Los autores agradecen a Robert Kelsh por compartir amablemente el reactivo de pez cebra promotor SOX10.

1. Embrión Collection para Fotoconversión <ol><li> Establecer un mínimo de diez pares de SOX10: pez cebra transgénico Kaede entre el 5 y 6 de la tarde. </li><li> A la mañana siguiente, tire divisores y recoger los huevos alrededor del mediodía. Transferirlos a placas de Petri y ponerlas en 28,5 ° C incubadora. </li><li> Alrededor de las 24 h después de la fecundación, limpie las placas de Petri eliminando embriones muertos. Compruebe el estado de desarrollo de los embriones de 13 utilizando microscopía de campo de luz y el microscopio de fluorescencia con mayor proteína verde fluorescente (EGFP) de filtro. Kaede será visible a través de este filtro. </li><li> Cuando los embriones llegan a 60 HPF transferencia de cinco embriones fluorescentes brillantes en una placa de Petri y dechorionate separada si es necesario. </li></ol> 2. El montaje de los embriones para Fotoconversión <ol><li> Montar el embrión utilizando un microscopio fluorescente normal. Montaje de los embriones en el microscopio confocal es extremadamente difícil. </li><li> Utilice soporteagua ard E3 o preparar E3 fresca con la siguiente receta: Agregue la siguiente por un litro de agua destilada dH 2 O añadir lo siguiente para hacer 1x E3L: 0,292 g de NaCl 5,0 mM 0.013 g KCl 0,17 mm 0,044 g de CaCl 2 0,33 mm (desecante, 96%, reactivo ACS) 0,081 g 0,33 mM de MgSO4 (anhidra) Opcional: añadir 200 l de 0,05% de azul de metileno para 1X E3 como fungicida y revuelva. Medio mantiene a temperatura ambiente durante 1 semana. </li><li> Utilice tricaína estándar o preparar tricaína usando siguiente receta: Para 5 L de 0,2% tricaína mezclar los siguientes ingredientes: 45 ml de Tris-Cl (1M pH 9,5) 5 LH 2 O 10 g tricaína </li><li> Preparar &#39;jugo de película&#39; (50 ml E3, solución tricaína 0,015%), mediante la adición de 3,75 ml de 0,2% de tricaína a 46,25 ml de E3. </li><li> Preparar agarosa mezclando 46,25 ml de agua E3 con 46,35 mg de bajo punto de fusión (LMP) de agarosa en un matraz de vidrio de 50 ml y disolver las partículas sólidas en el microondas. A continuación, agregue3,73 ml 0,2% tricaína a agarosa y poner matraz de vidrio con agarosa en 50 ° C baño de agua hasta que el uso. </li></ol> 3. Preparación y montaje de Embriones <ol><li> Anestesie embriones con solución tricaína E3/0.015% hecho en el paso 1.1.2 y la transferencia de embriones a la diapositiva cámara. </li><li> Disfrute todo el exceso de E3 con un pañuelo de papel y la opción de venta de agarosa en embrión. </li><li> Posición embrión dorsal perfectamente con consejos Microloader. Asegúrese de que todo el embrión no flota en la parte superior de la agarosa, pero empujar todo el cuerpo hacia abajo. A continuación, vierta la solución tricaína E3/0.015% sobre embrión agarosa incrustada hasta la cámara está totalmente llena de líquido. </li></ol> 4. Ajuste de la configuración de software en Microscopio Confocal <ol><li> Utilice 20X objetivo aire sumergible (apertura numérica 0.75, el tamaño del agujero de alfiler 20) para enfocar el embrión. A continuación, pulse el botón de luz L100 en el microscopio y seleccionar los canales de software. Controls/A1settings abrir la vista de iconos / adquisición y click el botón de configuración confocal. Siguiente, haga clic en &#39;auto&#39; y seleccionar los canales siguientes antes de cerrar la ventana de nuevo: Ch1: DAPI canal 403.4 nm (longitud de onda entre 350-410 nm se pueden utilizar) Ch2: 488,0 nm Ch3: 561,9 nm </li></ol> 5. Fotoconversión <ol><li> Apague Ch1 y 3 y encienda Ch2 antes de pulsar el botón de eliminación de bloqueo y escaneo. </li><li> Centrarse en las células de interés para fotoconversión, que es mejor hacerlo partiendo de tensión 1frame/sec y alto (HV) 200 y luego subir en el cuadros / seg y bajar el HV en consecuencia hasta llegar a 1/32 imágenes / seg y HV alrededor 100-120, dependiendo del ruido de fondo. </li><li> Cuando el embrión está en el foco, mira en el borde derecho de la pantalla, tome el borde verde y reducir su tamaño para que quepa en el área de interés para fotoconversión y haga clic derecho con el ratón. Más pequeño es mejor porque el fotoconversión es irreversible. </li><li> Gire cuadros / seg a 1 y HV a 200 y pulseescanear. Una imagen ampliada en el área de fotoconversión es ahora visible. </li><li> Células Photoconvert girando sobre Ch1 (403,4 nm). La exposicion al láser photoconverting cualquier lugar 5-60 segundos, dependiendo del tamaño de la región hasta Kaede verde como se ve a través del canal de GFP está casi ausente. </li><li> Cuando la señal de Kaede verde es casi ausente, gire Ch1 apagado. Gire Ch2 y 3 en. Luego, haga clic a la derecha en la ventana en A1 campo de área de exploración y pulsa &#39;retorno a tamaño original&#39; para comprobar si se photoconverted células deseadas. </li></ol> 6. Haciendo una pila Z <ol><li> Busque el icono &quot;captura de la serie Z &#39;en la barra de menú y ajuste los límites superior e inferior de la Z-stack antes de iniciarlo. Establecer LUT. </li></ol> 7. Software-Configuración de Time-lapse <ol><li> Ir a la caja A1Simple GUI y marque siguientes ajustes: 1/32 cuadros / seg tamaño de 1024 promedio de 4 u 8 veces, dependiendo del número de Z-pilas que tiene que ser tomada en un bucle </li><li> Ir a:controles de la vista / adquisición / ND adquisición secuencia y marco de tiempo establecido (8-30min), haga clic en continuo y haga clic en Z-stack. Establecer fronteras como probado antes y comenzar la película. </li><li> Mantenga la adición E3/0.015% solución tricaína diapositiva cámara durante todo el día. Por la noche, se llenan la cámara completamente. Que durará hasta la mañana siguiente. </li><li> A la mañana siguiente, vuelva a llenar solución tricaína E3/0.015% y comprobar si el embrión está todavía vivo. La pérdida de fluorescencia indica la muerte. Continuar recambios y controles durante el día. </li><li> Cuando la estructura se ha terminado la formación o el embrión ha muerto, terminar la película. </li></ol> 8. Procesamiento post-producción <ol><li> Haga clic en &quot;mostrar proyección máxima intensidad&quot; en la barra de herramientas, a continuación, haga clic derecho sobre la imagen y crear nuevo documento. El vídeo se puede ver en la mejor calidad posible. Vamos a eliminar los marcos que no son necesarios y ajustar LUT. Finalmente guardar como. Avi. Este formato estará bien para Windows. Para Mac software de descarga to convertir. mov o. wmv. </li></ol>

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B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., 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+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, 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=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., 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=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. 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Los autores declaran que no tienen intereses financieros en competencia.

Aquí se muestra un nuevo método para la visualización del desarrollo craneofacial en el modelo de pez cebra. Los SOX10: Kaede línea de pez cebra transgénico se ha utilizado con éxito para describir el patrón migratorio de CNCC en detalle se utiliza como un organismo modelo 5. Estudios previos han utilizado puntos de referencia brutos como el ojo a las células diana y se han basado en la inyección de ARNm Kaede, ensayos fotoconversión o dextrano fluoresceína enjaulado pa...

Hendiduras orofaciales representan la deformidad craneofacial más frecuente, con 1/700-1, 000 entregas afectadas 1. La interrupción del desarrollo temprano embriológico craneofacial puede conducir a la formación de labio leporino y paladar hendido (CL / P). Si bien las causas de hendidura sindrómica se ha demostrado en gran parte, aún deben ser descubiertos 2-4 las bases genéticas y epigenéticas de las formas no sindrómicas de hendidura orofacial. Con el fin de entender la etiología y la patogénesis de estas malformaciones, es necesario aclarar el desarrollo de estructuras craneofaciales sobre una base celular. En todas las especies de vertebrados células de la cresta neural craneal (CNCC) migran desde el tubo neural dorsal para rellenar los arcos faríngeos, lo que contribuirá a la formación de las estructuras orofaciales. La interrupción del desarrollo temprano de la cresta neural embrionaria puede conducir a la formación de las malformaciones craneofaciales, incluyendo CL / P 5-7. En anunciocondición a las similitudes estructurales entre el pez cebra y el desarrollo craneofacial mamíferos (CNCCs residen en regiones homólogas), la red de regulación de genes es altamente conservada. También se ha demostrado que CNCCs desarrollar de la misma manera entre las especies amniote y pez cebra 8, haciendo que el pez cebra un organismo de gran alcance para el estudio de la base del desarrollo y genética de CL / P. Tiene muchas ventajas, incluyendo tamaño pequeño, rápido y ex utero desarrollo embrionario, y las altas tasas de reproducción. Por otra parte, el embrión es ópticamente transparente, por lo que es susceptible a la observación de eventos de desarrollo complejos bajo el microscopio 9. Es un modelo animal ideal para el estudio de la migración y la diferenciación de células de la cresta neural craneal. Ampliando previamente publicado trabajos 8, 10, 11, el patrón migratorio de CNCC se describió en detalle el uso de los SOX10: Kaede modelo transgénico 5. Kaede es una proteína de la foto convertible que turns de verde a rojo después de la activación de fotos y hace que sea posible rastrear CNCCs precisión. Durante esta transformación la cadena principal del péptido se escinde, lo que sugiere que la conversión es estable, es decir, las células pueden ser rastreados a su destino final 12. Las líneas transgénicas marcados con Kaede bajo el control transcripcional de SOX10 mostraron que el paladar amniota y la placa etmoidal de pez cebra se forman de manera homóloga por la fusión de las prominencias maxilares bilaterales (MXP) con la prominencia frontonasal (FNP) y que la costura de fusión en forma de Y es análoga entre especies. Entre otras aplicaciones, los SOX10: Kaede modelo de pez cebra transgénico se utilizó para generar videos de embriones de pez cebra en diferentes etapas de desarrollo para mostrar la formación de estructuras craneofaciales normales y anormales. Fotoconversión de grupos específicos de células hace posible el seguimiento de su desarrollo. Con este método, un enfoque para crear imágenes en vivo del desarrollo de craniofacestructuras ial en el pez cebra se introduce, por lo que es fácil de demostrar visualmente este complejo proceso de desarrollo. Este protocolo tiene como objetivo compartir la experiencia de la generación de estos vídeos con el desarrollo normal de la placa etmoidal en SOX10: pez cebra transgénico Kaede como ejemplo. Este protocolo adicional se puede aplicar a la realización de vídeos time-lapse de cualquier estructura derivada de células de la cresta neural craneal en el pez cebra.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>sox10: kaede transgenic zebrafish line</td>
			<td>MGH</td>
			<td></td>
			<td>Available via the Liao lab</td>
		</tr>
		<tr>
			<td>Petri dishes 100x15 mm</td>
			<td>BD Falcon</td>
			<td>351029</td>
			<td></td>
		</tr>
		<tr>
			<td>Petri dishes 35 mmx10 mm</td>
			<td>BD Falcon</td>
			<td>351008</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultrapure Low melting point (LMP) Agarose</td>
			<td>Invitrogen</td>
			<td>15517022</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Tek 2 Chamber SlideSystem</td>
			<td>LabTek</td>
			<td>154453</td>
			<td></td>
		</tr>
		<tr>
			<td>Microloaders 200/pk</td>
			<td>Fisher</td>
			<td>E5242956003</td>
			<td></td>
		</tr>
		<tr>
			<td>Nikon A1R Si Confocal Ti series</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
		<tr>
			<td>NIS Elements Software AR3.2 64-bit</td>
			<td>Nikon</td>
			<td>No Catalog number</td>
			<td></td>
		</tr>
	</tbody>
</table>

visualization of craniofacial development in the sox10: kaede transgenic zebrafish line using time-lapse confocal microscopy

Palatogénesis vertebrado es un proceso de desarrollo altamente coreografía y complejo, que implica la migración de la cresta neural craneal (CNC) las células, la convergencia y la extensión de prominencias faciales, y la maduración del esqueleto craneofacial. Para estudiar la contribución de la cresta neural craneal a regiones específicas del paladar pez cebra un SOX10: Línea de pez cebra transgénico Kaede se generó. Sox10 proporciona restricción linaje de la proteína indicadora Kaede a la cresta neural, con lo que la célula etiquetado de un proceso más preciso que el tinte tradicional o reportero de la inyección de ARNm. Kaede es una proteína de la foto convertible que cambia de verde a rojo después de la activación de la foto y hace posible seguir con precisión las células. Los SOX10: Línea transgénica Kaede se utilizó para realizar el análisis de linaje para delinear las poblaciones de células CNC que dan lugar a maxilar contra elementos mandibulares e ilustran la homología de las prominencias faciales para amniotas. Este protocolo describe el pasos para generar un video time-lapse en vivo de un SOX10: embrión de pez cebra Kaede. Desarrollo de la placa etmoidal servirá como un ejemplo práctico. Este protocolo se puede aplicar a realizar una grabación confocal de lapso de tiempo de cualquier Kaede o proteína reportera fotoconvertible similar en el pez cebra transgénico. Además, puede ser utilizado para capturar no sólo normal, pero también anormal desarrollo de estructuras craneofaciales en los mutantes de pez cebra.

En el SOX10: CNCCs línea transgénica Kaede, migratorias y post migratorios están etiquetados con fluorescencia verde. Las células CNCC marcados con Kaede verde fluorescente recapitular endógena mRNA SOX10 expresión 5. Entre otras aplicaciones, se utilizó este modelo animal para visualizar mejor el desarrollo de las estructuras craneofaciales dependientes CNCC. El desarrollo normal de las estructuras específicas y también el desarrollo patológico de las malformaciones cra...

Watch this Scientific Journal Video about Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy at JoVE.com

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

La visualización de los datos experimentales se ha convertido en un elemento clave en la presentación de resultados a la comunidad científica. Generación de grabación a intervalos regulares en vivo de embriones en crecimiento contribuye a la mejor presentación y la comprensión de los procesos de desarrollo complejos. Este protocolo es una guía paso a paso para el etiquetado de células a través de la proteína fotoconversión Kaede en el pez cebra.

Visualización de Desarrollo Craneofacial en los SOX10: Kaede pez cebra transgénico Línea Usando Time-lapse microscopía confocal

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K vistas.  Massachusetts General Hospital.  La visualización de los datos experimentales se ha convertido en un elemento clave en la presentación de resultados a la comunidad científica. Generación de grabación a intervalos regulares en vivo de embriones en crecimiento contribuye a la mejor presentación y la comprensión de los procesos de desarrollo complejos. Este protocolo es una guía paso a paso para el etiquetado de células a través de la proteína fotoconversión Kaede en el pez cebra. 

Video: Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoreo de actina desmontaje con la Microscopía de Time-lapse

Investigación

Biología

Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo. Here we describe a method to mount zebrafish embryos for long-term imaging and demonstrate how to automate the capture of time-lapse images using a confocal microscope. We also describe a method to create controlled, precise damage to individual branches of peripheral sensory axons in zebrafish using the focused power of a femtosecond laser mounted on a two-photon microscope. The parameters for successful two-photon axotomy must be optimized for each microscope. We will demonstrate two-photon axotomy on both a custom built two-photon microscope and a Zeiss 510 confocal/two-photon to provide two examples.

Zebrafish trigeminal sensory neurons can be visualized in a transgenic line expressing GFP driven by a sensory neuron specific promoter 1. We have adapted this zebrafish trigeminal model to directly observe sensory axon regeneration in living zebrafish embryos. Embryos are anesthetized with tricaine and positioned within a drop of agarose as it solidifies. Immobilized embryos are sealed within an imaging chamber filled with phenylthiourea (PTU) Ringers. We have found that embryos can be continuously imaged in these chambers for 12-48 hours. A single confocal image is then captured to determine the desired site of axotomy. The region of interest is located on the two-photon microscope by imaging the sensory axons under low, non-damaging power. After zooming in on the desired site of axotomy, the power is increased and a single scan of that defined region is sufficient to sever the axon. Multiple location time-lapse imaging is then set up on a confocal microscope to directly observe axonal recovery from injury.

Here we describe a method for mounting zebrafish embryos for long-term imaging, two-photon imaging and tissue-damage techniques, and time-lapse confocal imaging.

De dos fotones axotomía y de lapso de tiempo de imagen confocal en embriones de pez cebra en vivo

Zebrafish have long been utilized to study the cellular and molecular mechanisms of development by time-lapse imaging of the living transparent embryo.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

Imágenes en vivo del cerebro del embrión de pez cebra mediante microscopía confocal

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Time-lapse Imaging of Mitosis After siRNA Transfection

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and cellular content. Hallmark events of mitosis, such as chromosome movement, can be readily tracked on an individual cell basis using time-lapse fluorescence microscopy of mammalian cell lines expressing specific GFP-tagged proteins. In combination with RNAi-based depletion, this can be a powerful method for pinpointing the stage(s) of mitosis where defects occur after levels of a particular protein have been lowered. In this protocol, we present a basic method for assessing the effect of depleting a potential mitotic regulatory protein on the timing of mitosis. Cells are transfected with siRNA, placed in a stage-top incubation chamber, and imaged using an automated fluorescence microscope. We describe how to use software to set up a time-lapse experiment, how to process the image sequences to make either still-image montages or movies, and how to quantify and analyze the timing of mitotic stages using a cell-line expressing mCherry-tagged histone H2B. Finally, we discuss important considerations for designing a time-lapse experiment. This strategy is complementary to other approaches and offers the advantages of 1) sensitivity to changes in kinetics that might not be observed when looking at cells as a population and 2) analysis of mitosis without the need to synchronize the cell cycle using drug treatments. The visual information from such imaging experiments not only allows the sub-stages of mitosis to be assessed, but can also provide unexpected insight that would not be apparent from cell cycle analysis by FACS.

Here we describe a basic protocol to image and quantify the mitotic timing of live mammalian tissue culture cells after siRNA transfection.

Time-lapse de imágenes de mitosis después de la transfección de siRNA

Changes in cellular organization and chromosome dynamics that occur during mitosis are tightly coordinated to ensure accurate inheritance of genomic and ...

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 ...

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes. The transparency of the 
embryos, the genetic resources, and the relative ease of transformation are qualities that make C. elegans an excellent model for early embryogenesis. Laser-based 
confocal microscopy and fluorescently labeled tags allow researchers to follow specific cellular structures and proteins in the developing embryo. For example, 
one can follow specific organelles, such as lysosomes or mitochondria, using fluorescently labeled dyes. These dyes can be delivered to the early embryo by means 
of microinjection into the adult gonad. Also, the localization of specific proteins can be followed using fluorescent protein tags. Examples are presented here 
demonstrating the use of a fluorescent lysosomal dye as well as fluorescently tagged histone and ubiquitin proteins. The labeled histone is used to visualize 
the DNA and thus identify the stage of the cell cycle. GFP-tagged ubiquitin reveals the dynamics of ubiquitinated vesicles in the early embryo. Observations 
of labeled lysosomes and GFP:: ubiquitin can be used to determine if there is colocalization between ubiquitinated vesicles and lysosomes. A technique for 
the microinjection of the lysosomal dye is presented. Techniques for generating transgenenic strains are presented elsewhere (1, 2). For imaging, embryos 
are cut out of adult hermaphrodite nematodes and mounted onto 2% agarose pads followed by time-lapse microscopy on a standard laser scanning confocal 
microscope or a spinning disk confocal microscope. This methodology provides for the high resolution visualization of early embryogenesis.

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

This article describes a technique for the visualization of the early events of embryogenesis in the nematode Caenorhabditis elegans.

Time-lapse microscopía de embriogénesis temprana en Caenorhabditis elegans

Caenorhabditis elegans has often been used as a model system in studies of early developmental processes.  The transparency of the 
embryos, the genetic ...

Electroporation of Craniofacial Mesenchyme

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. For example, electroporation has been used with great success to study neural and retinal development in Xenopus, chicken and mouse 1-10. However, it is important to note that in all of these studies, investigators were not targeting soft tissues. Because we are interested in craniofacial development, we adapted a method to target facial mesenchyme.
When we searched the literature, we found, to our surprise, very few reports of successful gene transfer into cartilaginous tissue. The majority of these studies were gene therapy studies, such as siRNA or protein delivery into chondrogenic cell lines, or, animal models of arthritis 11-13. In other systems, such as chicken or mouse, electroporation of facial mesenchyme has been challenging (personal communications, Dept of Craniofacial Development, KCL). We hypothesized that electroporation into procartilaginous and cartilaginous tissues in Xenopus might work better. In our studies, we show that gene transfer into the facial cartilages occurs efficiently at early stages (28), when the facial primordium is still comprised of soft tissue prior to cartilage differentiation.
Xenopus is a very accessible vertebrate system for analysis of craniofacial development. Craniofacial structures are more readily visible in Xenopus than in any other vertebrate model, primarily because Xenopus embryos are fertilized externally, allowing analyses of the earliest stages, and facilitating live imaging at single cell resolution, as well as reuse of the mothers 14. Among vertebrate models developing externally, Xenopus is more useful for craniofacial analysis than zebrafish, as Xenopus larvae are larger and easier to dissect, and the developing facial region is more accessible to imaging than the equivalent region in fish. In addition, Xenopus is evolutionarily closer to humans than zebrafish (&tilde;100 million years closer) 15. Finally, at these stages, Xenopus tadpoles are transparent, and concurrent expression of fluorescent proteins or molecules will allow easy visualization of the developing cartilages. We anticipate that this approach will allow us to rapidly and efficiently test candidate molecules in an in vivo model system.

Craniofacial cartilages develop in close contact with other tissues and are difficult to manipulate in live animals. We are using electroporation to deliver molecular tools during growth of the craniofacial skeleton while bypassing early embryonic effects. This approach will allow us to efficiently test candidate molecules in vivo.

Electroporación de mesénquima craneofacial

Electroporation is an efficient method of delivering DNA and other charged macromolecules into tissues at precise time points and in precise locations. ...

Quantitative Analysis of Random Migration of Cells Using Time-lapse Video Microscopy

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During directional migration, cells move rapidly in response to an extracellular chemotactic signal, or in response to intrinsic cues 3 provided by the basic motility machinery. Random migration occurs when a cell possesses low intrinsic directionality, allowing the cells to explore their local environment.
Cell migration is a complex process, in the initial response cell undergoes polarization and extends protrusions in the direction of migration 2. Traditional methods to measure migration such as the Boyden chamber migration assay is an easy method to measure chemotaxis in vitro, which allows measuring migration as an end point result. However, this approach neither allows measurement of individual migration parameters, nor does it allow to visualization of morphological changes that cell undergoes during migration.
Here, we present a method that allows us to monitor migrating cells in real time using video - time lapse microscopy. Since cell migration and invasion are hallmarks of cancer, this method will be applicable in studying cancer cell migration and invasion in vitro. Random migration of platelets has been considered as one of the parameters of platelet function 4, hence this method could also be helpful in studying platelet functions. This assay has the advantage of being rapid, reliable, reproducible, and does not require optimization of cell numbers. In order to maintain physiologically suitable conditions for cells, the microscope is equipped with CO2 supply and temperature thermostat. Cell movement is monitored by taking pictures using a camera fitted to the microscope at regular intervals. Cell migration can be calculated by measuring average speed and average displacement, which is calculated by Slidebook software.

This method allows monitoring of cells in real time and quantitative measurements of different cell migration parameters such as speed, displacement, and velocity. Unlike the traditional methods, this real time approach is not based on endpoint quantitative migration measurements; instead it allows monitoring and calculating different parameters continuously.

Análisis cuantitativo de la migración desordenada de las células mediante microscopía de Time-lapse vídeo

Cell migration is a dynamic process, which is important for embryonic development, tissue repair, immune system function, and tumor invasion 1, 2. During ...

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue patterning. During animal development, key cell proliferation and patterning events occur very quickly. For instance, in Caenorhabditis elegans all cell divisions required for the larval body plan are completed within six hours after fertilization, with seven mitotic cycles1; the sixteen or more mitoses of Drosophila embryogenesis occur in less than 24 hr2. In contrast, cell divisions during plant development are slow, typically on the order of a day 3,4,5 . This imposes a unique challenge and a need for long-term live imaging for documenting dynamic behaviors of cell division and differentiation events during plant organogenesis. Arabidopsis epidermis is an excellent model system for investigating signaling, cell fate, and development in plants. In the cotyledon, this tissue consists of air- and water-resistant pavement cells interspersed with evenly distributed stomata, valves that open and close to control gas exchange and water loss. Proper spacing of these stomata is critical to their function, and their development follows a sequence of asymmetric division and cell differentiation steps to produce the organized epidermis (Fig. 1).
This protocol allows observation of cells and proteins in the epidermis over several days of development. This time frame enables precise documentation of stem-cell divisions and differentiation of epidermal cells, including stomata and epidermal pavement cells. Fluorescent proteins can be fused to proteins of interest to assess their dynamics during cell division and differentiation processes. This technique allows us to understand the localization of a novel protein, POLAR6, during the proliferation stage of stomatal-lineage cells in the Arabidopsis cotyledon epidermis, where it is expressed in cells preceding asymmetric division events and moves to a characteristic area of the cell cortex shortly before division occurs. Images can be registered and streamlined video easily produced using public domain software to visualize dynamic protein localization and cell types as they change over time.

Long-term, High-resolution Confocal Time Lapse Imaging of Arabidopsis Cotyledon Epidermis during Germination

We describe a protocol using chamber slides and media to immobilize plant cotyledons for confocal imaging of the epidermis over several days of development, documenting stomatal differentiation. Fluorophore-tagged proteins can be tracked dynamically by expression and subcellular localization, increasing understanding of their possible roles during cell division and cell-type differentiation.

A largo plazo, de alta resolución de imagen confocal Time Lapse de<em&gt; Cotiledones Arabidopsis</em&gt; Epidermis durante la germinación

Imaging in vivo dynamics of cellular behavior throughout a developmental sequence can be a powerful technique for understanding the mechanics of tissue ...

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical clarity and amenability to genetic manipulation, the zebrafish embryo has become a popular model organism with which to perform time-lapse analysis of morphogenesis in living embryos. Confocal imaging of a live zebrafish embryo requires that a tissue of interest is persistently labeled with a fluorescent marker, such as a transgene or injected dye. The process demands that the embryo is anesthetized and held in place in such a way that healthy development proceeds normally. Parameters for imaging must be set to account for three-dimensional growth and to balance the demands of resolving individual cells while getting quick snapshots of development. Our results demonstrate the ability to perform long-term in vivo imaging of fluorescence-labeled zebrafish embryos and to detect varied tissue behaviors in the cranial neural crest that cause craniofacial abnormalities. Developmental delays caused by anesthesia and mounting are minimal, and embryos are unharmed by the process. Time-lapse imaged embryos can be returned to liquid medium and subsequently imaged or fixed at later points in development. With an increasing abundance of transgenic zebrafish lines and well-characterized fate mapping and transplantation techniques, imaging any desired tissue is possible. As such, time-lapse in vivo imaging combines powerfully with zebrafish genetic methods, including analyses of mutant and microinjected embryos.

Time-lapse confocal imaging is a powerful technique useful for characterizing embryonic development. Here, we describe the methodology and characterize craniofacial morphogenesis in wild-type, as well as pdgfra, smad5, and smo mutant embryos.

Analizando Craneofacial en la morfogénesis de pez cebra Utilizando 4D Microscopía Confocal

Time-lapse imaging is a technique that allows for the direct observation of the process of morphogenesis, or the generation of shape. Due to their optical ...