Este trabalho foi financiado pelo NIH R01 NS042228 para MCH O FV1000 Olympus confocal foi adquirido com uma instrumentação compartilhada NIH conceder S10RR023717 ao Departamento de Zoologia UW (PI Bement Bill). Erica Andersen, Namrata Asuri e Mateus Barro contribuíram igualmente para este papel.

1. Montagem de slides injeção e slides de imagens Slides injecção: <ol><li> Prepare Sylgard elastômero de silicone de acordo com instruções do fabricante. </li><li> Use o Sylgard de vínculo de vidro para microscópio padrão três slides juntos pelo empilhamento de um slide em cima do cruzamento de outras duas lâminas, que são dispostas lado a lado nas bordas longas. O slide superior cria um ângulo recto, ou &quot;muro&quot; entre o topo eo fundo slides. </li><li> Deixe Sylgard set durante a noite. Estes slides são reutilizáveis. </li></ol> Slides de imagens: <ol><li> Nós usamos retângulos de aço inoxidável cortado para o mesmo tamanho de um slide padrão de vidro de microscópio (75mm x 25mm x 1mm) para a câmara se encaixa no suporte de slides no palco microscópio. Um buraco de 1,5 cm é cortada no centro do retângulo. </li><li> Apor a 22 mm 2 lamela para o retângulo de metal com Sylgard. Use apenas o suficiente para Sylgard completamente selar a lamela. A imagem em um microscópio invertido, embriões será montado neste lamínulas e fotografada do fundo. </li><li> Deixe Sylgard set durante a noite. Estes slides são reutilizáveis ​​e limpas com etanol a 70% entre os usos. </li></ol> 2. Preparação de construções de DNA Para expressar um transgene em um tecido específico maneira, o gene de interesse é clonado por trás de um promotor de células específicas. Nós usamos um elemento cis-reguladora do gene neurogenin1 zebrafish (-3.1ngn1) 2 ou o gene SOX10 (-4.9sox10) 3 a unidade expressão em neurônios sensoriais ou células da crista neural, respectivamente. Para visualizar a dinâmica da membrana celular e, expressamos citoplasmática ou membrana localizada fluoróforos. A distribuição de imagens F-actina, expressamos DNA que codifica o domínio de homologia calponin de utrophin fundido a mCherry (UtrCH-mCherry). O UtrCH-mCherry sonda biossensor permite a visualização de F-actina, sem interferir com a dinâmica da actina 1. Nós gerar esses DNA construções usando o Gateway Invitrogen recombinação estratégia baseada em quatro clonagem e vetores fornecidas no Tol2kit zebrafish 5. <ol><li> Gerar vetor de expressão (s) utilizando Multisite-Gateway Tecnologia e vetores da Tol2kit zebrafish 5. Plasmídeos contém a espinha dorsal da Tol2 pT2KXIGDin. </li><li> Purificar DNA plasmídeo com QIAfilter Plasmid Midi Prep kit (QIAGEN Inc.) e eluir em água estéril. DNA não precisa ser linearizada para a injecção. </li></ol> 3. Injeção de DNA construções <ol><li> Diluir o DNA para uma concentração final de 10-50 DNA mcg / ml em 0,2% de vermelho de fenol (para a visualização durante a injeção) em água. </li><li> Encher e calibrar agulha: Back-fill puxou uma agulha capilar com cerca de 1 mL de solução de DNA e inseri-lo no porta-agulha do aparelho de injeção (usamos um Picospritzer). Usando uma pinça fina, quebra a ponta fora a agulha de modo que uma abertura é de aproximadamente 10 mm de diâmetro. Medir o diâmetro da gota de óleo mineral com um micrômetro ocular. Ajustar a configuração duração pressão para obter uma gota de aproximadamente 1 volume nl. </li><li> Coletar uma célula embriões em estágio embrionário em meio E3 (NaCl 5mM, 0.17mm KCl, CaCl 2 0,33 mm, 0,33 mm MgSO 4 * 7H 2 O, pH 7,0). </li><li> Transferência de cerca de 30-60 embriões para o slide de injeção ea posição dos embriões no canto ângulo reto formado entre o topo eo fundo slides. Remover a maioria da E3 de modo que os embriões são mantidos para o slide pela tensão superficial. </li><li> Use um alfinete dobrado dissecando embriões para rodar assim que o celular é contra a parede do slide injeção. </li><li> Use um micromanipulador para guiar a agulha através do córion do embrião, através da gema e na única célula do embrião. 0,5-1 injetar DNA nl solução para dentro da célula. </li><li> Transferir os embriões em placas de Petri na E3 e incubar a 28,5 ° C. Se necessário, o desenvolvimento pode ser retardado por incubação de embriões em temperatura mais baixa. </li></ol> 4. Embriões de montagem para imagens <ol><li> Remover o córions dos embriões com uma pinça fina cerca de uma hora antes de embriões atingem a idade desejada para a imagem latente (cerca de 14-17 horas pós-fertilização para os nossos propósitos). </li><li> Selecionar embriões com células marcadas utilizando um microscópio de dissecação equipados com fluorescência. Usamos um objetivo 4X (aumento de 40 vezes) em um escopo AZ100 Nikon devido à sua combinação de distância de trabalho longa e alta ampliação. </li><li> Coloque um embrião em um tubo de microcentrífuga com cerca de 50 E3 l contendo 0,2% tricaina anestésico (concentração 10X). </li><li> Adicionar 500 mL 1% agarose de baixo ponto de fusão na E3 com 10 mM HEPES (mantida a 42 ° C), diluindo o tricaina para uma concentração final de 0,02%. </li><li> Transferir imediatamente o embrião em uma queda de agarose àlamela do slide de imagens usando uma pipeta de vidro grande furo. </li><li> Antes da agarose endurece, a posição do embrião para a região com células marcadas é voltada para baixo contra a lamela inferior (baixo dorsal para as células da crista neural e dorsolateral para baixo para os neurônios sensoriais espinhais). </li><li> Montagem de câmara de imagem: Revestir um lado de um anel de plástico (2 cm de diâmetro externo, 3 mm de altura) com graxa de silicone a vácuo e apor ao slide de imagens de metal. Brasão do outro lado do ringue com graxa. </li><li> Encher a câmara com E3 contendo 10 mM HEPES e tricaina 0,02%. Seal no topo da câmara com a aposição de 22 mm 2 lamela para a superfície superior do anel untada. </li></ol> 5. 4D imagem de comportamentos célula em Olympus IX81 FV1000 confocal com microscópio Os detalhes da aquisição de imagem vai depender das especificidades de seu sistema de microscópio. Aqui nós descrevemos o processo de aquisição de lapso de tempo para o FV1000 Olympus microscópio confocal. Ver JOVE protocolo anterior para aquisição de lapso de tempo com o sistema confocal Zeiss LSM 510 6. <ol><li> Coloque a câmara de imagens no suporte de slides sobre o estágio do microscópio e se concentrar no embrião através de um ou 10X 20X objetivo sob luz transmitida. </li><li> Mudar para um objetivo 60X (NA de 1.2 ou superior) e se concentrar em células marcadas com epifluorescência. Nós usar um óleo de imersão (UPLSAPO 60xO NA 1,35) ou água de imersão (UPLSAPO 60xW NA 1,20) lente. </li><li> No software FV (Ver 1.7c), clique em XY repetir para começar a varredura a laser. </li><li> Ajustar as configurações de aquisição e controle de aquisição de imagem (laser de saída, velocidade de digitalização, HV, ganho, e os níveis de offset) para otimizar o sinal, mantendo a potência do laser, no mínimo (25% ou menos) para evitar danos nos tecidos e reduzir a fotodegradação. </li><li> Para adquirir um Z-series, definir os limites superior e inferior da pilha z-, juntamente com o tamanho do passo. Verifique se o ícone de profundidade está engajada para que o ícone de varredura XY XY e Z tem destaque. Iniciar aquisição, clicando no ícone de varredura XYZ. </li><li> Para adquirir uma série de lapso de tempo, defina o intervalo de tempo e número de pontos no tempo sob o título TimeScan na janela Configurações de Aquisição. Clique no ícone de tempo na janela de Controle de Aquisição de Imagens para que o ícone de varredura XY tem XY (Z) e T destaque. Iniciar aquisição, clicando no XY (Z) de varredura ícone T. </li><li> Alternativamente, o TimeController pode ser usado para configurar um lapso de tempo. Esta programação é útil ao gerar grandes conjuntos de dados, já que não exceda a alocação de memória. Em dispositivo, abrir a janela TimeController. Criar uma imagem nova tarefa. A janela de parâmetros de imagem será aberta. Clique no botão Status FV para atualizar as configurações de aquisição. Certifique-se de Salvar e Excluir são verificadas sob o título Terminate. Definir o nome do arquivo de saída e clique em OK para salvar as configurações e fechar a janela. Use a função Loop para definir o intervalo de tempo desejado eo número de pontos do tempo. Clique e Pronto Iniciar para começar a aquisição. O Pause e shift-Z funções pode ser usado para redirecionar enquanto imagem. </li></ol> 6. Resultados representante Os números e os filmes retratam exemplos de neurônios sensoriais e células da crista neural rotulados com membrana-alvo fluoróforos ou a sonda biosensor actina. <img src="/files/ftp_upload/1726/1726fig1.jpg" alt="Figura 1" /> Figura 1 imagens Confocal (z-projeções) de um (ngn1: GFP-caax) Tg. Embrião transgênico injetados com 25 pg ngn1: mCherry-caax DNA. Os rótulos mCherry-CAAX um neurônio vermelho (A) em um fundo com todos os neurônios Rohon Beard-sensorial marcadas de verde (B). C da imagem) Merged de ambos os canais verde e vermelho. Barra de escala = 50 mm. <img src="/files/ftp_upload/1726/1726fig2.jpg" alt="Figura 2" /> Figura 2 Confocal z-projeções de neurônios em embriões injetados com ~ 20 pg ngn1:.. MCherry-UtrCH DNA A) neurônio Rohon Beard-sensorial em 16,5 embrião hpf. F-actina sinal é forte em cones de crescimento (setas) e saliências ao longo dos axônios recém-formado. B) Neuron em 20 hpf embrião mostrando sinal de F-actina forte em cones de crescimento de axônio periférico ramificada (setas) e sinal fraco no centro mais maduro axônios (setas). Barra de escala = 50 mm.

<ol>
	<li>Burkel, B. M., von Dassow, G., Bement, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Versatile+fluorescent+probes+for+actin+filaments+based+on+the+actin-binding+domain+of+utrophin.">Versatile fluorescent probes for actin filaments based on the actin-binding domain of utrophin.</a> Cell motility and the cytoskeleton. 64, 822-832 (2007).</li><li>Blader, P., Plessy, C., Strahle, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+regulatory+elements+with+spatially+and+temporally+distinct+activities+control+neurogenin1+expression+in+primary+neurons+of+the+zebrafish+embryo.">Multiple regulatory elements with spatially and temporally distinct activities control neurogenin1 expression in primary neurons of the zebrafish embryo.</a> Mech Dev. 120, 211-218 (2003).</li><li>Wada, N., Javidan, Y., Nelson, S., Carney, T. J., Kelsh, R. N., Schilling, T. F. <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, 3977-3988 (2005).</li><li>Cheo, D. L., Titus, S. A., Byrd, D. R., Hartley, J. L., Temple, G. F., Brasch, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Concerted+assembly+and+cloning+of+multiple+DNA+segments+using+in+vitro+site-specific+recombination:+functional+analysis+of+multi-segment+expression+clones.">Concerted assembly and cloning of multiple DNA segments using in vitro site-specific recombination: functional analysis of multi-segment expression clones.</a> Genome Res. 14, 2111-2120 (2004).</li><li>Kwan, K. M., Fujimoto, E., Grabher, C., Mangum, B. D., Hardy, M. E., Campbell, D. S., Parant, J. M., Yost, H. J., Kanki, J. P., Chien, C. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Tol2kit:+a+multisite+gateway-based+construction+kit+for+Tol2+transposon+transgenesis+constructs.">The Tol2kit: a multisite gateway-based construction kit for Tol2 transposon transgenesis constructs.</a> Dev Dyn. 236, 3088-3099 (2007).</li><li>O'Brien, G. S., Rieger, S., Martin, S. M., Cavanaugh, A. M., Portera-Cailliau, C., Sagasti, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two-photon+axotomy+and+time-lapse+confocal+imaging+in+live+zebrafish+embryos.">Two-photon axotomy and time-lapse confocal imaging in live zebrafish embryos.</a> J Vis Exp. 24, (2009).</li><li>Berndt, J. D., Clay, M. R., Langenberg, T., Halloran, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rho-kinase+and+myosin+II+affect+dynamic+neural+crest+cell+behaviors+during+epithelial+to+mesenchymal+transition+in+vivo.">Rho-kinase and myosin II affect dynamic neural crest cell behaviors during epithelial to mesenchymal transition in vivo.</a> Dev Biol. 324, 236-244 (2008).</li></ol>

A concentração ótima de DNA injetado irá variar dependendo do tamanho e da força do promotor construir e deve ser determinada empiricamente. Injeção de DNA em excesso pode levar a embriões doentia com a morte celular extensa, enquanto que muito pouco irá resultar em uma proporção muito pequena de embriões injetados expressando o transgene. O nível de expressão de DNA se correlaciona com a força do sinal fluoróforo, que varia de célula para célula. Embora a triagem de embriões sob epifluorescência, ex...

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<td>Tricaine (Ethyl 3-aminobenzoate methanesulfonate)</td>
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<td>Sylgard Silicone Elastomer Kit</td>
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<td>Low melting point agarose</td>
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live imaging of cell motility and actin cytoskeleton of individual neurons and neural crest cells in zebrafish embryos

O peixe-zebra é um modelo ideal para comportamentos imagens de células durante o desenvolvimento in vivo. Embriões fertilizados peixe-zebra são externamente e, portanto, facilmente acessível em todas as fases de desenvolvimento. Além disso, sua claridade óptica permite imagens de alta resolução de celular e dinâmica molecular no ambiente natural do embrião intacto. Estamos usando uma abordagem imagens ao vivo para analisar comportamentos durante a migração de células da crista neural celular e no crescimento e orientação dos axônios dos neurônios. Imagens ao vivo é particularmente útil para a compreensão dos mecanismos que regulam os processos de motilidade celular. Para visualizar detalhes da motilidade celular, como a atividade protrusiva e dinâmica molecular, é vantajoso para rotular células individuais. Em peixes-zebra, a injeção de DNA plasmídeo produz um padrão de expressão transitória mosaico e oferece benefícios distintos sobre os métodos de rotulagem outras células. Por exemplo, linhagens transgênicas muitas vezes rótulo populações de células inteiras e, portanto, pode obscurecer a visualização das saliências finas (ou mudanças na distribuição molecular) em uma única célula. Além disso, a injeção de DNA na fase unicelular é menos invasivo e mais preciso do que injeções de corante em fases posteriores. Aqui nós descrevemos um método para a rotulagem de neurônios individuais em desenvolvimento ou células da crista neural e imagem o seu comportamento in vivo. Nós injetar DNA plasmídeo em uma célula de embriões palco, o que resulta em expressão do transgene mosaico. Os vetores contêm células específicas promotores que a expressão da unidade de um gene de interesse em um subconjunto de neurônios sensoriais ou células da crista neural. Nós fornecemos exemplos de células marcadas com GFP membrana alvo ou com uma sonda biosensor que permite a visualização de F-actina em células vivas 1. Erica Andersen, Namrata Asuri e Mateus Barro contribuíram igualmente para este trabalho.

Watch this Scientific Journal Video about Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos at JoVE.com

Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Este protocolo descreve imagens de neurônios individuais ou de células da crista neural em embriões vivos zebrafish. Este método é usado para examinar comportamentos e localização celular actina microscopia de fluorescência usando lapso de tempo confocal.

Imagens ao vivo da motilidade celular e citoesqueleto de actina de neurônios individuais e células da crista neural em embriões Zebrafish

The zebrafish is an ideal model for imaging cell behaviors during development in vivo. Zebrafish embryos are externally fertilized and thus easily ...

live-imaging-cell-motility-actin-cytoskeleton-individual-neurons

Research

JoVE Journal

Biology

13.3K visualizações.  University of Wisconsin-Madison. Este protocolo descreve imagens de neurônios individuais ou de células da crista neural em embriões vivos zebrafish. Este método é usado para examinar comportamentos e localização celular actina microscopia de fluorescência usando lapso de tempo confocal.

Vídeo: Live Imaging of Cell Motility and Actin Cytoskeleton of Individual Neurons and Neural Crest Cells in Zebrafish Embryos

Mouse Epidermal Neural Crest Stem Cell (EPI-NCSC) Cultures

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the physiological property to generate a wide array of differentiated cell types, including neurons, nerve supporting cells, smooth muscle cells, bone/cartilage cells and melanocytes. EPI-NCSC are easily accessible in the hairy skin and can be isolated as a highly pure population of stem cells. This video provides a detailed protocol for preparing mouse EPI-NCSC cultures from whisker follicles. The whisker pad of an adult mouse is removed, and whisker follicles dissected. The follicles are then cut longitudinally and subsequently transversely above and below the bulge region. The bulge is removed from the collagen capsule and placed in a culture plate. EPI-NCSC start to emigrate from the bulge explants 3 to 4 days later.

Mouse Epidermal Neural Crest Stem Cell EPI-NCSC Cultures

Here we show our method to isolate mouse epidermal neural crest stem cells (EPI-NCSC). Technique involves micro-dissecting whisker follicles, isolating the bulge and placeing it into tissue culture. EPI-NCSC start to emigrate from bulge explants onto the substratum within 3 - 4 days.

Rato Epidérmico Neural Stem Cell Crest (EPI-NCSC) Culturas

EPI-NCSC are remnants of the embryonic neural crest in an adult location, the bulge of hair follicles. They are multipotent stem cells that have the ...

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.  ...

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique advantage of FSM is its ability to capture the movement and turnover kinetics (assembly/disassembly) of the F-actin network within living cells. This technique is particularly useful in deriving quantitative measurements of F-actin dynamics when paired with computer vision software (qFSM).  We describe  the selection, microinjection and visualization of fluorescent actin probes in living cells. Importantly, similar procedures are applicable to visualizing other macomolecular assemblies. FSM has been demonstrated for microtubules, intermediate filaments, and adhesion complexes.  

Live Cell Imaging of F-actin Dynamics via Fluorescent Speckle Microscopy FSM

Selection, microinjection, and imaging of fluorescently-labeled F-actin via fluorescent speckle microscopy (FSM).

Imagens de células vivas de F-actina Dynamics via Microscopia fluorescente Speckle (FSM)

In this protocol we describe the use of Fluorescent Speckle Microscopy (FSM) to capture high-resolution images of actin dynamics in PtK1 cells. A unique ...

Two-Photon-Based Photoactivation in Live Zebrafish Embryos

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

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

Two-Photon-Based fotoativação no Live Embriões Zebrafish

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

Transplantation of GFP-expressing Blastomeres for Live Imaging of Retinal and Brain Development in Chimeric Zebrafish Embryos

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their environment. Chimeric embryos, which are composed of cells of different genetic background, are great tools to study the cell-cell interactions mediated by genes of interest. The embryonic transparency of zebrafish at early developmental stages permits direct visualization of the morphogenesis of tissues and organs at the cellular level. Here, we demonstrate a protocol to generate chimeric retinas and brains in zebrafish embryos and to perform live imaging of the donor cells. The protocol covers the preparation of transplantation needles, the transplantation of GFP-expressing donor blastomeres to GFP-negative hosts, and the examination of donor cell behavior under live confocal microscopy. With slight modifications, this protocol can also be used to study the embryonic development of other tissues and organs in zebrafish. The advantages of using GFP to label donor cells are also discussed. 

We demonstrate a protocol to generate chimeric zebrafish embryos for live imaging cellular behavior during embryogenesis.

Transplante de GFP-expressando Blastômeros for Imaging ao vivo de Desenvolvimento da retina e do cérebro em embriões Zebrafish Quimérico

Cells change extensively in their locations and property during embryogenesis. These changes are regulated by the interactions between the cells and their ...

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes in cell surface area and cell height, and where cells undergo mitosis and migrate. Furthermore, epithelial cells can also regulate morphogenetic movements in adjacent tissues1. A traditional method to study epithelial cells and tissues involve chemical fixation and histological methods to determine cell morphology or localization of particular proteins of interest. These approaches continue to be useful and provide "snapshots" of cell shapes and tissue architecture, however, much remains to be understood about how cells acquire specific shapes, how various proteins move or localize to specific positions, and what paths cells follow toward their final differentiated fate. High resolution live imaging complements traditional methods and also allows more direct investigation into the dynamic cellular processes involved in the formation, maintenance, and morphogenesis of multicellular epithelial sheets. Here we demonstrate experimental methods from the isolation of animal cap tissues from Xenopus laevis embryos to confocal imaging of epithelial cells and simple measurement approaches that together can augment molecular and cellular studies of epithelial morphogenesis.

Live-cell Imaging and Quantitative Analysis of Embryonic Epithelial Cells in Xenopus laevis

Xenopus embryonic epithelia are an ideal model system to study cell behaviors such as polarity development and shape change during epithelial morphogenesis. Traditional histology of fixed samples is increasingly being complemented by live-cell confocal imaging. Here we demonstrate methods to isolate frog tissues and visualize live epithelial cells and their cytoskeleton using live-cell confocal microscopy.

Em células vivas, Imagem e Análise Quantitativa de células embrionárias epiteliais em Xenopus laevis</em

Embryonic epithelial cells serve as an ideal model to study morphogenesis where multi-cellular tissues undergo changes in their geometry, such as changes ...

Study of the Actin Cytoskeleton in Live Endothelial Cells Expressing GFP-Actin

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial cells regulate permeability of the endothelium, and many studies have indicated the important contribution of the actin cytoskeleton to determining junctional integrity1-5. A cortical actin belt is thought to be important for the maintenance of stable junctions1, 2, 4, 5. In contrast, actin stress fibers are thought to generate centripetal tension within endothelial cells that weakens junctions2-5. Much of this theory has been based on studies in which endothelial cells are treated with inflammatory mediators known to increase endothelial permeability, and then fixing the cells and labeling F-actin for microscopic observation. However, these studies provide a very limited understanding of the role of the actin cytoskeleton because images of fixed cells provide only snapshots in time with no information about the dynamics of actin structures5. 
Live-cell imaging allows incorporation of the dynamic nature of the actin cytoskeleton into the studies of the mechanisms determining endothelial barrier integrity. A major advantage of this method is that the impact of various inflammatory stimuli on actin structures in endothelial cells can be assessed in the same set of living cells before and after treatment, removing potential bias that may occur when observing fixed specimens. Human umbilical vein endothelial cells (HUVEC) are transfected with a GFP-&beta;-actin plasmid and grown to confluence on glass coverslips. Time-lapse images of GFP-actin in confluent HUVEC are captured before and after the addition of inflammatory mediators that elicit time-dependent changes in endothelial barrier integrity. These studies enable visual observation of the fluid sequence of changes in the actin cytoskeleton that contribute to endothelial barrier disruption and restoration. 
Our results consistently show local, actin-rich lamellipodia formation and turnover in endothelial cells. The formation and movement of actin stress fibers can also be observed. An analysis of the frequency of formation and turnover of the local lamellipodia, before and after treatment with inflammatory stimuli can be documented by kymograph analyses. These studies provide important information on the dynamic nature of the actin cytoskeleton in endothelial cells that can used to discover previously unidentified molecular mechanisms important for the maintenance of endothelial barrier integrity.

Microscopic imaging of live endothelial cells expressing GFP-actin allows characterization of dynamic changes in cytoskeletal structures. Unlike techniques that use fixed specimens, this method provides a detailed assessment of temporal changes in the actin cytoskeleton in the same cells before, during, and after various physical, pharmacological, or inflammatory stimuli.

Estudo do citoesqueleto de actina em Live Células Endoteliais Expressando GFP-Actina

The microvascular endothelium plays an important role as a selectively permeable barrier to fluids and solutes. The adhesive junctions between endothelial ...

4D Imaging of Protein Aggregation in Live Cells

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental conditions and an intracellular environment that is tightly packed, sticky, and hazardous to protein stability1. The ability to dynamically balance protein production, folding and degradation demands highly-specialized quality control machinery, whose absolute necessity is observed best when it malfunctions. Diseases such as ALS, Alzheimer's, Parkinson's, and certain forms of Cystic Fibrosis have a direct link to protein folding quality control components2, and therefore future therapeutic development requires a basic understanding of underlying processes. Our experimental challenge is to understand how cells integrate damage signals and mount responses that are tailored to diverse circumstances.
The primary reason why protein misfolding represents an existential threat to the cell is the propensity of incorrectly folded proteins to aggregate, thus causing a global perturbation of the crowded and delicate intracellular folding environment1. The folding health, or "proteostasis," of the cellular proteome is maintained, even under the duress of aging, stress and oxidative damage, by the coordinated action of different mechanistic units in an elaborate quality control system3,4. A specialized machinery of molecular chaperones can bind non-native polypeptides and promote their folding into the native state1, target them for degradation by the ubiquitin-proteasome system5, or direct them to protective aggregation inclusions6-9.
In eukaryotes, the cytosolic aggregation quality control load is partitioned between two compartments8-10: the juxtanuclear quality control compartment (JUNQ) and the insoluble protein deposit (IPOD) (Figure 1 - model). Proteins that are ubiquitinated by the protein folding quality control machinery are delivered to the JUNQ, where they are processed for degradation by the proteasome. Misfolded proteins that are not ubiquitinated are diverted to the IPOD, where they are actively aggregated in a protective compartment.
Up until this point, the methodological paradigm of live-cell fluorescence microscopy has largely been to label proteins and track their locations in the cell at specific time-points and usually in two dimensions. As new technologies have begun to grant experimenters unprecedented access to the submicron scale in living cells, the dynamic architecture of the cytosol has come into view as a challenging new frontier for experimental characterization. We present a method for rapidly monitoring the 3D spatial distributions of multiple fluorescently labeled proteins in the yeast cytosol over time. 3D timelapse (4D imaging) is not merely a technical challenge; rather, it also facilitates a dramatic shift in the conceptual framework used to analyze cellular structure.
We utilize a cytosolic folding sensor protein in live yeast to visualize distinct fates for misfolded proteins in cellular aggregation quality control, using rapid 4D fluorescent imaging. The temperature sensitive mutant of the Ubc9 protein10-12 (Ubc9ts) is extremely effective both as a sensor of cellular proteostasis, and a physiological model for tracking aggregation quality control. As with most ts proteins, Ubc9ts is fully folded and functional at permissive temperatures due to active cellular chaperones. Above 30 &deg;C, or when the cell faces misfolding stress, Ubc9ts misfolds and follows the fate of a native globular protein that has been misfolded due to mutation, heat denaturation, or oxidative damage. By fusing it to GFP or other fluorophores, it can be tracked in 3D as it forms Stress Foci, or is directed to JUNQ or IPOD.

Cellular viability depends on timely and efficient management of protein misfolding. Here we describe a method for visualizing the different potential fates of a misfolded protein: refolding, degradation, or sequestration in inclusions. We demonstrate the use of a folding sensor, Ubc9ts, for monitoring proteostasis and aggregation quality control in live cells using 4D microscopy.

Formação de imagens 4D de agregação de proteínas em células vivas

One of the key tasks of any living cell is maintaining the proper folding of newly synthesized proteins in the face of ever-changing environmental ...

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

The proper elimination of unwanted or aberrant cells through apoptosis and subsequent phagocytosis (apoptotic cell clearance) is crucial for normal development in all metazoan organisms. Apoptotic cell clearance is a highly dynamic process intimately associated with cell death; unengulfed apoptotic cells are barely seen in vivo under normal conditions. In order to understand the different steps of apoptotic cell clearance and to compare 'professional' phagocytes - macrophages and dendritic cells to 'non-professional' - tissue-resident neighboring cells, in vivo live imaging of the process is extremely valuable. Here we describe a protocol for studying apoptotic cell clearance in live Drosophila embryos. To follow the dynamics of different steps in phagocytosis we use specific markers for apoptotic cells and phagocytes. In addition, we can monitor two phagocyte systems in parallel: 'professional' macrophages and 'semi-professional' glia in the developing central nervous system (CNS). The method described here employs the Drosophila embryo as an excellent model for real time studies of apoptotic cell clearance.

Live Imaging of Apoptotic Cell Clearance during Drosophila Embryogenesis

Here we describe an effective method for studying dynamics of apoptotic cell clearance in vivo. This method employs live Drosophila embryos as a powerful model for monitoring phagocytosis of apoptotic cells using specific labeling of apoptotic cells and phagocytes.

Imagens ao vivo de Apuramento celular por apoptose durante<em&gt; Drosophila</em&gt; Embriogênese

The  proper elimination of unwanted or aberrant cells through apoptosis and  subsequent phagocytosis (apoptotic cell clearance) is crucial for normal  ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Profiling Individual células estaminais embrionárias humanas por RT-PCR quantitativo

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...