Agradecemos a Bernard Kulemaka y miembros del laboratorio de Smith por sus comentarios útiles y orientación de reactivo, Sam Connell y Brent Redford de 3i para enviar imágenes preguntas y Deborah Bang, Karen Heed y Kay Stewart para el cuidado del pez cebra. Este trabajo fue financiado por la Universidad de Notre Dame, la Elizabeth y Michael Gallagher Family, el Alfred P. Sloan Foundation, centro de investigación de pez cebra en la Universidad de Notre y centro de las células madre y medicina regenerativa en la Universidad de Notre Dame. Todos los estudios en animales se realizaron en cumplimiento de la Universidad de Notre Dame IACUC Dr. Cody Smith.

Todos los estudios en animales fueron aprobados por la Universidad de Notre Dame Animal cuidado institucional y Comité de uso.
1. preparación de la muestra de pez cebra
<ol><li>Coloque un macho adulto y una Tg femenino adulto (proteína convertible) en una cámara de acoplamiento por procedimientos estándar13. En este manuscrito, utilice Tg(sox10:eos) de pescado9 por acceso pero otras líneas transgénicas con proteínas de photoconvertible puede ser utilizado igualmente. Configurar más de una cámara en caso de que no ponga el pescado. Permita que el pescado permanezca en la cámara durante la noche.</li>
	<li>A la mañana siguiente, recoger los huevos en cajas Petri de 100 mm. Permiten huevos madurar a la fertilización después de 24 h (hpf) antes de la proyección.</li>
	<li>Si arriba los animales eran heterocigotos para el transgen, platos de pantalla 24 hpf embriones Tg(sox10:eos) + con una fuente de luz nm y GFP 488 filtran de conjuntos en un microscopio de disección. Aislar Tg(sox10:eos) + embriones y dejar para madurar hasta 48 hpf.</li>
	<li>Dechorionate embriones manualmente con una aguja o unas pinzas.</li>
	<li>Preparar y microondas 5 mL de solución de agarosa de punto de bajo punto de fusión de 0,8%.<ol>
<li>Una vez que la agarosa es fría al tacto, lugar Tg anestesiados de 3-4 (sox10:eos) + 48 peces hpf en el centro de un cubreobjetos de cristal 10 mm plato de Petri de fondo.</li>
		<li>Añadir suficiente agarosa para cubrir la superficie del cubreobjetos, aproximadamente 1 mL. Utilice una aguja de punta de prueba para arreglar pescado en sus lados. Permita que la agarosa se solidifique para asegurar el montaje de los animales. Puede ser necesario reorganizar continuamente el pez cebra hasta que el agar solidifica14. Solidificación tarda aproximadamente 2 minutos.</li>
	</ol>
</li>
	<li>Después de la agarosa ha solidificado por 2 min, lentamente agregar medio embrión que contiene 0.02% éster del ácido paraaminobenzoico (Metanosulfonato) a plato hasta que la superficie del fondo del agar y plato quede sumergida. Este debe ser aprox. 3mL.</li>
</ol>2. microscopio montaje y proyección de imagen de conversión previa
<ol><li>Confocal software abierto y seleccione las ventanas de captura y enfoque [figura 1]. Debajo de la ventana de captura, seleccione conversión específica de laboratorio de imagen bajo la captura ajuste desplegable pestaña [figura 1]. Aquí, el ajuste del laboratorio específico se llama "proyección de imagen de peces".</li>
	<li>Coloque el espécimen en ámbito confocal y enfocar con el curso y las perillas de ajuste fino.</li>
	<li>Abra la ventana de foco y localizar la región deseada de interés (es decir, los ganglios de raíz dorsal).</li>
	<li>Seleccione el láser c488 bajo el menú filtro. Ajustar la exposición a 300 m, laser de potencia a 5 e intensificar a 75 [figura 2].</li>
	<li>Marque la casilla 3D en la sección tipo de captura . En la sección 3D de captura , seleccione posición actual y Compruebe la gama alrededor de corriente. En la misma sección, establecer el rango a 35, el número de aviones a 36 y el tamaño de paso 1. La gama puede aumentarse o disminuirse para acomodar la profundidad de la zona de proyección de imagen. Para la médula espinal un valor entre 35-40 pilas normalmente es suficiente [figura 5].</li>
	<li>Seleccione la ubicación actual [figura 5].</li>
	<li>Haga clic en Inicio en la parte inferior de la ventana de captura para adquirir imagen.</li>
</ol>3. single-cell Photoconversion
<ol><li>Confocal software abierto y seleccione las ventanas de captura y enfoque [figura 1]. Debajo de la ventana de captura, seleccione conversión específica de laboratorio de imagen bajo la captura ajuste desplegable pestaña [figura 1]. Aquí, el ajuste del laboratorio específico se llama "Pescado ablar chip completo".</li>
	<li>Seleccione el láser c488 y c541 bajo el menú filtro. Si no utiliza el mismo software del microscopio, encontrar el menú para seleccionar diferentes láseres y seleccione el 488 nm y láseres de 541 nm. Establecer las exposiciones a 300 m, laser de potencia a 5 e intensificar a 75 [figura 2]. Estos ajustes del láser son seleccionados basado en la producción de suficiente señal fluorescente sin causar fotoblanqueo o toxicidad. Si se visualiza la toxicidad o el fotoblanqueo, reducir la potencia del láser o la exposición.</li>
	<li>Abra la ventana de foco y haga clic en la ficha de Fotomanipulación en la ventana de foco. Ajustar los parámetros del láser en consecuencia. Cambiar la potencia de la pila de láser a 2 y a continuación, haga clic en ir. Cambiar el tamaño de bloque de trama 1 y haga clic en establecer. Cambiar el tamaño de doble clic 4. Cambia la línea láser a v405 [figura 3].</li>
	<li>Abra la configuración avanzada de la captura en la ventana de captura. Seleccione la ficha de Fotomanipulación y cambiar las repeticiones haga doble clic en 2. Haga clic en aceptar [figura 4].</li>
	<li>Seleccione la ficha XY en la ventana de foco. Verifique los parámetros del láser del paso 2 en la ficha de Fotomanipulación [figura 3, figura 4]. Configuración de láser para photoconvert la célula de interés sin photoconversion de las células circundantes.<ol>
<li>Si hay photoconversion de las células adyacentes, reducir la potencia del láser. Si no se produce photoconversion de las células, se pueden aumentar potencias láser. Ajuste óptimo, energía del laser a photoconvert sólo la región de interés y alrededores no.</li>
	</ol>
</li>
	<li>Marque la casilla de timelapse con tipo de captura. A continuación, haga clic en Inicio [figura 6A].</li>
	<li>Una vez que el timelapse en la ventana, seleccione la herramienta de círculo en la barra de herramientas superior [figura 7]</li>
	<li>Dibuja un círculo en la región más central de la célula. Haga clic con el botón derecho del círculo dibujado, seleccione región FRAPy espere 3 segundos. El área seleccionada debe convertirse en regulador [figura 8]. Haga clic en Detener captura.</li>
	<li>Si la proyección de imagen más de un animal, volver a la ficha XY en el menú de enfoque y posición seleccione 2. A continuación, repita los pasos 4.1-4.6.</li>
	<li>Para convertir a una población de células, siga los parámetros de protocolo y láser para pasos 4.1-4.4 excepto en lugar de dibujar un círculo para FRAP la región de interés, utilice la herramienta de línea. Trace una línea en la región de interés y FRAP la región utilizando los mismos parámetros mencionados anteriormente.</li>
</ol>4. post-photoconversion la proyección de imagen
<ol><li>Una vez que todos los puntos son photoconverted. Seleccionar la pila de imagen estándar de laboratorio específica ajuste descrito en el paso 3 la proyección de imagen de precoversion. Esto se encuentra bajo la captura ajuste desplegable pestaña en la ventana de captura [figura 5].</li>
	<li>Seleccione el láser c488 y ajustar la exposición a 300ms, laser de potencia a 5 e intensificar a 75 [figura 2].</li>
	<li>Seleccione el c541 láser en el mismo menú que el láser c488. Ajustar la exposición a 500 ms, laser de potencia de 10 e intensificar a 75 [figura 9].</li>
	<li>Marque la casilla 3D en la sección tipo de captura . En la sección 3D de captura , seleccione posición actual y Compruebe la gama alrededor de corriente. En la misma sección, establecer el rango a 35, el número de aviones a 36 y el tamaño de paso 1. El número de rango puede aumentar o disminuir para acomodar la profundidad de imagen deseada [figura 5].</li>
	<li>Si hay múltiples puntos en la ficha de atención XY, seleccione la opción lista multipunto en la ventana de captura. Si no, seleccione la ubicación actual.</li>
	<li>Haga clic en Inicio en la parte inferior de la ventana de captura para adquirir imagen.</li>
</ol>

<ol>
	<li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263 (5148), 802-805 (1994).</li><li>Livet, J., Weissman, T. 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=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-62 (2007).</li><li>Goins, W. F., Krisky, D., 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=Herpes+simplex+virus+vectors+for+gene+transfer+to+the+nervous+system.">Herpes simplex virus vectors for gene transfer to the nervous system.</a> J Neurovirol. 3 Suppl 1, S80-S88 (1997).</li><li>Boevink, P., Cruz, S., Hawes, C., Harris, N., Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-mediated+delivery+of+the+green+fluorescent+protein+to+the+endoplasmic+reticulum+of+plant+cells.">Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells.</a> Plant J. 10 (5), 935-941 (1996).</li><li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chem Soc Rev. 38 (10), 2887-2921 (2009).</li><li>Wiedenmann, J., Ivanchenko, S., 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=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101 (45), 15905-15910 (2004).</li><li>Maurel, D., Banala, S., Laroche, T., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+and+Photoconvertible+Fluorescent+Probes+for+Protein+Labeling.">Photoactivatable and Photoconvertible Fluorescent Probes for Protein Labeling.</a> ACS Chem Biol. 5 (5), 507-516 (2010).</li><li>Terskikh, A., Fradkov, 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=&amp;#34;Fluorescent+timer&amp;#34;:+protein+that+changes+color+with+time.">&amp;#34;Fluorescent timer&amp;#34;: protein that changes color with time.</a> Science. 290 (5496), 1585-1588 (2000).</li><li>McGraw, H. 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Los autores no tienen nada que revelar.

En tejidos complejos, distintos tipos de células se organizan en dominios específicos. Recientemente se han utilizado técnicas a las células individuales de la etiqueta dentro de estos tejido grandes estructuras1,2,3. Aquí muestran dos técnicas que pueden utilizarse igualmente para visualizar las interacciones de la célula y las interacciones de población de células dentro de tejidos complejos. La ventaja de la técnica...

Varias celdas distintas interactúan para construir y mantener los tejidos animales y vegetales complejos. Estas células son a menudo intercalados y difíciles de distinguir de los vecinos a un nivel unicelular sin microscopía de alta resolución que requieren fijación del tejido. Sin embargo, para entender cómo estas forman los tejidos, se mantiene y se convierten en enfermos, ha sido esencial para investigar cómo solo las células dentro del tejido están interactuando con el tiempo. Idealmente, estos experimentos requieren el etiquetado de células individuales dentro de un tejido de una manera no invasiva sin necesidad de fijación. Los científicos ahora han desarrollado numerosas técnicas para lograr esta tarea1,2,3,4.
El descubrimiento y la aplicación de la proteína fluorescente de Medusa verde (GFP) es un enfoque interesante que permitió para el etiquetado de distintas células en un tejido medio ambiente1. Utilizando promotores específicos de la célula, es posible seleccionar genéticamente un subconjunto de las células que llevan la etiqueta1. Alternativamente, viral inducido expresión de GFP puede ser utilizado para la expresión seleccionada por el usuario de GFP3,4. Aunque muy útil, expresión génica mediada de GFP no permite expresión seleccionada por el usuario dentro de un subconjunto de las células en el tejido; y viral expresión de GFP, aunque ventajosa, puede ser invasivo. Con la llegada de los derivados GFP y técnicas inteligentes como Brainbow para expresar distintas proteínas fluorescentes más escasamente en los tejidos, es posible visualizar las células y las interacciones entre ellos en el complejo tejido2, 5. sin embargo, estos enfoques etiqueta de células al azar. Si el experimento deseado requiere la visualización de una sola célula o población de células que se define por el experimentador, por lo tanto son limitados. Con tales experimentos, sería ventajoso tener una proteína fluorescente genéticamente expresa que puede ser manipulada para distinguir, en forma unicelular, de otras células fluorescentes y no fluorescentes.
Para alcanzar esta meta y visualizar la biología celular de las células en un tejido complejo de la vida, la comunidad científica utiliza photoconversion unicelular de diferentes proteínas fluorescentes6,7,8. Utilizando genéticamente controlada expresión de una proteína photoconvertible (es decir, eos, kaede, etc.) que las transiciones de un verde a rojo estado fluorescente cuando se exponen a los rayos UV (488 nm) luz, podemos distinguir una sola célula de su fluorescencia marcada vecinos6,7,8. Este enfoque utiliza un aparato conectado a nuestro microscopio confocal que puede dirigir la luz de una pila de láser a una región limitada de la difracción de interés. Con esta técnica, o bien podemos etiquetar las células o poblaciones más grandes en una manera definida por el usuario9,10,11. La técnica es mínimamente invasiva en comparación con las inyecciones de célula de GFP viral. Como una prueba de concepto, demostramos que podemos photoconvert las células dentro de un ganglio en el sistema nervioso periférico y photoconvert poblaciones más grandes como células situadas en el lado ventral de la médula espinal9,10, 11,12. Entonces podemos visualizar estas poblaciones de células de la photoconverted 24 h más tarde para ganar la penetración en su movimiento y diferenciación durante el desarrollo.

<table border="0" cellpadding="0" cellspacing="0" width="1846">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt;width:320pt" width="427">Tg(sox10:eos) zebrafish animals</td>
			<td style="width:125pt" width="167"></td>
			<td style="width:85pt" width="113"></td>
			<td style="width:854pt" width="1139">Fish were obtained and crossed from the fish facility at the University of Notre Dame</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">100 X 15 mm petri dish</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Embryo medium</td>
			<td></td>
			<td></td>
			<td>Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.8% Low Melting Point Agarose</td>
			<td>dot scientific inc</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">35 X 10 mm glass-coverslip bottom petri dish</td>
			<td>Ted Pella Inc.</td>
			<td>14021-20</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Needle dissecting probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.002% 3-aminobenzoic acid ester (Tricaine)</td>
			<td>Fluka analytical</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Fluorescent Dissecting Microscope with GFP filters</td>
			<td>Zeiss Axiozoom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Confocal microscope with lasers to excite GFP and RFP filter sets</td>
			<td>3i spinning disk confocal</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">UV light source (laser) for photoconversion</td>
			<td>405 nm laser</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Slidebook software</td>
			<td>3i</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Methylene blue</td>
			<td>Kordon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell photoconversion in living intact zebrafish

Tejido animal y vegetal se compone de distintas poblaciones de células. Estas células interactúan en el tiempo para construir y mantener los tejidos y pueden causar enfermedad cuando interrumpió. Los científicos han desarrollado técnicas inteligentes para investigar las características y dinámica natural de estas células dentro del tejido intacto por expresión de proteínas fluorescentes en subconjuntos de células. Sin embargo, a veces, los experimentos requieren más seleccionado visualización de células dentro del tejido, a veces en la forma unicelular o población de células. Para lograr esto y visualizar las células dentro de una población de células, los científicos han utilizado sola célula photoconversion de proteínas fluorescentes. Para demostrar esta técnica, aquí mostramos cómo a la luz UV a una celda de Eos-expresión de interés en un intacto, vive de zebrafish. Entonces a la imagen de esas células de Eos+ photoconverted 24 h más tarde para determinar cómo ha cambiado en el tejido. Se describen dos técnicas: solo celular photoconversion y photoconversions de las poblaciones de la célula. Estas técnicas pueden utilizarse para visualizar las interacciones célula-célula, destino celular y diferenciación y migraciones celulares, lo que es una técnica que es aplicable en muchas de las preguntas biológicas.

Photoconversion de proteínas fluorescentes puede utilizarse para etiquetar las distintas células dentro de un tejido6. Para demostrar esto, Tg(sox10:eos) pescado9 fueron utilizados para expresar la proteína photoconvertible Eos en las secuencias reguladoras de sox10. Los animales Tg(sox10:eos) en 48 hpf fueron montados primero y luego reflejada para detectar cualquier photoconversion no específicos que puede ha...

Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

Single-cell Photoconversion in Living Intact Zebrafish

Aquí, presentamos un protocolo para mostrar cómo photoconversion celular se logra a través de la exposición Ultravioleta a áreas específicas que expresan la proteína fluorescente, Eos, en animales vivos.

Sola célula Photoconversion en pez cebra intacto de la vida

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

single-cell-photoconversion-in-living-intact-zebrafish

Research

JoVE Journal

Developmental Biology

5.9K vistas.  University of Notre Dame. Aquí, presentamos un protocolo para mostrar cómo photoconversion celular se logra a través de la exposición Ultravioleta a áreas específicas que expresan la proteína fluorescente, Eos, en animales vivos.

Video: Single-cell Photoconversion in Living Intact Zebrafish

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Medición de la estabilidad de proteínas en la sala de embriones de pez cebra Utilizando fluorescencia Decay Después Fotoconversión (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Investigación

Biología del Desarrollo

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análisis de pez cebra larvas esquelético Integridad del músculo con el colorante azul de Evans

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Potencial de membrana de la neurona motora de Biosensing en los embriones vivos de pez cebra

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Inducción de la hipoxia en los sapos vivos y los embriones de pez cebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Siguientes movimientos de tejido endocárdico vía Photoconversion de la célula en el embrión de pez cebra

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análisis transcriptómico de unicelular de células endocrinas pancreáticas de ratón

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinyección de ADN en los oculares en embriones de Xenopus laevis e imágenes de gFP que expresan los árboles axonales ópticos en los renacuajos de Xenopus vivos

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Visualización del cerebro en desarrollo en el pez cebra vivo mediante imágenes confocales de barco iris y lapso de tiempo

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Extirpación de ojos en larvas vivas de pez cebra para examinar el crecimiento dependiente de la inervación y el desarrollo del sistema visual

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...