Nos gustaría expresar nuestro agradecimiento por los aportes y comentarios de los miembros del Laboratorio de Padilla. Cepas de nematodos en este trabajo fueron proporcionados por el Centro de Genética Caenorhabditis, que es financiado por el NIH Centro Nacional para Recursos de Investigación (CNRR). Reconocemos y agradecemos el Dr. Lon Turnbull de asistencia técnica con microscopía confocal. Este trabajo ha sido financiado por una beca de la Fundación Nacional de Ciencia (NSF-IOS, CARRERA) para PAP

1. Preparación de la muestra <ol><li> Producir u obtener apropiado C. transgénico elegans cepa de interés utilizando metodologías transgénicas o de Genética Caenorhabditis Stock Center (CGC) o colega. En este caso estamos usando la cepa TH32 (pastel-1 :: TBG-1 :: GFP; pastel-1 :: GFP :: H2B) 5 para visualizar los cromosomas y los centrosomas como marcadores para la división celular. </li><li> Generar una población sincronizado mediante uno de tres métodos: 1) se desintegran adultos grávidas en una solución de hipoclorito y el uso de embriones sobrantes 6, 2) recoger los adultos grávidas en un plato sin semillas y dejar que ellos se extendía embriones durante 1-2 horas, o 3) recoger L4 larvas de una población mixta y pasar a una nueva placa sembrada. </li><li> Crecer nematodos a 20 ° C hasta la edad adulta joven embarazada, que durará aproximadamente 96 horas de incubación, 72 horas a partir de larvas L1 ó 24 horas después de la muda L4. Embriones (2-20 celular) en el útero contener blastómeras grandes y núcleos que son óptimas para imagción sub-celulares y sub nucleares cambios, respectivamente. Esta metodología proporciona un medio para generar una población de adultos jóvenes que contienen un número abundante de embriones tempranos. </li></ol> 2. Preparación de diapositivas y la Cámara Anoxia Configuración <ol><li> Hacer una cámara húmeda para retener muestras preparadas mediante la colocación de una toalla de papel húmeda en el interior de una gran placa de Petri o compartimiento cubierto con una tapa de tamaño suficiente. </li><li> Preparar fundido agarosa al 2% en H2O desionizada, calentando la mezcla en recipientes de vidrio a través de microondas o baño de agua. </li><li> Ponga 1-3 gotas de cálida agarosa sobre un portaobjetos de microscopio de vidrio limpio. Colocar inmediatamente una segunda corredera invertida perpendicularmente en la parte superior de la gota de agarosa (s), presiona ligeramente de modo que la almohadilla resultante será delgada y libre de burbujas de aire. </li><li> Dar tiempo a que se enfríe lentamente y desmontar los dos portaobjetos dejando intacta la almohadilla de agarosa en una de las diapositivas. Es importante asegurar la almohadilla de agarosa es lo suficientemente delgada como para no interfevolver con la capacidad de enfocar el microscopio después. Capacidad de visualizar la muestra es también depende de la distancia óptica específica del objetivo en uso, que puede variar entre los microscopios. </li><li> Use una hoja de afeitar limpia para dar forma almohadilla de agarosa en una pequeña plaza. Almacenar en la cámara húmeda preparada hasta que esté listo para usar. </li><li> Con cuidado, utilizando la hoja de afeitar limpia mismo, tomar el borde de la almohadilla de agarosa y transferirla desde el portaobjetos sobre un cubreobjetos redondo 25 mm micro, evitando la acumulación de burbujas de aire debajo de la almohadilla. El cubreobjetos redondo seleccionado para usar encaja adecuadamente en el microincubator. </li><li> Añadir una gota de anestésico (0,5% tricaína, 0,05% tetramisol en M9 buffer) en la almohadilla de agarosa para uso como anestésico. Elige gusanos 5-10 y colóquelos en la gota de anestésico. Permita 1-3 min para los gusanos de cesar el movimiento. </li><li> El fundamento para observar los embriones en el útero adulto, en vez de embriones disecados, es reducir al mínimo el potencial de embrión desecaciónción y el movimiento debido al flujo de gas nitrógeno a través del embrión. </li><li> Inmediatamente se añade una gota de aceite de halocarbono en la parte superior de los gusanos para evitar que los gusanos de la deshidratación de gas se hace fluir a través de la cámara. Dado que el aceite de halocarbono es viscoso se puede usar una punta de pipeta o punta de un gusano de selección para recoger el petróleo y caen sobre los gusanos. </li><li> Una vez que el cubreobjetos circular que contiene los gusanos está listo, las dos mitades de la Leiden cerrado perfusión microincubator se separan, y el cubreobjetos es entonces cuidadosamente colocados de pie sobre la parte inferior del anillo. Los dos lados de la cámara se cierran herméticamente juntos. </li><li> La cámara se conecta entonces al depósito de gas nitrógeno a través de tubo de plástico flexible y se coloca en el lugar apropiado en la platina del microscopio (Figura 1C). En este momento el tubo y microincubator deben verificarse cuidadosamente para detectar cualquier fuga, garantizando una fijación segura de la tubería y de las clavijas de puerto a lo largo del lado de la cámara. Uno puede altan ligeramente extendió una pequeña cantidad de agua de jabón sobre el tubo en busca de burbujas, los cuales formarán si existe una fuga. </li></ol> 3. Microscopía en Anoxia <ol><li> Localice los animales a un menor aumento, entonces, pasar a la magnificación superior que les corresponda (64x para esta aplicación) para imágenes de tejido o células de interés, tales como blastómeros de embriones u ovocitos en los adultos. Encender el láser de 488 nm para ver la señal del GFP y GFP localizar proteína de fusión en la célula (s) de interés. </li><li> Para visualizar la detención en la fase específica de la detención del ciclo celular (por ejemplo profase tardía) identificar un blastómeros en una fase anterior de la división celular (por ejemplo, interfase profase temprana o tardía). Marcadores celulares tales como la posición de centríolos, la iniciación de la condensación de cromosomas, localización cromosómica y la presencia o ausencia de la envoltura nuclear ayudará a identificar la etapa del ciclo celular (Figura 2A). </li><li> Entonces la cámara se perfundió con gas nitrógeno (99,999%N 2; &lt;2 ppm O 2). En este caso se utiliza una presión de 6 psi, sin embargo la presión puede necesitar ajustarse y optimizarse en función del tamaño de cada microincubator y el diámetro del tubo en uso. Continuar controlando el blastómero (s) como el nitrógeno llena la cámara, ajustar el plano focal cuando sea necesario. </li><li> En este punto, imagen de lapso de tiempo ha comenzado. Formación de imágenes se llevó a cabo durante tanto tiempo como sea necesario para capturar fenómeno de interés, en este caso detención profase y acoplamiento de cromosomas a la membrana nuclear interna. Las imágenes son tomadas una vez cada 10 seg durante no más de 30 min. Las frecuencias de captura de imagen y ajustes tales como la ganancia y la potencia de láser debe ser ajustado como sea necesario. </li><li> El tiempo en el que los embriones de responder al medio ambiente anóxico puede variar dependiendo de la fase del ciclo celular después de la exposición anoxia. Típicamente anoxia inducida detención profase se produce dentro de 20-30 min de flujo de nitrógeno a partir de gas. Hemos observado anoxia inducida ciclo celular ocupación detenciónrencia en &lt;20 min. Las imágenes se pueden obtener durante este marco de tiempo, o imágenes específicas se pueden aislar después de una serie de lapso de tiempo. Una opción adicional es el uso de microscopía de vídeo para visualizar las estructuras subcelulares en los embriones. </li><li> Para documentar la recuperación del estado anoxia inducida por el detenido, el gas se apaga y la cámara se le permitió regresar a normoxia por difusión durante la grabación de una serie de lapso de tiempo. La reanudación de la progresión del ciclo celular y el desacoplamiento de los cromosomas se produce normalmente dentro de 5-20 minutos de convertir el gas nitrógeno apagado. </li><li> Tenga en cuenta que en experimentos separados un indicador anoxia, resazurina, se colocó en la micro-cámara de flujo a través, se determinó que la cámara se vuelve anóxico en promedio de aproximadamente 19 min después de flujo de gas nitrógeno se ha iniciado. </li><li> Las imágenes y los vídeos se procesan utilizando Imaris, J Imagen o Photoshop e importados en QuickTime para la visualización. </li></ol>

<ol>
	<li>Powell-Coffman, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hypoxia+signaling+and+resistance+in+C.+elegans.">Hypoxia signaling and resistance in C. elegans.</a> Trends Endocrinol. Metab. 21 (10), 435-440 (2010).</li><li>Padilla, P. A., Ladage, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Suspended+animation,+diapause+and+quiescence:+Arresting+the+cell+cycle+in+C.+elegans.">Suspended animation, diapause and quiescence: Arresting the cell cycle in C. elegans.</a> Cell Cycle. 11, (2012).</li><li>Hu, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dauer.">Dauer.</a> WormBook. , 1-19 (2007).</li><li>Zhou, K. I., Pincus, Z., Slack, F. 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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=.">.</a> The Nematode Caenorhabditis elegans. , 587-606 (1988).</li><li>Padilla, P. A., Nystul, T. G., Zager, R. A., Johnson, A. C., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dephosphorylation+of+Cell+Cycle-regulated+Proteins+Correlates+with+Anoxia-induced+Suspended+Animation+in+Caenorhabditis+elegans.">Dephosphorylation of Cell Cycle-regulated Proteins Correlates with Anoxia-induced Suspended Animation in Caenorhabditis elegans.</a> Mol. Biol Cell. 13, 1473-1483 (2002).</li><li>Hajeri, V. A., Trejo, J., Padilla, P. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+sub-nuclear+changes+in+Caenorhabditis+elegans+embryos+exposed+to+brief,+intermediate+and+long-term+anoxia+to+analyze+anoxia-induced+cell+cycle+arrest.">Characterization of sub-nuclear changes in Caenorhabditis elegans embryos exposed to brief, intermediate and long-term anoxia to analyze anoxia-induced cell cycle arrest.</a> BMC Cell Biol. 6, 1471-2121 (2005).</li><li>Padilla, P. A., Goy, J. M., Hajeri, P. A., Padilla, <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Anoxia. , (2012).</li><li>Hajeri, V. A., Little, B. A., Ladage, M. L., Padilla, P. 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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=Suspended+animation+in+C.+elegans+requires+the+spindle+checkpoint.">Suspended animation in C. elegans requires the spindle checkpoint.</a> Science. 302, 1038-1041 (2003).</li><li>Margalit, A., Vlcek, S., Gruenbaum, Y., Foisner, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breaking+and+making+of+the+nuclear+envelope.">Breaking and making of the nuclear envelope.</a> J. Cell Biochem. 95, 454-465 (2005).</li><li>Maddox, A. S., Maddox, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-resolution+imaging+of+cellular+processes+in+Caenorhabditis+elegans.">High-resolution imaging of cellular processes in Caenorhabditis elegans.</a> Methods Cell Biol. 107, 1-34 (2012).</li><li>Miller, D. L., Roth, M. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=C.+elegans+are+protected+from+lethal+hypoxia+by+an+embryonic+diapause.">C. elegans are protected from lethal hypoxia by an embryonic diapause.</a> Curr. Biol. 19, 1233-1237 (2009).</li></ol>

No hay conflictos de interés declarado.

La privación de oxígeno y la animación suspendida Aunque la exposición a la privación de oxígeno severa puede ser fatal para algunos organismos, algunos organismos son capaces de sobrevivir a la exposición a la anoxia. En el caso de C. elegans, la supervivencia de la exposición anoxia depende de la etapa de desarrollo y respuesta a la anoxia es la entrada en un estado de animación suspendida reversible en el que se detuvo a varios procesos biológicos observables. Los procesos...

<table border="1"><tbody><tr><td> Reactivos / Equipo </td><td></td><td></td><td> Composición </td></tr><tr><td> Solución de hipoclorito </td><td></td><td></td><td> 0,7 g de KOH, 12 ml de NaOCl al 5%, llevar a 50 ml con ddH 2 0 </td></tr><tr><td> M9 buffer de </td><td></td><td></td><td> 3 g de KH 2 PO 4, 11 g de Na 2 HPO 4, 5 g de NaCl, 1 ml (1 M) MgSO 4 por 1 L ddH 2 O </td></tr><tr><td> Portaobjetos de vidrio </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; x1.0 mm </td></tr><tr><td> Ronda micro cubreobjetos </td><td> Microscopía Electrónica de Ciencias </td><td> 72223-01 </td><td> Diámetro 25 mm </td></tr><tr><td> Halocarbono 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Anestésico </td><td></td><td></td><td> 0,5% tricaine, 0,05% tetramisol </td></tr><tr><td> Leiden Cerrado perfusión Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> UHP Nitrógeno </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N 2 &lt;2 ppm de O 2 </td></tr><tr><td> Los tubos de plástico </td><td> VWR </td><td> 89068-468 </td><td> 0,062 &quot;ID x 0.125&quot; OD </td></tr><tr><td> Microscopio Confocal Spinning disco </td><td> McBain Sistemas </td><td></td><td> Zeiss invertido microscopio óptico del sistema, iluminación epifluorescente, CSU-10 Yokogawa escáner confocal, Hamamatsu multiplicador de electrones CCD cámara. </td></tr></tbody></table>

use of time lapse microscopy to visualize anoxia-induced suspended animation in c. elegans embryos

Caenorhabdits elegans se ha usado extensivamente en el estudio de la resistencia a la tensión, que se ve facilitado por la transparencia de las fases adultas y el embrión así como por la disponibilidad de mutantes genéticos y cepas transgénicas que expresan una miríada de 1-4 proteínas de fusión. Además, los procesos dinámicos tales como la división celular se puede ver con la etiqueta fluorescente proteínas indicadoras. El estudio de la mitosis se puede facilitar mediante el uso de experimentos con lapso de tiempo en los diferentes sistemas, incluyendo organismos intactos, por lo que la temprana C. embrión elegans es muy adecuado para este estudio. Aquí se presenta una técnica mediante la cual la formación de imágenes in vivo de estructuras subcelulares en respuesta a anóxica (99,999% N 2; &lt;2 ppm O 2) el estrés es posible con un flujo de gas a través de la configuración sencilla en un microscopio de alta potencia. Una cámara de microincubation se utiliza en conjunción con el flujo de gas de nitrógeno a través de un microscopio y disco giratorio confocalpara crear un ambiente controlado en el que los animales pueden obtener imágenes in vivo. Uso de GFP-etiquetados tubulina gamma y la histona, la dinámica y la detención de la división celular puede ser controlada antes, durante y después de la exposición a un ambiente privado de oxígeno. Los resultados de esta técnica son de alta resolución, videos detalladas y las imágenes de las estructuras celulares dentro de blastómeros de embriones expuestos a privación de oxígeno.

C. elegans embriones expuestos a privación de oxígeno grave (anoxia) son capaces de sobrevivir al detener procesos biológicos, incluyendo el desarrollo y la división celular 7. La detención inducida por anoxia de la división celular se puede monitorizar, en un nivel sub-celular, mediante el uso de una cámara de microincubation en conjunción con flujo de nitrógeno y gas a través de un microscopio confocal de disco giratorio para crear un ambiente controlado en el que los animales pueden obte...

Watch this Scientific Journal Video about Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos at JoVE.com

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

Descrito aquí es un<em&gt; In vivo</em&gt; Técnica de imagen sub-estructuras celulares en animales expuestos a la anoxia usando un flujo de gas a través de la cámara de microincubation en conjunción con un microscopio confocal de disco giratorio. Este método es sencillo y lo suficientemente flexible como para adaptarse a una variedad de parámetros experimentales y los sistemas del modelo.

El uso de microscopía lapso de tiempo para Visualizar anoxia inducida animación suspendida en<em&gt; C. elegans</em&gt; Los embriones

Caenorhabdits elegans has been used extensively in the study of stress resistance, which is facilitated by the transparency of the adult and embryo stages ...

use-time-lapse-microscopy-to-visualize-anoxia-induced-suspended

Research

JoVE Journal

Biology

Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

11.5K vistas.  University of North Texas. Descrito aquí es un In vivo Técnica de imagen sub-estructuras celulares en animales expuestos a la anoxia usando un flujo de gas a través de la cámara de microincubation en conjunción con un microscopio confocal de disco giratorio. Este método es sencillo y lo suficientemente flexible como para adaptarse a una variedad de parámetros experimentales y los sistemas del modelo.

Video: Use of Time Lapse Microscopy to Visualize Anoxia-induced Suspended Animation in C. elegans Embryos

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.  ...

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 ...

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using fluorescent-labeled reporter proteins or differential interference contrast (DIC) microscopy, allows for the examination of the temporal progression of these dynamic events which is otherwise inferred from analysis of fixed samples1,2. Moreover, the study of the developmental regulations of cellular processes necessitates conducting time-lapse experiments on an intact organism during development. The Caenorhabiditis elegans embryo is light-transparent and has a rapid, invariant developmental program with a known cell lineage3, thus providing an ideal experiment model for studying questions in cell biology4,5and development6-9. C. elegans is amendable to genetic manipulation by forward genetics (based on random mutagenesis10,11) and reverse genetics to target specific genes (based on RNAi-mediated interference and targeted mutagenesis12-15). In addition, transgenic animals can be readily created to express fluorescently tagged proteins or reporters16,17. These traits combine to make it easy to identify the genetic pathways regulating fundamental cellular and developmental processes in vivo18-21. In this protocol we present methods for live imaging of C. elegans embryos using DIC optics or GFP fluorescence on a compound epifluorescent microscope. We demonstrate the ease with which readily available microscopes, typically used for fixed sample imaging, can also be applied for time-lapse analysis using open-source software to automate the imaging process.

Imaging C. elegans Embryos using an Epifluorescent Microscope and Open Source Software

The C. elegans embryo is a powerful system for studying cell biology and development. We present a protocol for live imaging of C. elegans embryos utilizing DIC optics or fluorescence using readily available epifluorescent microscopes and open-source software.

Imágenes C. elegans Los embriones utilizando un microscopio de epifluorescencia y software de código abierto

Cellular processes, such as chromosome assembly, segregation and cytokinesis,are inherently dynamic. Time-lapse imaging of living cells, using ...

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, messages are carried by neurotransmitters released from presynaptic terminals and received by receptors on postsynaptic cells. Our previous research in Caenorhabditis elegans has shown that VSM-1 negatively regulates exocytosis. Additionally, analysis of synapses in vsm-1 mutants showed that animals lacking a fully functional VSM-1 have increased synaptic connectivity. Based on these preliminary findings, we hypothesized that C. elegans VSM-1 may play a crucial role in synaptogenesis. To test this hypothesis, double-labeled microarray analysis was performed, and gene expression profiles were determined. First, total RNA was isolated, reversely transcribed to cDNA, and hybridized to the DNA microarrays. Then, in-silico analysis of fluorescent probe hybridization revealed significant induction of many genes coding for members of the major sperm protein family (MSP) in mutants with enhanced synaptogenesis. MSPs are the major component of sperm in C. elegans and appear to signal nematode oocyte maturation and ovulation . In fruit flies, Chai and colleagues 1 demonstrated that MSP-like molecules regulate presynaptic bouton number and size at the neuromuscular junction. Moreover, analysis performed by Tsuda and coworkers 2 suggested that MSPs may act as ligands for Eph receptors and trigger receptor tyrosine kinase signaling cascades. Lastly, real time PCR analysis corroborated that the gene coding for MSP-32 is induced in vsm-1(ok1468) mutants. Taken together, research performed by our laboratory has shown that vsm-1 mutants have a significant increase in synaptic density, which could be mediated by MSP-32 signaling.

Determining Genetic Expression Profiles in C. elegans Using Microarray and Real-time PCR

Microarray analysis was conducted to determine genetic expression profiles in C. elegans, and real-time PCR was used to validate and quantify microarray data.

La determinación de perfiles genéticos de expresión en C. elegans Utilización de microarrays y PCR en tiempo real

Synapses are composed of a presynaptic active zone in the signaling cell and a postsynaptic terminal in the target cell. In the case of chemical synapses, ...

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 ...

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated tools and resources for studies in this system are facilitating continued discovery of genes with more subtle phenotypes or roles. 
Here we present a generalized protocol we adapted for identifying C. elegans genes with postembryonic phenotypes of interest using RNAi2. This procedure is easily modified to assay the phenotype of choice, whether by light or fluorescence optics on a dissecting or compound microscope. This screening protocol capitalizes on the physical assets of the organism and molecular tools the C. elegans research community has produced. As an example, we demonstrate the use of an integrated transgene that expresses a fluorescent product in an RNAi screen to identify genes required for the normal localization of this product in late stage larvae and adults. First, we used a commercially available genomic RNAi library with full-length cDNA inserts. This library facilitates the rapid identification of multiple candidates by RNAi reduction of the candidate gene product. Second, we generated an integrated transgene that expresses our fluorecently tagged protein of interest in an RNAi-sensitive background. Third, by exposing hatched animals to RNAi, this screen permits identification of gene products that have a vital embryonic role that would otherwise mask a post-embryonic role in regulating the protein of interest. Lastly, this screen uses a compound microscope equipped for single cell resolution.

RNAi Screening to Identify Postembryonic Phenotypes in C. elegans

We describe a sensitized method to identify postembryonic regulators of protein expression and localization in C. elegans using an RNAi-based genomic screen and an integrated transgene that expresses a functional, fluorescently tagged protein.

RNAi de detección para identificar fenotipos postembrionarios en C. elegans</em

C. elegans has proven to be a valuable model system for the discovery and functional characterization of many genes and gene pathways1. More sophisticated ...

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 ...

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

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

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, ...

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age are significant public health burdens. We currently understand little about the genetic regulation of muscle health with disease or age. The nematode C. elegans is an established model for understanding the genomic regulation of biological processes of interest. This worm&#8217;s body wall muscles display a large degree of homology with the muscles of higher metazoan species. Since C. elegans is a transparent organism, the localization of GFP to mitochondria and sarcomeres allows visualization of these structures in vivo. Similarly, feeding animals cationic dyes, which accumulate based on the existence of a mitochondrial membrane potential, allows the assessment of mitochondrial function in vivo. These methods, as well as assessment of muscle protein homeostasis, are combined with assessment of whole animal muscle function, in the form of movement assays, to allow correlation of sub-cellular defects with functional measures of muscle performance. Thus, C. elegans provides a powerful platform with which to assess the impact of mutations, gene knockdown, and/or chemical compounds upon muscle structure and function. Lastly, as GFP, cationic dyes, and movement assays are assessed non-invasively, prospective studies of muscle structure and function can be conducted across the whole life course and this at present cannot be easily investigated in vivo in any other organism.

Methods to Assess Subcellular Compartments of Muscle in C. elegans

Skeletal muscle is essential for locomotion and is the bodies&#8217; main protein store. Muscle health measurements within C. elegans are described. Prospective changes to muscle structure and function are assessed using localized GFP and cationic dyes.

Métodos para evaluar subcelulares compartimentos de músculo en<em&gt; C. elegans</em

Muscle is a dynamic tissue that responds to changes in nutrition, exercise, and disease state. The loss of muscle mass and function with disease and age ...