Nós gostaríamos de expressar o nosso apreço pela entrada e comentários dos membros do Laboratório de Padilla. Cepas de nematóides neste trabalho foram fornecidos pelo Centro de Genética Caenorhabditis, que é financiado pelo NIH National Center for Research Resources (NCRR). Nós reconhecer e agradecer o Dr. Lon Turnbull de assistência técnica com a microscopia confocal. Este trabalho foi financiado por uma bolsa da National Science Foundation (NSF-IOS, carreira) para PAP

1. Preparação da Amostra <ol><li> Produzir ou obter apropriado transgênico C. elegans estirpe de interesse, utilizando metodologias de transgênicos ou de Caenorhabditis Genética da Center (CGC) ou colega. Neste caso estamos usando tensão TH32 (torta-1 :: TBG-1 :: GFP; torta-1 :: GFP :: H2B) 5 para visualizar cromossomos e centrossomas como marcadores para a divisão celular. </li><li> Gerar uma população sincronizada usando um dos três métodos: 1) se desintegrar adultos grávidas em solução de hipoclorito e usar embriões restantes 6, 2) escolher adultos grávidas em uma placa sem sementes e deixá-los colocar embriões para 1-2 horas, ou 3) escolher L4 larvas de uma população mista e mudança para um novo prato semeado. </li><li> Nematóides crescer a 20 ° C a gravid idade adulta jovem, que terá cerca de 96 horas de incubação, 72 horas a partir de larvas L1 ou 24 horas pós muda L4. Embriões (2-20 células) no útero conter blastômeros grandes e núcleos que são ideais para imagalterações profundas sub-celular e sub-nuclear, respectivamente. Esta metodologia proporciona um meio para gerar uma população de adultos jovens que contêm um número abundante de embriões precoces. </li></ol> 2. Preparação de slides e anoxia Câmara Configurar <ol><li> Fazer uma câmara húmida para segurar lâminas preparadas pela colocação de uma toalha de papel húmido no interior de uma caixa de Petri grande ou bin coberto com uma tampa de tamanho suficiente. </li><li> Prepare fundido agarose a 2% em H2O desionizada aquecendo mistura em recipientes de vidro por meio de banho de ondas ou água. </li><li> Coloque gotas 1-3 de agarose quente sobre uma lâmina de vidro limpa microscópio. Colocar imediatamente uma segunda lâmina invertida perpendicularmente por cima da gota de agarose (s), pressione ligeiramente de modo que a almofada resultante será fina e livre de bolhas de ar. </li><li> Dê tempo para esfriar e, lentamente, desmontar as duas lâminas de microscópio, deixando o bloco de agarose intacto em um dos slides. É importante assegurar a almofada de agarose é suficientemente fino para não interferindore com a capacidade de focar o microscópio depois. Capacidade de visualizar de amostra é também dependente da distância óptica específica do objectivo de utilização, que pode variar entre os microscópios. </li><li> Use uma lâmina de barbear limpa para moldar almofada de agarose em um pequeno quadrado. Armazenar na câmara preparado úmido até que esteja pronto para uso. </li><li> Com cuidado, utilizando a mesma lâmina limpa, tomar a borda do bloco de agarose e transferi-lo a partir da corrediça para uma lamela 25 mm de diâmetro, micro, evitando a acumulação de bolhas de ar por baixo da almofada. A lamela rodada selecionado para usar apropriadamente se encaixa no microincubator. </li><li> Adicionar uma gota de anestésico (0,5% tricaina, tetramisole 0,05% em tampão M9) sobre a almofada de agarose para o uso como um anestésico. Escolha vermes 5-10 e coloque na gota de anestésico. Permitir 1-3 min para vermes para cessar o movimento. </li><li> O racional para a observação de embriões no útero de adultos, em vez de embriões dissecados, é o de minimizar o potencial de embrião desiccação e de movimento, devido ao fluxo de gás de azoto através do embrião. </li><li> Imediatamente adicionar uma gota de óleo halocarbono em cima dos vermes para prevenir os vermes de desidratação como o gás flui através da câmara. Uma vez que o óleo é viscoso halocarbono pode-se usar uma ponta de pipeta ou ponta de um verme escolher para recolher o óleo e cair sobre os vermes. </li><li> Uma vez que a lamela circular contendo as minhocas está pronta, as duas metades do Leiden fechado perfusão microincubator são separadas, e a lamela é então cuidadosamente colocada de pé sobre o anel inferior. Os dois lados da câmara são então fechada hermeticamente em conjunto. </li><li> A câmara é então ligada ao tanque de gás de azoto através do tubo de plástico flexível e colocado no local adequado na platina do microscópio (Figura 1C). Neste momento, o tubo e microincubator devem ser cuidadosamente verificados para identificar qualquer fuga ao assegurar uma fixação segura do tubo e dos tampões de porta ao longo do lado da câmara. Pode-se altão levemente espalhar uma pequena quantidade de água de sabão ao longo do tubo de olhar para as bolhas, as quais irão formar um vazamento, se estiver presente. </li></ol> 3. Microscopia em Anoxia <ol><li> Localize animais com menor ampliação, então, mudar-se para a ampliação adequada superior (64x para esta aplicação) para o tecido de imagem ou células de interesse, como blastômeros embrionárias ou ovócitos em adultos. Ligar o laser de 488 nm para visualizar o sinal GFP e localizar a proteína GFP na célula (s) de interesse. </li><li> Para visualizar a prisão na fase específica de parada do ciclo celular (por exemplo, prófase tarde) identificar um blastômero em um estágio antes da divisão celular (interfase tardio, ou prófase). Marcadores celulares, tais como a posição de centríolos, a iniciação da condensação cromossômica, localização cromossoma ea presença ou ausência do envelope nuclear permitirá identificar estágio do ciclo celular (Figura 2A). </li><li> A câmara é, então, submetido a perfusão com gás azoto (99,999%N 2; &lt;2 ppm de O 2). Neste caso, usamos uma pressão de 6 psi, mas a pressão pode necessitar de ser ajustado e optimizado em função do tamanho de cada microincubator e diâmetro do tubo a ser utilizado. Continuar a monitorizar o blastômero (s) como o azoto enche a câmara, ajustando o plano focal conforme necessário. </li><li> Neste ponto, o lapso de tempo de imagem tiver começado. Formação de imagens é realizada durante o tempo necessário para capturar fenómeno de interesse, neste caso prisão prófase e acoplagem de cromossomas para a membrana nuclear interna. Imagens são tomadas uma vez a cada 10 segundos por não mais do que 30 minutos. Freqüências de captura de imagem e definições, como ganho e potência do laser deve ser ajustada conforme necessário. </li><li> O tempo em que os embriões respondem ao ambiente anóxico pode variar dependendo da fase do ciclo celular após exposição anoxia. Tipicamente anóxia induzida prisão prófase ocorre dentro de 20-30 min a partir do fluxo de gás de azoto. Temos observado anóxia induzida por células ocu parada do ciclorring em &lt;20 min. As imagens podem ser obtidas durante este período de tempo, ou imagens específicas pode ser isolado depois de uma série de lapso de tempo. Uma opção adicional é a utilização de microscopia de vídeo para visualização de estruturas sub-celulares em embriões. </li><li> Para documentar a recuperação do estado de anóxia induzida preso, o gás está desligado ea câmara é permitida para retornar à normóxia por difusão durante a gravação de uma série de lapso de tempo. Reinício da progressão do ciclo celular e desencaixe de cromossomos normalmente ocorre dentro de 5-20 minutos de transformar o gás nitrogênio fora. </li><li> Note-se que em experiências separadas indicador anoxia, resazurina, foi colocado na microcâmara flow-through, determinou-se que a câmara anóxica torna-se, em média, de cerca de 19 min após o fluxo de gás azoto foi iniciada. </li><li> Imagens e vídeos são processadas utilizando Imaris, Imagem J ou Photoshop e importados para o QuickTime para exibição. </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. 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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. 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Privação de oxigênio e Suspended Animation Embora a exposição à privação de oxigênio grave pode ser fatal para alguns organismos, alguns organismos são capazes de sobreviver à exposição a anoxia. No caso de C. elegans, a sobrevivência de exposição anoxia depende estágio de desenvolvimento e de resposta à anoxia é a entrada para um estado de animação suspensa reversível no qual um certo número de processos biológicos observáveis ​​são presos. Processos de d...

<table border="1"><tbody><tr><td> Reagentes / Equipamentos </td><td></td><td></td><td> Composição </td></tr><tr><td> Solução de hipoclorito </td><td></td><td></td><td> 0,7 g de KOH, 12 ml de NaOCl a 5%, levar a 50 ml com DDQ 2 0 </td></tr><tr><td> Tampão M9 </td><td></td><td></td><td> 3 g 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> Lâminas de vidro de microscópio </td><td> Fisher Scientific </td><td> 12-550-343 </td><td> 3 &quot;x1&quot; mm X1.0 </td></tr><tr><td> Rodada micro lamínula </td><td> Electron Microscopy Sciences </td><td> 72223-01 </td><td> 25 mm de diâmetro </td></tr><tr><td> Halocarbon óleo 700 </td><td> Sigma </td><td> H8898-100ml </td><td></td></tr><tr><td> Anestésico </td><td></td><td></td><td> Tric 0,5%aine, tetramisole 0,05% </td></tr><tr><td> Leiden Perfusão Fechado Microincubator </td><td> Harvard Apparatus </td><td> 650041 </td><td></td></tr><tr><td> Nitrogênio UHP </td><td> Calgaz (Air Liquide) </td><td></td><td> &gt; 99,9990% N 2 &lt;2 ppm de O2 </td></tr><tr><td> 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> Spinning microscópio confocal de disco </td><td> McBain Sistemas </td><td></td><td> Invertido Zeiss microscópio óptico, o sistema de iluminação de epifluorescência, CSU-10 Yokogawa digitalizador confocal, Hamamatsu multiplicador de electrões de câmara CCD. </td></tr></tbody></table>

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

Caenorhabdits elegans tem sido amplamente utilizado no estudo da resistência a fadiga, o que é facilitado pela transparência das fases adultas e de embriões, assim como pela disponibilidade de mutantes genéticos e estirpes transgénicas que expressam uma variedade de proteínas de fusão 1-4. Além disso, os processos dinâmicos, tais como a divisão celular pode ser visualizado utilizando proteínas repórter fluorescente etiquetado. O estudo da mitose pode ser facilitado através da utilização de experiências de lapso de tempo de vários sistemas, incluindo organismos intactos, assim a C. precoce embrião elegans é bem adequado para este estudo. Apresentado aqui é uma técnica pela qual imagem in vivo de sub-estruturas celulares em resposta a anóxica (99,999% N 2; &lt;2 ppm O 2) o estresse é possível através de um fluxo de gás através de simples instalação em um microscópio de alta potência. A câmara microincubation é usado em conjunto com um fluxo de azoto e de gás através de um microscópio confocal de disco giratóriopara criar um ambiente controlado no qual os animais podem ser visualizados in vivo. Usando GFP-tagged tubulina gama e histona, a dinâmica ea prisão de divisão celular podem ser monitorados antes, durante e após a exposição a um ambiente privado de oxigênio. Os resultados desta técnica são de alta resolução, vídeos e imagens detalhadas das estruturas celulares dentro blastômeros de embriões expostos à privação de oxigênio.

Embriões C. elegans expostas à privação de oxigênio grave (anoxia) são capazes de sobreviver com a prisão de processos biológicos, incluindo desenvolvimento e divisão celular 7. A prisão anóxia induzida da divisão celular pode ser monitorizada, a um nível sub-celular, por meio de uma câmara de microincubation em conjunto com um fluxo de azoto e de gás através de um microscópio confocal de disco giratório para criar um ambiente controlado no qual os animais podem ser visualizados <em...

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 aqui é um<em&gt; In vivo</em&gt; A técnica de sub-imagem estruturas celulares em animais expostos a anoxia usando um fluxo de gás através da câmara microincubation em conjunto com um microscópio confocal de disco giratório. Este método é simples e suficientemente flexível para satisfazer uma variedade de parâmetros experimentais e sistemas modelo.

Uso de Microscopia Time Lapse para Visualize Anoxia induzida Suspended Animation em<em&gt; C. elegans</em&gt; Embriões

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

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Research

JoVE Journal

Biology

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

11.5K visualizações.  University of North Texas. Descrito aqui é um In vivo A técnica de sub-imagem estruturas celulares em animais expostos a anoxia usando um fluxo de gás através da câmara microincubation em conjunto com um microscópio confocal de disco giratório. Este método é simples e suficientemente flexível para satisfazer uma variedade de parâmetros experimentais e sistemas modelo.

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

Monitoring Actin Disassembly with Time-lapse Microscopy

Monitoramento Actina Desmontagem com a Time-lapse Microscopia

Biologia

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.

Dois fótons axotomia e lapso de tempo de imagem confocal em embriões de peixe-zebra ao 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.

Lapso de tempo de imagens de Mitose Depois de Transfection 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.

Imagem C. elegans Os embriões utilizando um microscópio de epifluorescência e Software de Código Aberto

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.

Determinar perfis de expressão genética em C. elegans Usando Microarray e PCR em tempo 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.

Lapso de tempo Microscopia de embriogênese inicial em 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 triagem para identificar fenótipos pós-embrionário em 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.

Análise Quantitativa de Migração aleatória de células usando microscopia de lapso de tempo 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.

Visualização de Desenvolvimento Craniofacial nas SOX10: Kaede Transgênicos Zebrafish Linha Usando Time-lapse microscopia 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 avaliar Subcelulares Compartimentos de músculo em<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 ...