la sonda anti-sentido. Precalentar esta solución a 50 ° C, y el tampón de aspirado de la hibridación. </li><li> Mezclar embriones pre-hibridado en 100 l de la solución de la sonda, el sello con una lámina adhesiva, y se hibrida a 50 ° C durante 2 a 7 días, dependiendo de la sonda (la sonda foxq2 se puede incubar durante 2 días). NOTA: Los tiempos de incubación puede variar en función de la sonda. Algunos genes expresados ​​en niveles bajos requieren hasta una incubación de 7 días. placas de 96 pocillos se pueden colocar en una caja de humedad como un seguro contra la evaporación si los ADHESive hoja no sellar adecuadamente. </li><li> Lavar 5 veces a 50 ° C con 180 l de MOPS recién hechos tampón de lavado para un total de 3 h. A continuación, lavar 3 veces (15 min) con 180 l de MOPS de tampón de lavado a temperatura ambiente. Recuerde mezclar embriones en el tampón de lavado cada vez con la pipeta suavemente varias veces. </li></ol></li><li> La incubación de anticuerpos <ol><li> Aspirar el MOPS tampón de lavado y se mezclan los embriones en 180 l de tampón de bloqueo que consiste en 10% de suero de oveja normal y 5 mg / ml de BSA en MOPS tampón de lavado y se incuba durante al menos 45 minutos a temperatura ambiente o 4 ° C durante la noche. </li><li> Eliminar el tampón de bloqueo y luego mezclar embriones en tampón de bloqueo que contiene una dilución 1: 1500 de anticuerpo anti-digoxigenina conjugado con fosfatasa alcalina diluido en tampón de bloqueo. Incubar toda la noche a temperatura ambiente en una placa de sellado para evitar la evaporación. NOTA: No deje de anticuerpos en más de una noche. </li><li> Lavar los embriones de 6 veces (5 min o hasta que caen embriones) en MOPS tampón de lavado a temperatura ambiente. Los embriones puedenser almacenados durante la noche a 4 ° C. </li></ol></li><li> En desarrollo in situ <ol><li> Lavar embriones 3 veces (10 min) a temperatura ambiente con tampón de pH 9,5 recién hecho que consiste en 0,1 M Tris pH 9,5, NaCl 100 mM, MgCl 2 mM 50 mM, levamisol 1, y 0,1% de Tween-20. <ol><li> Por 20 ml pH 9,5 tampón uso 2 ml de Tris 1 M pH 9,5, 400 l de NaCl 5 M, 1 ml 1 M MgCl 2, 20 l de Tween-20, 16,6 ml dH2O, y 0,0048 g levamisol. </li></ol></li><li> Incubar los embriones a 37 ° C en tampón de tinción protegido de la luz. tampón de tinción se compone de 10% dimetil formamida, 4,5 l / mL 4-Nitro cloruro de azul tetrazolio (NBT) y 3,5 l / ml 5-Bromo-4-cloro-3-indolil-fosfato (BCIP) en recién hecho tampón de pH 9,5 . <ol><li> Para 1 ml de tampón de tinción utilizar 100 l dimetil formamida, 4,5 l NBT y BCIP 3,5 l. </li></ol></li><li> Detener la reacción de la fosfatasa alcalina mediante el lavado de 3 a 5 veces en tampón de lavado MOPS. La reacción wiº la sonda foxq2 normalmente tarda 30 minutos a 1 hora. Algunas sondas pueden necesitar de incubación durante la noche a RT, ya sea o 4 ° C. </li><li> Mezclar los embriones en una solución de lavado MOPS 70% y 30% de glicerol. El glicerol proporciona un índice de refracción necesario para microscopía. Los embriones se pueden almacenar en esta solución durante varias semanas. Sellar las placas con parafina de plástico para evitar la evaporación. </li></ol></li><li> Preparación de los portaobjetos y la captura de imágenes <ol><li> Preparar un portaobjetos mediante la disposición de tres pequeñas tiras de cinta adhesiva de doble cara en un triángulo con pequeños espacios entre las tiras en un portaobjetos. </li><li> La transferencia de los embriones en el lavado MOPS 70% y solución de glicerol al 30% hasta el centro del triángulo y cubrir con un cubreobjetos. </li><li> Sellar el cubreobjetos mediante la aplicación de una capa de esmalte de uñas alrededor de los bordes del cubreobjetos. </li><li> Capturar imágenes utilizando un microscopio de luz compuesto con un objetivo de 20X y una cámara adjunta. </li></ol></li></ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=LvNumb+works+synergistically+with+Notch+signaling+to+specify+non-skeletal+mesoderm+cells+in+the+sea+urchin+embryo.">LvNumb works synergistically with Notch signaling to specify non-skeletal mesoderm cells in the sea urchin embryo.</a> Development. 135, 2445-2454 (2008).</li><li>Range, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cis-regulatory+analysis+of+nodal+and+maternal+control+of+dorsal-ventral+axis+formation+by+Univin,+a+TGF-beta+related+to+Vg1.">Cis-regulatory analysis of nodal and maternal control of dorsal-ventral axis formation by Univin, a TGF-beta related to Vg1.</a> Development. 134, 3649-3664 (2007).</li><li>Warner, J. F., Miranda, E. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+hedgehog+signaling+to+the+establishment+of+left-right+asymmetry+in+the+sea+urchin.">Contribution of hedgehog signaling to the establishment of left-right asymmetry in the sea urchin.</a> Dev Biol. 411, 314-324 (2016).</li><li>Rottinger, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=FGF+signals+guide+migration+of+mesenchymal+cells,+control+skeletal+morphogenesis+[corrected]+and+regulate+gastrulation+during+sea+urchin+development.">FGF signals guide migration of mesenchymal cells, control skeletal morphogenesis [corrected] and regulate gastrulation during sea urchin development.</a> Development. 135, 353-365 (2008).</li><li>Warner, J. F., McCarthy, A. M., Morris, R. L., McClay, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+requires+motile+cilia+in+the+sea+urchin.">Hedgehog signaling requires motile cilia in the sea urchin.</a> Mol Biol Evol. 31, 18-22 (2014).</li><li>Technau, U., Steele, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolutionary+crossroads+in+developmental+biology.">Evolutionary crossroads in developmental biology.</a> Cnidaria. Development. 138, 1447-1458 (2011).</li><li>Yaguchi, J., Takeda, N., Inaba, K., Yaguchi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cooperative+Wnt-Nodal+Signals+Regulate+the+Patterning+of+Anterior+Neuroectoderm.">Cooperative Wnt-Nodal Signals Regulate the Patterning of Anterior Neuroectoderm.</a> PLoS Genet. 12, e1006001 (2016).</li><li>Duboc, V., Rottinger, E., Besnardeau, L., Lepage, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nodal+and+BMP2/4+signaling+organizes+the+oral-aboral+axis+of+the+sea+urchin+embryo.">Nodal and BMP2/4 signaling organizes the oral-aboral axis of the sea urchin embryo.</a> Dev Cell. 6, 397-410 (2004).</li><li>Bradham, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chordin+is+required+for+neural+but+not+axial+development+in+sea+urchin+embryos.">Chordin is required for neural but not axial development in sea urchin embryos.</a> Dev Biol. 328, 221-233 (2009).</li><li>Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+regulatory+networks+for+ectoderm+specification+in+sea+urchin+embryos.">Gene regulatory networks for ectoderm specification in sea urchin embryos.</a> Biochimica et biophysica acta. 1789, 261-267 (2009).</li><li>Lin, C. Y., Su, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genome+editing+in+sea+urchin+embryos+by+using+a+CRISPR/Cas9+system.">Genome editing in sea urchin embryos by using a CRISPR/Cas9 system.</a> Dev Biol. 409, 420-428 (2016).</li></ol>

The authors have nothing to disclose.

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 ...

The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the signaling pathways and the gene regulatory networks that they activate in an in vivo developmental context.Demonstrating this procedure will be Marina Martinez-Bartolom

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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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7.8K vistas.  Mississippi State University. The overall goal of these experiments is to illustrate the advantages that simple organisms offer in the study of complex interactions among gene regulatory networks that direct developmental processes.This methodology can help answer key questions in evolutionary and developmental biology, such as how signaling pathways govern cell fate specification along the major embryonic axes in metazoans.One of the main advantages of this methodology is it can be used to efficiently characterize the si...

Video: The Power of Simplicity: Sea Urchin Embryos as in Vivo Developmental Models for Studying Complex Cell-to-cell Signaling Network Interactions

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by pre-globular, globular, heart, torpedo and cotyledon stages. This progressive development is under the tight regulation of a complex molecular network. Harvesting sufficient early embryos at a similar stage of development is essential for investigating the cellular and molecular regulation of early embryogenesis. This is not straightforward since early embryogenesis undergoes rapid morphogenesis in a short while e.g. 8 days for Medicago truncatula to reach the early cotyledon stage. Here, we address the issue by two approaches. The first one establishes a linkage between embryo development and pod morphology in helping indicate the stage of the zygotic embryo. This is particularly based on the number of pod spirals and development of the spines. An alternative way to complement the in vivo studies is via culturing leaf explants to produce somatic embryos. The medium includes an unusual hormone combination - an auxin (1-naphthaleneacetic acid), a cytokinin (6-benzylaminopurine), abscisic acid and gibberellic acid. The different stages can be discerned growing out of the callus without dissection.

Protocols for Obtaining Zygotic and Somatic Embryos for Studying the Regulation of Early Embryo Development in the Model Legume Medicago truncatula

The goal is to illustrate that the model legume Medicago truncatula can be readily utilized to investigate the regulation of early plant embryogenesis to complement the non-legume Arabidopsis model. Pod morphology is linked to zygotic embryogenesis stages and a protocol to collect embryos using tissue culture is also provided.

Protocolos para la obtención de embriones somáticos y cigóticos para el estudio de la regulación del desarrollo embrionario temprano en el Modelo de Leguminosas<em&gt; Medicago truncatula</em

Early embryogenesis starting from a single cell zygote goes through rapid cell division and morphogenesis, and is morphologically characterized by ...

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Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T cell replacement in lymphopenic patients. We studied thymic settling progenitors from murine embryonic day 13 and 18 thymi by two complementary in vitro and in vivo techniques, both based on the &#8220;hanging drop&#8221; method. This method allowed colonizing irradiated fetal thymic lobes with E13 and/or E18 thymic progenitors distinguished by CD45 allotypic markers and thus following their progeny. Colonization with mixed populations allows analyzing cell autonomous differences in biologic properties of the progenitors while colonization with either population removes possible competitive selective pressures. The colonized thymic lobes can also be grafted in immunodeficient male recipient mice allowing the analysis of the mature T cell progeny in vivo, such as population dynamics of the peripheral immune system and colonization of different tissues and organs. Fetal thymic organ cultures revealed that E13 progenitors developed rapidly into all mature CD3+ cells and gave rise to the canonical &#947;&#948; T cell subset, known as dendritic epithelial T cells. In comparison, E18 progenitors have a delayed differentiation and were unable to generate dendritic epithelial T cells. The monitoring of peripheral blood of thymus-grafted CD3-/- mice further showed that E18 thymic settling progenitors generate, with time, larger numbers of mature T cells than their E13 counterparts, a feature that could not be appreciated in the short term fetal thymic organ cultures.

Characterization of Thymic Settling Progenitors in the Mouse Embryo Using In Vivo and In Vitro Assays

This article describes in vivo and in vitro methodology to characterize the thymic settling progenitors by the analysis of the kinetics of generation, phenotype and numbers of their T cell progeny.

Caracterización de tímico Instalarse Progenitores en el embrión de ratón Uso<em&gt; En Vivo</em&gt; Y<em&gt; In Vitro</em&gt; Ensayos

Characterizing thymic settling progenitors is important to understand the pre-thymic stages of T cell development, essential to devise strategies for T ...

Studying Wnt Signaling During Patterning of Conducting Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis controlled by Wnt signaling is required to understand how tissues are patterned during normal development. This knowledge is also critical to determine the etiology of birth defects such as lung hypoplasia and tracheobronchomalacia. Analysis of earliest stages of development of respiratory tract imposes challenges, as the limited amount of tissue prevents the performance of standard protocols better suited for postnatal studies. In this paper, we discuss methodologies to study cell differentiation and proliferation in the respiratory tract. We describe techniques such as whole mount staining, processing of the tissue for confocal microscopy and immunofluorescence in paraffin sections applied to developing tracheal lung. We also discuss methodologies for the study of tracheal mesenchyme differentiation, in particular cartilage formation. Approaches and techniques discussed in the current paper circumvent the limitation of material while working with embryonic tissue, allowing for a better understanding of the patterning process of developing conducting airways.

The use of reporter mice coupled to whole mount and section staining, microscopy and in vivo assays facilitates the analysis of mechanisms underlying the normal patterning of the respiratory tract. Here we describe how these techniques contributed to the analysis of Wnt signaling during tracheal development.

El estudio de la señalización Wnt Durante Patrones de llevar a cabo Airways

Wnt signaling pathways play critical roles during development of the respiratory tract. Defining precise mechanisms of differentiation and morphogenesis ...

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched modern biological inquiry through the work of Trembley more than 250 years ago. Presently the study of regeneration has seen a resurgence using both &#34;classic&#34; regenerative organisms, such as the Hydra, planaria and Urodeles, as well as a widening spectrum of species spanning the range of metazoa, from sponges through mammals. Besides its intrinsic interest as a biological phenomenon, understanding how regeneration works in a variety of species will inform us about whether regenerative processes share common features and/or species or context-specific cellular and molecular mechanisms. The starlet sea anemone, Nematostella vectensis, is an emerging model organism for regeneration. Like Hydra, Nematostella is a member of the ancient phylum, cnidaria, but within the class anthozoa, a sister clade to the hydrozoa that is evolutionarily more basal. Thus aspects of regeneration in Nematostella will be interesting to compare and contrast with those of Hydra and other cnidarians. In this article, we present a method to bisect, observe and classify regeneration of the aboral end of the Nematostella adult, which is called the physa. The physa naturally undergoes fission as a means of asexual reproduction, and either natural fission or manual amputation of the physa triggers re-growth and reformation of complex morphologies. Here we have codified these simple morphological changes in a Nematostella Regeneration Staging System (the NRSS). We use the NRSS to test the effects of chloroquine, an inhibitor of lysosomal function that blocks autophagy. The results show that the regeneration of polyp structures, particularly the mesenteries, is abnormal when autophagy is inhibited.

Inducing Complete Polyp Regeneration from the Aboral Physa of the Starlet Sea Anemone Nematostella vectensis

Here we demonstrate how to induce and monitor regeneration in the Starlet Sea Anemone Nematostella vectensis, a model cnidarian anthozoan. We demonstrate how to amputate and categorize regeneration using a morphological staging system, and we use this system to reveal a requirement for autophagy in regenerating polyp structures.

Inducir completa Pólipo regeneración de la aboral Physa de la anémona de mar Starlet<em&gt; Vectensis Nematostella</em

Cnidarians, and specifically Hydra, were the first animals shown to regenerate damaged or severed structures, and indeed such studies arguably launched ...

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Bone-forming osteoblasts interact with bone-resorbing osteoclasts to coordinate the turnover of bone matrix and to control skeletal homeostasis. Medaka and zebrafish larvae are widely used to analyze the behavior of bone cells during bone formation, degeneration, and repair. Their optical clarity allows the visualization of fluorescently labeled bone cells and fluorescent dyes bound to the mineralized skeletal matrix. Our lab has generated transgenic medaka fish that express the osteoclast-inducing factor Receptor Activator of Nuclear-factor &#954;B Ligand (RANKL) under the control of a heat&#160;shock-inducible promoter. Ectopic expression of RANKL results in the excess formation of activated osteoclasts, which can be visualized in reporter lines with nlGFP expression under the control of the cathepsin K (ctsk) promoter. RANKL induction and ectopic osteoclast formation leads to severe osteoporosis-like phenotypes. Compound transgenic medaka lines that express ctsk:nlGFP in osteoclasts, as well as mCherry under the control of the osterix (osx) promoter in premature osteoblasts, can be used to study the interaction of both cell types. This facilitates the in vivo observation of cellular behavior under conditions of bone degeneration and repair. Here, we describe the use of this system to test a drug commonly used in human osteoporosis therapy and describe a protocol for live imaging. The medaka model complements studies in cell culture and mice, and offers a novel system for the in vivo analysis of drug action in the skeletal system.

Drug Treatment and In Vivo Imaging of Osteoblast-Osteoclast Interactions in a Medaka Fish Osteoporosis Model

Small laboratory fish have become popular models for bone research on the mechanisms underlying human bone disorders and for the screening of bone-modulating drugs. In this report, we describe a protocol to assess the effect of alendronate on bone cells in medaka larvae with osteoporotic lesions.

El tratamiento de drogas y<em&gt; En Vivo</em&gt; Imagen de osteoblasto-osteoclastos Interacciones en un modelo Medaka pescado osteoporosis

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

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced in vivo throughout the rapid developmental period. Multiple processes can be studied including cell proliferation, gene expression, cell migration and morphogenesis. Importantly, the generation of chimeras through transplantations can be easily performed, allowing mosaic labeling and tracking of individual cells under the influence of the host environment. For example, by combining functional gene manipulations of the host embryo (e.g., through morpholino microinjection) and live imaging, the effects of extrinsic, cell nonautonomous signals (provided by the genetically modified environment) on individual transplanted donor cells can be assessed. Here we demonstrate how this approach is used to compare the onset of fluorescent transgene expression as a proxy for the timing of cell fate determination in different genetic host environments.
In this article, we provide the protocol for microinjecting zebrafish embryos to mark donor cells and to cause gene knockdown in host embryos, a description of the transplantation technique used to generate chimeric embryos, and the protocol for preparing and running in vivo time-lapse confocal imaging of multiple embryos. In particular, performing multiposition imaging is crucial when comparing timing of events such as the onset of gene expression. This requires data collection from multiple control and experimental embryos processed simultaneously. Such an approach can easily be extended for studies of extrinsic influences in any organ or tissue of choice accessible to live imaging, provided that transplantations can be targeted easily according to established embryonic fate maps.

In Vivo Imaging of Transgenic Gene Expression in Individual Retinal Progenitors in Chimeric Zebrafish Embryos to Study Cell Nonautonomous Influences

Live tracking of individual WT retinal progenitors in distinct genetic backgrounds allows for the assessment of the contribution of cell non-autonomous signaling during neurogenesis. Here, a combination of gene knockdown, chimera generation via embryo transplantation and in vivo time-lapse confocal imaging was utilized for this purpose.

En vivo de imágenes de transgénicos de la Expresión Génica en el Estimado de retina progenitores en embriones de pez cebra quiméricos para estudiar celulares Influencias no autónomos

The genetic and technical strengths have made the zebrafish vertebrate a key model organism in which the consequences of gene manipulations can be traced ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Imágenes en vivo de la fusión secundaria del paladar del ratón

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualización de la placa notocordal en embriones de ratón Gastrulating con microscopía electrónica e inmunofluorescencia de Monte todo y nodo

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

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

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

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

Evaluación del daño oxidativo en las células madre primarias del ratón/células madre en respuesta al daño ultravioleta-C (UV-C)

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

A Layered Mounting Method for Extended Time-Lapse Confocal Microscopy of Whole Zebrafish Embryos

Dynamics of development can be followed by confocal time-lapse microscopy of live transgenic zebrafish embryos expressing fluorescence in specific tissues or cells. A difficulty with imaging whole embryo development is that zebrafish embryos grow substantially in length. When mounted as regularly done in 0.3-1% low melt agarose, the agarose imposes growth restriction, leading to distortions in the soft embryo body. Yet, to perform confocal time-lapse microscopy, the embryo must be immobilized. This article describes a layered mounting method for zebrafish embryos that restrict the motility of the embryos while allowing for the unrestricted growth. The mounting is performed in layers of agarose at different concentrations. To demonstrate the usability of this method, whole embryo vascular, neuronal and muscle development was imaged in transgenic fish for 55 consecutive hours. This mounting method can be used for easy, low-cost imaging of whole zebrafish embryos using inverted microscopes without requirements of molds or special equipment.

This article describes a method to mount fragile zebrafish embryos for extended time-lapse confocal microscopy. This low-cost method is easy to perform using regular glass-bottom microscopy dishes for imaging on any inverted microscope. The mounting is performed in layers of agarose at different concentrations.

Un método de montaje en capas para la microscopía confocal de lapso de tiempo extendido de embriones enteros de pez cebra

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