We would like to thank C. Burns, C. McRae for outlining the basic principle of this purification protocol, and F. Priller for initial implementation of this method in our lab. S.A.-S. is supported by a Heisenberg professorship of the Deutsche Forschungsgemeinschaft (DFG). This work was supported by DFG grant SE2016/7-1.

Este protocolo sigue las pautas de cuidado de animales de la ley alemana y el estado de Berlín; manejo del pez cebra fue supervisado por la autoridad local para la protección de animales (LaGeSo, Berlin-Brandenburg). 1. La obtención de embriones de pez cebra para el Corazón de Extracción <ol><li> Cross pez cebra reportero cardiaca tales como la Tg (myl7: EGFP) twu34 línea transgénica 9 con el fin de obtener embriones con la expresión específica de GFP-corazón. </li><li> Mantener los embriones en el agua de huevo a 28,5 ° C hasta la etapa embrionaria deseada 10,11. Asegúrese de que la población de embriones recogidos es homogénea tanto genética como por la etapa de desarrollo para limitar las variaciones en la expresión génica. </li><li> Dechorionate los embriones manualmente. </li></ol> 2. Disección de pez cebra embrionarias Corazones NOTA: Mantenga todas las soluciones en hielo. Si es posible, hacer el trabajo en equipo: una persona disecciona los corazones de la embryos mientras que otra persona ordena los corazones bajo el microscopio estereoscópico de fluorescencia. Esto permite que el procesamiento de varios cientos de embriones en unas pocas horas. <ol><li> Transferir unos 100 embriones en un tubo de 1,5 ml centrifugación. Anestesiar a los embriones con tricaína (0,16 mg / ml en medio de E3). Proceda una vez que los embriones son claramente anestesiados (que no nadan y los sedimentos en el fondo del tubo, pero sus corazones siguen latiendo). </li><li> Eliminar la solución / E3 tricaína y lavar los embriones una vez con 1 ml L-15/10% de medio FBS y mantener en hielo. NOTA: El medio FBS L-15/10% es importante para mantener los órganos y células vivas durante todo el procedimiento de disección hasta que los corazones se han transferido en RNAlater o Trizol. </li><li> Añadir 1 ml de L-15/10% de medio FBS a los embriones y la pipeta hacia arriba y abajo 5-8 veces con una Ronda Gel Cargando Consejo hasta que la yema esté completamente interrumpido. Pipeta los embriones gota a gota sobre la superficie de la solución, por lo que la caída va a estallar ylos embriones se rompen suavemente. NOTA: Cómo vigorosamente y con qué frecuencia los embriones tienen que ser pipeta hacia arriba y abajo depende de la etapa de desarrollo de los embriones, se necesita decir más pipeteo en fases avanzadas (por ejemplo, a 56 HPF). </li><li> Para el primer lote de embriones disecados, evaluar la integridad de los embriones y la proporción de corazones disecados bajo un microscopio estereoscópico, ya que algunos corazones pueden permanecer unidos a los embriones si pipeteada demasiado suavemente. Ajuste el modo de pipeteo para las próximas rondas de disección en consecuencia. NOTA: Utilice puntas de pipeta de retención baja y tubos de microcentrífuga para minimizar los corazones que se pegan al plástico. </li><li> Aplicar la muestra sobre un filtro de 100 micras colocado en un tubo de centrifugación de 50 ml; enjuagar el tubo de 1,5 ml con 1 ml de medio L-15/10% de FBS y luego aplicar esto a el filtro con el fin de minimizar la pérdida de la muestra (algunos corazones pueden estar pegadas a las paredes del tubo). Lavar el filtro dos veces con 1 ml L-15 / 10% de FBS. En este paso los corazones pasan a través del filtro y se recogen en el tubo de 50 ml. </li><li> Aplicar el flujo a través sobre un filtro de 30 micras colocado sobre un tubo de centrifugación de 15 ml y enjuagar el filtro una vez con 1 ml L-15/10% de medio FBS. En esta segunda etapa de filtración, los corazones se retienen en el filtro y los residuos más pequeño se lava. </li><li> Girar el filtro al revés y vaciar los corazones en el filtro en un agarosa recubiertas de placa de Petri 1% mediante la aplicación de tres lavados consecutivos con 1 ml de L-15/10% de FBS. </li><li> Separar manualmente corazones GFP-positivas a partir de los restos de embriones no fluorescente (por ejemplo, lentes de ojo) con un par de pinzas bajo un microscopio estereoscópico de fluorescencia y se concentran en el centro del plato. Colecciónalos según la etapa 3. NOTA: Si los embriones son mayores de 24 HPF, los corazones deben latir, lo que indica que los tejidos están todavía fisiológicamente normal. </li></ol> 3. Aislamiento de ARNm de disecciónted Corazones <ol><li> Recoge los corazones en el menor volumen posible (por ejemplo, 10 l) y pipeta en un tubo de 1,5 ml contiene 0,75 ml RNAlater (en hielo). Verifique que no haya corazones permanecen en la punta de la pipeta viéndolo bajo un microscopio estereoscópico de fluorescencia. </li><li> Alternativamente, si una campana química está disponible en la vecindad inmediata del microscopio de fluorescencia, la transferencia de los corazones recogidos en Trizol (PRECAUCIÓN) bajo el capó química. Esto evita la posible pérdida de corazones de fricción en la punta de pipeta y también durante la centrifugación de los corazones (pasos 3.4, 3.5 y 3.7). Reúnase el corazón de varios ciclos de aislamiento en el mismo tubo con Trizol y vaya directamente al paso 3.8; el volumen final no debe superar el 10% del volumen original Trizol. NOTA: Lea la Hoja de Seguridad Trizol (MSDS) antes de usar. Maneje reactivo Trizol bajo una campana y el desgaste recomendado Equipo de Protección Personal. Evite el contacto con la piel, ojos y clothing. Retirar todas las fuentes de ignición. </li><li> Agrupar los corazones de varias rondas de aislamiento en el mismo tubo con RNAlater (en hielo), como la eficiencia de aislamiento de ARN mejora con la cantidad de tejido recogido. Si el volumen total de la muestra supera el 10% del volumen RNAlater original, utilizar un tubo adicional con 0,75 ml RNAlater para el almacenamiento (en hielo). </li><li> Centrifugar las muestras a 15.700 xg durante 20 min a 4 ° C para sedimentar los corazones. </li><li> Bajo un microscopio estereoscópico de fluorescencia, recoger cuidadosamente tanto sobrenadante como sea posible en un 1% de agarosa-placa de Petri recubierta que contiene 3 ml L-15/10% de medio FBS, pero no molestar los corazones sedimentadas en el fondo del tubo de centrifugación. Dado que hasta 15% de los corazones puede permanecer en el sobrenadante debido a la alta viscosidad de RNAlater, recuperarlos como se describe en el paso 3.7. </li><li> Bajo una campana química, añadir 0,5 ml de Trizol al tubo que contiene los corazones sedimentadas y mantener en hielo. </li><li> Bajo el microsco fluorescentepe, transferir los corazones (del paso 3.5) del plato de agarosa recubiertas de 1% FBS que contiene la solución RNAlater / L-15/10% en otro plato de agarosa recubiertas de 1% con 3 ml L-15/10% FBS para diluir el RNAlater. Recoge los corazones en el centro de la placa de Petri y transferirlos en un pequeño volumen en el tubo de corazones sedimentadas en Trizol bajo una campana química. </li><li> Vortex el tubo de 1,5 ml que contiene los corazones en Trizol para interrumpir los corazones e incubar durante 5 min a temperatura ambiente. </li><li> Bajo una campana química, añadir 100 l de cloroformo (ATENCIÓN), mezclar bien y pasar la solución / cloroformo Trizol en un 1,5 ml tubo de pre-hilado PLG (centrífuga 30 segundos a velocidad máxima antes de su uso). Incubar durante 3 min a temperatura ambiente. NOTA: Lea las MSDS cloroformo antes de su uso. Maneje reactivo cloroformo bajo una campana y el desgaste recomendado Equipo de Protección Personal. Evite el contacto con la piel, ojos y ropa. Mantener alejado de materiales incompatibles tales como los metales, los álcalis. </li><li> Centrifuge la muestra a 15.700 xg durante 15 min a 4 ° C y transferir la fase acuosa a un nuevo tubo de 1,5 ml. </li><li> Añadir 5-10 g de glucógeno y 250 l preenfriados (a -20 ° C) isopropanol; mezclar bien e incubar durante la noche a -20 ° C. </li><li> Centrifugar la muestra a 15.700 xg durante 30 min a 4 ° C y descartar el sobrenadante. </li><li> Lavar el sedimento con 1 ml de etanol 75%, se centrifuga a 15.700 xg durante 15 min a 4 ° C y descartar el sobrenadante. </li><li> Se deja evaporar el etanol mediante secado de la pastilla a temperatura ambiente hasta que se convierte en blanco (por ejemplo, durante 10-15 min). </li><li> Añadir 20 l RNasa libre de ddH 2 O al sedimento y se incuba durante 10-15 min a 55 ° C para disolver el ARN; a continuación, mantener en hielo. </li><li> Evaluar la concentración de ARN y la pureza por medición de la absorbancia. Evaluar la integridad del ARN mediante la ejecución de la muestra en un gel de agarosa al 1%. </li></ol>

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N., 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=Mutations+affecting+the+cardiovascular+system+and+other+internal+organs+in+zebrafish.">Mutations affecting the cardiovascular system and other internal organs in zebrafish.</a> Development. 123, 293-302 (1996).</li><li>Bakkers, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish+as+a+model+to+study+cardiac+development+and+human+cardiac+disease.">Zebrafish as a model to study cardiac development and human cardiac disease.</a> Cardiovasc Res. 91, 279-288 (2011).</li><li>Jowett, T., Lettice, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole-mount+in+situ+hybridizations+on+zebrafish+embryos+using+a+mixture+of+digoxigenin-+and+fluorescein-labelled+probes.">Whole-mount in situ hybridizations on zebrafish embryos using a mixture of digoxigenin- and fluorescein-labelled probes.</a> Trends Genet. 10, 73-74 (1994).</li><li>Gibson, U. 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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The zebrafish book. A guide for the laboratory use of zebrafish (Danio rerio). , (2000).</li><li>Veerkamp, J., 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=Unilateral+dampening+of+Bmp+activity+by+nodal+generates+cardiac+left-right+asymmetry).">Unilateral dampening of Bmp activity by nodal generates cardiac left-right asymmetry).</a> Dev Cell. 24, 660-667 (2013).</li><li>Jin, S. W., Beis, D., Mitchell, T., Chen, J. N., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cellular+and+molecular+analyses+of+vascular+tube+and+lumen+formation+in+zebrafish.">Cellular and molecular analyses of vascular tube and lumen formation in zebrafish.</a> Development. 132, 5199-5209 (2005).</li><li>Brownlie, 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=Characterization+of+embryonic+globin+genes+of+the+zebrafish.">Characterization of embryonic globin genes of the zebrafish.</a> Dev Biol. 255, 48-61 (2003).</li><li>Marvin, M., 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=Developmental+expression+patterns+of+the+zebrafish+small+heat+shock+proteins.">Developmental expression patterns of the zebrafish small heat shock proteins.</a> Dev Dyn. 237, 454-463 (2008).</li><li>Lam, C. S., Marz, M., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=gfap+and+nestin+reporter+lines+reveal+characteristics+of+neural+progenitors+in+the+adult+zebrafish+brain.">gfap and nestin reporter lines reveal characteristics of neural progenitors in the adult zebrafish brain.</a> Dev Dyn. 238, 475-486 (2009).</li><li>Wang, Y., 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=Moesin1+and+Ve-cadherin+are+required+in+endothelial+cells+during+in+vivo+tubulogenesis.">Moesin1 and Ve-cadherin are required in endothelial cells during in vivo tubulogenesis.</a> Development. 137, 3119-3128 (2010).</li><li>Heiman, M., 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=A+translational+profiling+approach+for+the+molecular+characterization+of+CNS+cell+types.">A translational profiling approach for the molecular characterization of CNS cell types.</a> Cell. 135, 738-748 (2008).</li><li>Tryon, R. 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The authors have nothing to disclose.

Este protocolo permite el rápido enriquecimiento de tejido del corazón embrionario de pez cebra para la expresión génica análisis. La cantidad y la calidad de la muestra específica cardiaca ARN depende en gran medida de unos pocos pasos cruciales: en primer lugar, la cantidad de la muestra se mejora en gran medida si la pérdida de corazones se impide a cada paso del protocolo, ya que la purificación del ARN sólo funcionará con suficiente material de partida. En segundo lugar, la pureza de la muestra, que depen...

El pez cebra (Danio rerio) es ampliamente utilizado en biología del desarrollo para estudiar la organogénesis in vivo debido a su desarrollo embrionario rápido, transparente y extrauterino, combinado con el pequeño tamaño y la disponibilidad de líneas reportero transgénicas con expresión específica de tejido de proteínas fluorescentes. Este pequeño vertebrado es especialmente adecuado para estudiar el desarrollo del corazón debido a la oxigenación de los primeros embriones de pez cebra no se basa en los latidos del corazón y el flujo sanguíneo; estas características han permitido la caracterización de un gran número de mutantes cardiovasculares 1,2 y el pez cebra es ahora un organismo modelo ampliamente reconocido para estudiar enfermedades del corazón 3. Para el estudio de la expresión génica durante el desarrollo embrionario, las transcripciones son comúnmente analizados por todo el montaje hibridación in situ (DESEOS) 4, RT-qPCR 5, 6, microarrays o secuenciación de próxima generación (RNA-Seq) 7. MientrasDESEO permite un análisis espacio-temporal de la expresión génica dentro de todo el embrión, los niveles de transcripción suelen ser evaluada por RT-qPCR, microarrays o enfoques de RNA-Seq. Sin embargo, estos métodos requieren el enriquecimiento de tejido para perfiles de expresión génica específica. Puesto que el corazón embrionario de pez cebra representa una pequeña fracción de todo el embrión, los estudios transcriptoma durante el desarrollo cardiaco requieren un protocolo para la disección y el enriquecimiento de los corazones. Además, para obtener datos fisiológicamente relevantes, es importante mantener el tejido cardíaco totalmente funcional hasta la extracción de ARN. Aquí se describe un protocolo para aislar rápidamente fisiológicamente normal y el corazón palpitante de cientos de embriones de pez cebra para obtener de manera eficiente las muestras de ARN de alta calidad para análisis posteriores. El presente método se basa en el protocolo reportado por Burns y MacRae, 2006 8. Para el enriquecimiento del tejido cardiaco, ambos métodos utilizan una línea reportero infarto transgénicoy tomar ventaja de las propiedades de adherencia diferencial de los corazones de embriones de pez cebra en comparación con otros tejidos. En pocas palabras, con la pipeta muchos embriones arriba y abajo a través de una punta de pipeta estrecho, corazones son liberados al mismo tiempo de los órganos embrionarios y posteriormente separados de escombros embrionaria en dos etapas de filtración rápida; entonces los corazones etiqueta fluorescente se ordenan de forma manual desde la basura y los recogen para su posterior procesamiento.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr >
			<td >Equipment for raising fish and collecting eggs&#160;</td>
			<td></td>
			<td></td>
			<td>see the Zebrafish Book11 for details</td>
		</tr>
		<tr >
			<td >Fluorescence stereomicroscope</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Refrigerated Microcentrifuge</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >UV-Spectrophotometer</td>
			<td></td>
			<td></td>
			<td>eg. Thermo Scientific Nanodrop 2000</td>
		</tr>
		<tr >
			<td >Nucleic acid electrophoresis chamber</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Petri dishes 4cm &#216;, coated with 1% agarose in E3 medium</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Micropipettes and tips (P20, P100, P1000)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1,5ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >15ml and 50ml centrifugation tubes</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Pair of Dumont #5 forceps</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >ExactaCruz&#8482; Round Gel Loading Tips in Sterile Rack, 1-200&#956;l&#160;</td>
			<td>Santa Cruz</td>
			<td>sc-201732</td>
			<td></td>
		</tr>
		<tr >
			<td >100 &#956;m filter (BD Falcon 100 mm Cell Strainer)</td>
			<td>BD Biosciences</td>
			<td>352360</td>
			<td></td>
		</tr>
		<tr >
			<td >30 &#956;m filter (Pre-Separation Filters-30 &#181;m)</td>
			<td>Miltenyi Biotec</td>
			<td>130-041-407</td>
			<td></td>
		</tr>
		<tr >
			<td >Phase lock gel, heavy, 1,5ml tubes&#160;</td>
			<td>Prime</td>
			<td>2302810</td>
			<td></td>
		</tr>
		<tr >
			<td >Egg water medium</td>
			<td></td>
			<td></td>
			<td>60&#956;g/ml Instant Ocean Sea Salts in ddH2O, 0.00001% (w/v) Methylene Blue</td>
		</tr>
		<tr >
			<td >E3 medium&#160;</td>
			<td></td>
			<td></td>
			<td>5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4</td>
		</tr>
		<tr >
			<td >Tricaine (3-amino benzoic acidethylester)</td>
			<td>Sigma-Aldrich</td>
			<td>A-5040</td>
			<td>4mg/ml Tricaine stock solution, pH 7</td>
		</tr>
		<tr >
			<td >1% agarose (in E3 medium)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >1% agarose gel (in TBE buffer)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Leibovitz&#180;s L-15 medium&#160;</td>
			<td>Gibco</td>
			<td>21083-027</td>
			<td></td>
		</tr>
		<tr >
			<td >FBS (Fetal Bovine Serum)</td>
			<td>Sigma</td>
			<td>F4135</td>
			<td></td>
		</tr>
		<tr >
			<td >RNAlater</td>
			<td>Ambion</td>
			<td>AM7020</td>
			<td></td>
		</tr>
		<tr >
			<td >Trizol</td>
			<td>Ambion</td>
			<td>1559606</td>
			<td></td>
		</tr>
		<tr >
			<td >Glycogen&#160;</td>
			<td>Invitrogen</td>
			<td>10814-010</td>
			<td>20 &#181;g/&#181;L in RNase-free water</td>
		</tr>
		<tr >
			<td >chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >isopropanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >75% ethanol (in DEPC-ddH2O)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nuclease-free water or sterilized DEPC treated ddH2O</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >Nucleic acid loading buffer</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr >
			<td >TBE (Tris/Borate/EDTA) buffer for electrophoresis</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

large-scale zebrafish embryonic heart dissection for transcriptional analysis

El corazón embrionario de pez cebra se compone de sólo unos pocos cientos de células, lo que representa sólo una pequeña fracción de todo el embrión. Por lo tanto, para evitar que el transcriptoma cardiaca de ser enmascarada por el transcriptoma embrionario global, es necesario reunir un número suficiente de corazones para nuevos análisis. Además, como el desarrollo cardiaco pez cebra procede rápidamente, colección del corazón y los métodos de extracción de ARN tienen que ser rápida a fin de asegurar la homogeneidad de las muestras. A continuación, presentamos un protocolo rápido disección manual para recopilar funcionales corazones / golpeando a partir de embriones de pez cebra. Este es un requisito esencial para la posterior extracción de RNA específica del corazón para determinar los niveles de expresión de genes cardiacos específicos por análisis del transcriptoma, tales como la reacción en cadena de la polimerasa cuantitativa en tiempo real (RT-qPCR). El método se basa en propiedades adhesivas diferenciales del corazón embrionario de pez cebra en comparación con otros tejidos; esto permite la rápida physeparación de SICAL cardiaca a partir de tejido extracardíaca por una combinación de fuerza de cizallamiento de fluido interrupción, filtración por etapas y la recogida manual de los corazones transgénicos marcados con fluorescencia.

A continuación, describimos un representante experimento disección cardíaco usando el Tg pez cebra (myl7: GFP) twu34 línea transgénica 9, que expresa la proteína verde fluorescente (GFP) exclusivamente dentro del miocardio (Fig. 1). Se recogieron tanto los corazones disecados y los embriones a partir de los cuales se derivaron para evaluar la pureza de la muestra del corazón. En pocas palabras, homocigotos Tg (myl7: GFP) pez cebra twu34 9 se outcrossed c...

Watch this Scientific Journal Video about Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis at JoVE.com

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Para analizar los perfiles de expresión de genes cardiacos durante el desarrollo del corazón del pez cebra, el ARN total tiene que ser extraído de corazones aislados. A continuación, presentamos un protocolo de recogida funcionales corazones / batiendo por disección manual de rápida a partir de embriones de pez cebra para obtener ARNm específico del corazón.

A gran escala de pez cebra embrionario del corazón Disección de Análisis transcripcional

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

large-scale-zebrafish-embryonic-heart-dissection-for-transcriptional

Research

JoVE Journal

Developmental Biology

14.3K vistas.  Max Delbrück Center for Molecular Medicine.  Para analizar los perfiles de expresión de genes cardiacos durante el desarrollo del corazón del pez cebra, el ARN total tiene que ser extraído de corazones aislados. A continuación, presentamos un protocolo de recogida funcionales corazones / batiendo por disección manual de rápida a partir de embriones de pez cebra para obtener ARNm específico del corazón. 

Video: Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

Analysis of Cardiomyocyte Development using Immunofluorescence in Embryonic Mouse Heart

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated discs, and costameres requires the coordinated assembly of multiple components in time and space. Disruption in assembly of these components leads to developmental heart defects. Immunofluorescent staining techniques are used commonly in cultured cardiomyocytes to probe myofibril maturation, but this ex vivo approach is limited by the extent to which myocytes will fully differentiate in culture, lack of normal in vivo mechanical inputs, and absence of endocardial cues. Application of immunofluorescence techniques to the study of developing mouse heart is desirable but more technically challenging, and methods often lack sufficient sensitivity and resolution to visualize sarcomeres in the early stages of heart development. Here, we describe a robust and reproducible method to co-immunostain multiple proteins or to co-visualize a fluorescent protein with immunofluorescent staining in the embryonic mouse heart and use this method to analyze developing myofibrils, intercalated discs, and costameres. This method can be further applied to assess cardiomyocyte structural changes caused by mutations that lead to developmental heart defects.

Mutations that lead to congenital heart defects benefit from in vivo investigation of cardiac structure during development, but high-resolution structural studies in the mouse embryonic heart are technically challenging. Here we present a robust immunofluorescence and image analysis method to assess cardiomyocyte-specific structures in the developing mouse heart.

Análisis para el Desarrollo de los cardiomiocitos mediante inmunofluorescencia en el corazón de embriones de ratón

During heart development, the generation of myocardial-specific structural and functional units including sarcomeres, contractile myofibrils, intercalated ...

Investigación

Biología del Desarrollo

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain&#39;s macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy Nanotomy of Healthy and Injured Zebrafish Brain

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution EM. Here we describe a universal method for nanotomy applied to investigate the zebrafish larval brain in health and upon non-invasive brain injury.

A gran escala de barrido Microscopía Electrónica de Transmisión (Nanotomy) de pez cebra cerebrales saludables y lesionados

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously ...

Large-Scale Production of Cardiomyocytes from Human Pluripotent Stem Cells Using a Highly Reproducible Small Molecule-Based Differentiation Protocol

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust methods for the large-scale production of functional cell types, including cardiomyocytes. Here we demonstrate that the temporal manipulation of WNT, TGF-&#946;, and SHH signaling pathways leads to highly efficient cardiomyocyte differentiation of single-cell passaged hPSC lines in both static suspension and stirred suspension bioreactor systems. Employing this strategy resulted in ~ 100% beating spheroids, consistently containing &#62; 80% cardiac troponin T-positive cells after 15 days of culture, validated in multiple hPSC lines. We also report on a variation of this protocol for use with cell lines not currently adapted to single-cell passaging, the success of which has been verified in 42 hPSC lines. Cardiomyocytes generated using these protocols express lineage-specific markers and show expected electrophysiological functionalities. Our protocol presents a simple, efficient and robust platform for the large-scale production of human cardiomyocytes.

Here, we present a robust, fast and scalable cardiomyocyte differentiation protocol for human pluripotent stem cells (hPSCs). Cardiomyocytes derived using this large-scale method can provide sufficient cell numbers for their effective use in human cardiovascular disease modeling, high-throughput drug screening, and potentially clinical applications.

La producción a gran escala de los cardiomiocitos de células madre pluripotentes utilizando un pequeño protocolo de diferenciación basada en la molécula altamente reproducible

Maximizing the benefit of human pluripotent stem cells (hPSCs) for research, disease modeling, pharmaceutical and clinical applications requires robust ...

Analysis of Coronary Vessels in Cleared Embryonic Hearts

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard microscopy techniques, due to the scattering of imaging light by the thick heart tissue, as these vessels are localized deep within the walls of the developing heart. As optical clearing of tissues using organic solvents such as BABB (1 part benzyl alcohol to 2 parts benzyl benzoate) has been shown to greatly improve the optical penetration depth that can be achieved, we combined clearance of whole, PECAM1-immunostained hearts, with laser-scanning confocal microscopy, in order to obtain high-resolution images of vessels throughout the entire heart. BABB clearance of embryonic hearts takes place rapidly and also acts to preserve the fluorescent signal for several weeks; in addition, samples can be imaged multiple times without loss of signal. This straightforward method is also applicable to imaging other types of blood vessels in whole embryos.

We present a protocol for the analysis of coronary vessels in whole embryonic murine hearts up to E15.5, using standard immunological staining methods followed by optical clearance and confocal microscopy. This technique enables visualization of blood vessels throughout the entire heart without the need for time-consuming analysis of serial sections.

Análisis de los vasos coronarios en los corazones embrionarias aclarados

Whole mount visualization of the embryonic coronary plexus from which the capillary and arterial networks will form is rendered problematic using standard ...

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

La disección de las larvas de pez cebra de tejidos gonadales

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The efficient in vivo visualization of blood vessels and the reliable quantification of their complexity are key to understanding the biology and disease of the vascular network. Here, we provide a detailed method to visualize blood vessels with a commercially available fluorescent dye, human plasma acetylated low density lipoprotein DiI complex (DiI-AcLDL), and to quantify their complexity in Xenopus tropicalis. Blood vessels can be labeled by a simple injection of DiI-AcLDL into the beating heart of an embryo, and blood vessels in the entire embryo can be imaged in live or fixed embryos. Combined with gene perturbation by the targeted microinjection of nucleic acids and/or the bath application of pharmacological reagents, the roles of a gene or of a signaling pathway on vascular development can be investigated within one week without resorting to sophisticated genetically engineered animals. Because of the well-defined venous system of Xenopus and its stereotypic angiogenesis, the sprouting of pre-existing vessels, vessel complexity can be quantified efficiently after perturbation experiments. This relatively simple protocol should serve as an easily accessible tool in diverse fields of cardiovascular research.

Visualization and Quantitative Analysis of Embryonic Angiogenesis in Xenopus tropicalis

This protocol demonstrates a fluorescence-based method to visualize the vasculature and to quantify its complexity in Xenopus tropicalis. Blood vessels can be imaged minutes after the injection of a fluorescent dye into the beating heart of an embryo after genetic and/or pharmacological manipulations to study cardiovascular development in vivo.

Visualización y análisis cuantitativo de la angiogénesis embrionaria<em&gt; Xenopus tropicalis</em

Blood vessels supply oxygen and nutrients throughout the body, and the formation of the vascular network is under tight developmental control. The ...

Dissection and Culture of Mouse Embryonic Kidney

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney development, the two progenitor tissues, the ureteric bud and the metanephric mesenchyme, communicate and reciprocally induce cellular mechanisms to eventually form the collecting system and the nephrons of the kidney. As mammalian embryos grow intrauterine and therefore are inaccessible to the observer, an organ culture has been developed. With this method, it is possible to study epithelial-mesenchymal interactions and cellular behavior during kidney organogenesis. Furthermore, the origin of congenital kidney and urogenital tract malformations can be investigated. After careful dissection, the metanephric rudiments are transferred onto a filter that floats on culture medium and can be kept in a cell culture incubator for several days. However, one must be aware that the conditions are artificial and could influence the metabolism in the tissue. Also, the penetration of test substances could be limited due to the extracellular matrix and basal membrane present in the explant.
One main advantage of organ culture is that the experimenter can gain direct access to the organ. This technology is cheap, simple, and allows a large number of modifications, such as the addition of biologically active substances, the study of genetic variants, and the application of advanced imaging techniques.

This protocol describes a method for isolating and culturing metanephric rudiments from mouse embryos.

Disección y cultivo de riñón embrionario de ratón

The goal of this protocol is to describe a method for the dissection, isolation, and culture of mouse metanephric rudiments. 
During mammalian kidney ...

Correlative Super-resolution and Electron Microscopy to Resolve Protein Localization in Zebrafish Retina

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and scanning electron microscopy. The sub-diffraction limit resolution capabilities of super-resolution light microscopes allow improving the accuracy of the correlated data. Briefly, 110 nanometer thick cryo-sections are transferred to a silicon wafer and, after immunofluorescence staining, are imaged by super-resolution light microscopy. Subsequently, the sections are preserved in methylcellulose and platinum shadowed prior to imaging in a scanning electron microscope (SEM). The images from these two microscopy modalities are easily merged using tissue landmarks with open source software. Here we describe the adapted method for the larval zebrafish retina. However, this method is also applicable to other types of tissues and organisms. We demonstrate that the complementary information obtained by this correlation is able to resolve the expression of mitochondrial proteins in relation with the membranes and cristae of mitochondria as well as to other compartments of the cell.

This protocol describes the necessary steps to obtain subcellular protein localization results on zebrafish retina by correlating super-resolution light microscopy and scanning electron microscopy images.

Resolución súper correlativo y la microscopia electrónica para resolver la localización de la proteína en la Retina del pez cebra

We present a method to investigate the subcellular protein localization in the larval zebrafish retina by combining super-resolution light microscopy and ...

Nanoparticle-mediated siRNA Gene-silencing in Adult Zebrafish Heart

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart after apex resection or cryoinjury, making it an important model organism for heart regeneration study. However, the lack of loss-of-function methods for adult organs has restricted insights into the mechanisms underlying heart regeneration. RNA interference via different delivery systems is a powerful tool for silencing genes in mammalian cells and model organisms. We have previously reported that siRNA-encapsulated nanoparticles successfully enter cells and result in a remarkable gene-specific knockdown in the regenerating adult zebrafish heart. Here, we present a simple, rapid, and efficient protocol for the dendrimer-mediated siRNA delivery and gene-silencing in the regenerating adult zebrafish heart. This method provides an alternative approach for determining gene functions in adult organs in zebrafish and can be extended to other model organisms as well.

It remains a major challenge to develop conditional gene-knockout or effective gene-knockdown in adult zebrafish organs. Here we report a protocol for performing nanoparticle-mediated siRNA gene-silencing in adult zebrafish heart, thus providing a new loss-of-function method for studying adult organs in zebrafish and other model organisms.

Nanopartícula mediada por siRNA silenciamiento génico en corazón de pez cebra adulto

Mammals have a very limited capacity to regenerate the heart after myocardial infarction. On the other hand, the adult zebrafish regenerates its heart ...

Microinjection-based System for In Vivo Implantation of Embryonic Cardiomyocytes in the Avian Embryo

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a general challenge in developmental biology research. In the embryonic heart, this can be particularly difficult as regional differences in the expression of transcriptional regulators, paracrine/juxtacrine signaling cues, and hemodynamic force are all known to influence cardiomyocyte maturation. A simplified method to alter a developing cardiomyocyte's molecular and biomechanical microenvironment would, therefore, serve as a powerful technique to examine how local conditions influence cell fate and function. To address this, we have optimized a method to physically transplant juvenile cardiomyocytes into ectopic locations in the heart or the surrounding embryonic tissue. This allows us to examine how microenvironmental conditions influence cardiomyocyte fate transitions at single cell resolution within the intact embryo. Here, we describe a protocol in which embryonic myocytes can be isolated from a variety of cardiac sub-domains, dissociated, fluorescently labeled, and microinjected into host embryos with high precision. Cells can then be directly analyzed in situ using a variety of imaging and histological techniques. This protocol is a powerful alternative to traditional grafting experiments that can be prohibitively difficult in a moving tissue such as the heart. The general outline of this method can also be adapted to a variety of donor tissues and host environments, and its ease of use, low cost, and speed make it a potentially useful application for a variety of developmental studies.

In this method, embryonic cardiac tissues are surgically microdissected, dissociated, fluorescently labeled, and implanted into host embryonic tissues. This provides a platform for studying the individual or tissue level developmental organization under ectopic hemodynamic conditions, and/or altered paracrine/juxtacrine environments.

Sistema basado en la microinyección en Vivo implantación de cardiomiocitos embrionarios en el embrión aviar

Interpreting the relative impact of cell autonomous patterning versus extrinsic microenvironmental influence on cell lineage determination represents a ...