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>

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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><tbody><tr><td>&lt;font class=&quot;font5&quot;&gt;Tg(&lt;em&gt;sox10:eos&lt;/em&gt;)&lt;/font&gt;&lt;font class=&quot;font0&quot;&gt; zebrafish animals&lt;/font&gt;</td><td></td><td></td><td>Fish were obtained and crossed from the fish facility at the University of Notre Dame</td></tr><tr><td>100 X 15 mm petri dish</td><td>VWR</td><td>25384-302</td><td></td></tr><tr><td>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><td>0.8% Low Melting Point Agarose</td><td>dot scientific inc</td><td>9012-36-6</td><td></td></tr><tr><td>35 X 10 mm glass-coverslip bottom petri dish</td><td>Ted Pella Inc.</td><td>14021-20</td><td></td></tr><tr><td>Needle dissecting probe</td><td></td><td></td><td></td></tr><tr><td>0.002% 3-aminobenzoic acid ester (Tricaine)</td><td>Fluka analytical</td><td>A5040-250G</td><td></td></tr><tr><td>Fluorescent Dissecting Microscope with GFP filters</td><td>Zeiss Axiozoom</td><td></td><td></td></tr><tr><td>Confocal microscope with lasers to excite GFP and RFP filter sets</td><td>3i spinning disk confocal</td><td></td><td></td></tr><tr><td>UV light source (laser) for photoconversion</td><td>405 nm laser</td><td></td><td></td></tr><tr><td>Slidebook software</td><td>3i</td><td></td><td></td></tr><tr><td>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

Universidad de Cartagena UDEC

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

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 ...

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

El seguimiento de las células en el pez cebra transgénico GFP-Uso del sistema de fotoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

Culture of Adult Transgenic Zebrafish Retinal Explants for Live-cell Imaging by Multiphoton Microscopy

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The M&#252;ller glia re-enter the cell cycle and produce neuronal progenitor cells that undergo subsequent rounds of cell divisions and differentiate into the lost neuronal cell types. Both M&#252;ller glia and neuronal progenitor cell nuclei replicate their DNA and undergo mitosis in distinct locations of the retina, i.e. they migrate between the basal Inner Nuclear Layer (INL) and the Outer Nuclear Layer (ONL), respectively, in a process described as Interkinetic Nuclear Migration (INM). INM has predominantly been studied in the developing retina. To examine the dynamics of INM in the adult regenerating zebrafish retina in detail, live-cell imaging of fluorescently-labeled M&#252;ller glia/neuronal progenitor cells is required. Here, we provide the conditions to isolate and culture dorsal retinas from Tg[gfap:nGFP]mi2004 zebrafish that were exposed to constant intense light for 35 h. We also show that these retinal cultures are viable to perform live-cell imaging experiments, continuously acquiring z-stack images throughout the thickness of the retinal explant for up to 8 h using multiphoton microscopy to monitor the migratory behavior of gfap:nGFP-positive cells. In addition, we describe the details to perform post-imaging analysis to determine the velocity of apical and basal INM. To summarize, we established conditions to study the dynamics of INM in an adult model of neuronal regeneration. This will advance our understanding of this crucial cellular process and allow us to determine the mechanisms that control INM.

Zebrafish retinal regeneration has mostly been studied using fixed retinas. However, dynamic processes such as interkinetic nuclear migration occur during the regenerative response and require live-cell imaging to investigate the underlying mechanisms. Here, we describe culture and imaging conditions to monitor Interkinetic Nuclear Migration (INM) in real-time using multiphoton microscopy.

Cultura de adulto pez cebra transgénico de retina explantes de Células vivas imágenes de microscopía multifotónica

An endogenous regeneration program is initiated by M&#252;ller glia in the adult zebrafish (Danio rerio) retina following neuronal damage and death. The ...

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 ...

Culture and Transfection of Zebrafish Primary Cells

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in an intact and developing vertebrate. However, the detailed imaging of the morphologies of distinct cell types and subcellular structures is limited in whole mounts. Therefore, we established an efficient and easy-to-use protocol to culture live primary cells from zebrafish embryos and adult tissue.
In brief, 2 dpf zebrafish embryos are dechorionated, deyolked, sterilized, and dissociated to single cells with collagenase. After a filtration step, primary cells are plated onto glass bottom dishes and cultivated for several days. Fresh cultures, as much as long term differenciated ones, can be used for high resolution confocal imaging studies. The culture contains different cell types, with striated myocytes and neurons being prominent on poly-L-lysine coating. To specifically label subcellular structures by fluorescent marker proteins, we also established an electroporation protocol which allows the transfection of plasmid DNA into different cell types, including neurons. Thus, in the presence of operator defined stimuli, complex cell behavior, and intracellular dynamics of primary zebrafish cells can be assessed with high spatial and temporal resolution. In addition, by using adult zebrafish brain, we demonstrate that the described dissociation technique, as well as the basic culturing conditions, also work for adult zebrafish tissue.

We present an efficient and easy-to-use protocol for preparing primary cell cultures of zebrafish embryos for transfection and live cell imaging as well as a protocol to prepare primary cells from adult zebrafish brain.

Cultura y transfección de células primarias de pez cebra

Zebrafish embryos are transparent and develop rapidly outside the mother, thus allowing for excellent in vivo imaging of dynamic biological processes in ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

Multiphoton microscopy allows control of low energy photons with deep optical penetration and reduced phototoxicity. We describe the use of this technology for live cell labeling in zebrafish embryos. This protocol can be readily adapted for photo-induction of various light-responsive molecules.

De dos fotones basado en fotoactivación en vivo embriones de pez cebra

Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic ...

Biología

Multicolor Time-lapse Imaging of Transgenic Zebrafish: Visualizing Retinal Stem Cells Activated by Targeted Neuronal Cell Ablation

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound transgenic fish which express different fluorescent reporters in neighboring cell types provide a means of following cellular interactions 2 and/or tissue-level responses to experimental manipulations over time. In this video, we demonstrate methods that can be used for imaging multiple transgenically labeled cell types serially in individual fish over time courses that can span from minutes to several days. The techniques described are applicable to any study seeking to correlate the "behavior" of neighboring cells types over time, including: 1) serial 'catch and release' methods for imaging a large number of fish over successive days, 2) simplified approaches for separating fluorophores with overlapping excitation/emission profiles (e.g., GFP and YFP), 3) use of hypopigmented mutant lines to extend the time window available for high-resolution imaging into late larval stages of development, 4) use of membrane targeted fluorescent reporters to reveal fine morphological detail of individual cells as well as cellular details in larger populations of cells, and 5) a previously described method for chemically-induced ablation of transgenically targeted cell types; i.e., nitroreductase (NTR) mediated conversion of prodrug substrates, such as metronidazole (MTZ), to cytotoxic derivatives 3,5.
As an example of these approaches, we will visualize the ablation and regeneration of a subtype of retinal bipolar neuron within individual fish over several days. Simultaneously we will monitor several other retinal cell types, including neighboring non-targeted bipolar cells and potential degeneration-stimulated retinal stem cells (i.e., M&#971;ller glia). This strategy is being applied in our lab to characterize cell- and tissue-level (e.g., stem cell niche) responses to the selective loss and regeneration of targeted neuronal cell types.

In this video, techniques for multicolor confocal time-lapse imaging and targeted cell ablation are provided. Time-lapse imaging is used to monitor the behavior of multiple cell types of interest in vivo. Targeted cell ablation facilitates the study neural circuit function and cell-specific neuronal regeneration paradigms.

Multicolor Time-lapse de imágenes de pez cebra transgénico: Visualización de células madre retinianas activado por la ablación selectiva de células neuronales

High-resolution time-lapse imaging of living zebrafish larvae can be utilized to visualize how biological processes unfold (for review see 1). Compound ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Sola célula de mapeado de destino en el pez cebra

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Celular seguimiento mediante fotoconvertible proteínas durante el desarrollo de pez cebra

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...