Os autores agradecem Robert Kelsh por gentilmente compartilhar o reagente SOX10 promotor peixe-zebra.

1. De colheita de embriões para Fotoconversão <ol><li> Instituído pelo menos dez pares de SOX10: zebrafish transgênicos Kaede entre 5 e 6:00 da noite. </li><li> Na manhã seguinte, puxe divisores e recolher os ovos em torno de meio-dia. Transfira-os para placas de Petri e colocá-los em 28,5 ° C incubadora. </li><li> Por volta das 24 horas pós fertilização, limpe as placas de Petri, removendo os embriões mortos. Para verificar o estágio de desenvolvimento de embriões 13 usando microscopia de campo claro e qualquer microscópio de fluorescência com maior proteína verde fluorescente (EGFP) filtro. Kaede será visível através deste filtro. </li><li> Quando chegar a 60 embriões transferência hpf cinco embriões fluorescentes brilhantemente em um prato separado Petri e dechorionate-los, se necessário. </li></ol> 2. Montagem do embrião para Fotoconversão <ol><li> Montar o embrião usando um microscópio fluorescente normal. Montagem de embriões em microscópio confocal é extremamente desafiador. </li><li> Use estandeágua ard E3 ou E3 preparar fresco utilizando a seguinte receita: Adicione o seguinte por um litro de água destilada dH 2 O, adicione o seguinte para fazer 1x E3L: 0,292 g de 5,0 mM de NaCl 0,013 g 0.17mm KCl 0,044 g 0,33 mm de CaCl2 (dessecante, 96%, ACS reagente) 0,081 g 0,33 mM de MgSO4 (anihidro) Opcional: adicionar 200 mL de 0,05% de azul de metileno a 1X E3 como fungicida e mexa. Meio mantém à temperatura ambiente durante 1 semana. </li><li> Use tricaina padrão ou preparar tricaina usando seguinte receita: Para 5 L de 0,2% tricaina misturar os seguintes ingredientes: 45 ml de Tris-Cl (pH 9,5 1M) 5 LH H2O 10 g tricaina </li><li> Prepare &#39;suco filme&#39; (50 ml E3, solução tricaina 0,015%), pela adição de 3,75 ml de 0,2% para 46,25 ml tricaina E3. </li><li> Prepare agarose através da mistura de 46,25 ml de água E3 com 46,35 mg de baixo ponto de fusão (LMP) de agarose em um frasco de vidro de 50 ml e dissolver partículas sólidas por microondas. Em seguida, adicionar3,73 ml de 0,2% de agarose tricaina e colocar frasco de vidro com agarose em 50 ° C banho-maria até o uso. </li></ol> 3. Preparação e montagem de Embrião <ol><li> Anestesiar embriões com solução tricaina E3/0.015% feita no passo 1.1.2 e transferência de embriões para slides câmara. </li><li> Deixe-se envolver todo o excesso de E3 com lenço de papel ea colocou de agarose para embrião. </li><li> Posição embrião perfeitamente dorsal com dicas Microloader. Certifique-se de todo o embrião não flutua em cima da agarose, mas empurrar todo o corpo para baixo. Em seguida, despeje solução tricaina E3/0.015% em relação ao embrião agarose incorporado até câmara está totalmente preenchido com fluido. </li></ol> 4. Como ajustar as configurações de software em Microscópio Confocal <ol><li> Use 20X ar objetivo submergível (abertura numérica 0,75; pinhole tamanho 20) para focar o embrião. Em seguida, pressione o botão da luz L100 no microscópio e selecionar canais de software. Controls/A1settings abrir a ícones ver / aquisição e click o botão de configuração confocal. Em seguida clique em &quot;auto&quot; e selecione os canais seguintes antes de fechar a janela novamente: Ch1: DAPI canal de 403,4 nm (comprimentos de onda entre 350-410 nm pode ser usado) CH2: 488,0 nm CH3: 561,9 nm </li></ol> 5. Fotoconversão <ol><li> Desligue Ch1 e 3 e ligue Ch2 antes de clicar no botão remover interlock e digitalização. </li><li> Concentre-se em células de interesse para fotoconversão, que é o melhor feito por começando com 1frame/sec e alta tensão (HV) 200 e, em seguida, subir no frames / seg e diminuindo o HV em conformidade até chegar a 1/32 frames / seg e HV em torno 100-120, de acordo com o ruído de fundo. </li><li> Quando o embrião está em foco, olhar para a borda direita da tela, pegue a borda verde e torná-lo menor para que caiba a área de interesse para fotoconversão e clique direito com o mouse. Menor é melhor porque o fotoconversão é irreversível. </li><li> Vire frames / seg a 1 e HV para 200 e baterdigitalizar. Um zoom na imagem da área de fotoconversão é agora visível. </li><li> Photoconvert células ativando Ch1 (403,4 nm). Expor ao laser photoconverting em qualquer lugar 5-60 segundos, dependendo do tamanho da região até Kaede verde como visto através do canal de GFP é quase ausente. </li><li> Quando o sinal de Kaede verde é quase ausente, vire Ch1 fora. Vire Ch2 e 3 no. Em seguida, clique direito sobre a janela no campo área de digitalização A1 e clique em &#39;retorno ao tamanho original&#39; para verificar se as células pretendidas foram photoconverted. </li></ol> 6. Fazendo uma pilha Z <ol><li> Encontre o ícone &quot;captura Z-series &#39;na barra de menu e definir os limites superior e inferior do Z-stack antes de iniciá-lo. Definir LUTs. </li></ol> 7. Software-Configurações Time-lapse <ol><li> Vá para a caixa A1Simple GUI e marque seguintes definições: 1/32 frames / seg Tamanho 1024 média de 4 ou 8 X, dependendo do número de Z-pilhas que tem de ser feita em um circuito </li><li> Vá para:controles view / aquisição / aquisição seqüência ND e definir intervalo de tempo (8-30min), clique em contínua e clique Z-stack. Definir fronteiras como testado antes e começar filme. </li><li> Continue adicionando E3/0.015% tricaina solução câmara de slides durante todo o dia. À noite, encher câmara completamente. Isso vai durar até a manhã seguinte. </li><li> Na manhã seguinte, encher solução tricaina E3/0.015% e verifique se embrião ainda está vivo. A perda de fluorescência indica morte. Continuar recargas e verificações ao longo do dia. </li><li> Quando a estrutura de terminar a formação, ou o embrião tenha morrido, o filme terminar. </li></ol> 8. Processamento de pós-produção <ol><li> Clique &#39;show projeção intensidade máxima&#39; na barra de ferramentas, em seguida, clique direito sobre a imagem e criar um novo documento. O vídeo é agora visível na melhor qualidade possível. Vá em frente para eliminar quadros que não são necessários e ajustar LUTs. Finalmente salvar como. Avi. Este formato será bom para o Windows. Para download de software Mac to converter. mov ou. wmv. </li></ol>

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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breakthroughs+in+the+genetics+of+orofacial+clefting.">Breakthroughs in the genetics of orofacial clefting.</a> Trends Mol. Med. 17 (12), 725-733 (2011).</li><li>Kimmel, C. B., Miller, C. T., Moens, 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=Specification+and+morphogenesis+of+the+zebrafish+larval+head+skeleton.">Specification and morphogenesis of the zebrafish larval head skeleton.</a> Dev. Biol. 233 (2), 239-257 (2001).</li><li>Dougherty, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+Fate+Map+of+First+Pharyngeal+Arch+Structures+in+the+sox10:+kaede+Zebrafish+Transgenic+Model.">Embryonic Fate Map of First Pharyngeal Arch Structures in the sox10: kaede Zebrafish Transgenic Model.</a> J. Craniofac. Surg. 23 (5), 1333-1337 (2012).</li><li>Trainor, P. A., Krumlauf, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hox+genes,+neural+crest+cells+and+branchial+arch+patterning.">Hox genes, neural crest cells and branchial arch patterning.</a> Curr. Opin. Cell Biol. 13 (6), 698-705 (2001).</li><li>Schilling, T. F., Kimmel, 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=Segment+and+cell+type+lineage+restrictions+during+pharyngeal+arch+development+in+the+zebrafish+embryo.">Segment and cell type lineage restrictions during pharyngeal arch development in the zebrafish embryo.</a> Development. 120 (3), 483-494 (1994).</li><li>Swartz, M. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+a+palatogenic+gene+program+in+zebrafish.">Examination of a palatogenic gene program in zebrafish.</a> Dev. Dyn. 240 (9), 2204-2220 (2011).</li><li>McCollum, C. W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Developmental+toxicity+screening+in+zebrafish.+Birth+Defects+Res.">Developmental toxicity screening in zebrafish. Birth Defects Res.</a> C. Embryo Today. 93 (2), 67-114 (2011).</li><li>Wada, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hedgehog+signaling+is+required+for+cranial+neural+crest+morphogenesis+and+chondrogenesis+at+the+midline+in+the+zebrafish+skull.">Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.</a> Development. 132 (17), 3977-3988 (2005).</li><li>Eberhart, J. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Early+Hedgehog+signaling+from+neural+to+oral+epithelium+organizes+anterior+craniofacial+development.">Early Hedgehog signaling from neural to oral epithelium organizes anterior craniofacial development.</a> Development. 133 (6), 1069-1077 (2006).</li><li>Ando, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+optical+marker+based+on+the+UV-induced+green-to-red+photoconversion+of+a+fluorescent+protein.">An optical marker based on the UV-induced green-to-red photoconversion of a fluorescent protein.</a> Proc. Natl. Acad. Sci. U S A. 99 (20), 12651-12656 (2002).</li><li>Kimmel, C. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev. Dyn. 203 (3), 253-310 (1993).</li><li>Kawakami, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+zebrafish-secreted+matrix+protein+you/scube2+is+implicated+in+long-range+regulation+of+hedgehog+signaling.">The zebrafish-secreted matrix protein you/scube2 is implicated in long-range regulation of hedgehog signaling.</a> Curr. Biol. 15 (5), 480-488 (2005).</li><li>Lombardo, V. A., Sporbert, A., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+tracking+using+photoconvertible+proteins+during+zebrafish+development.">Cell tracking using photoconvertible proteins during zebrafish development.</a> J. Vis. Exp. (67), e4350 (2012).</li></ol>

Os autores declaram que não têm interesses financeiros concorrentes.

Aqui, um novo método para a visualização de desenvolvimento craniofacial no modelo do peixe-zebra é mostrado. Os SOX10: kaede linha de peixes-zebra transgénicos tem sido usado com sucesso para descrever o padrão de migração de CNCC em detalhe é usado como um organismo modelo 5. Estudos anteriores já haviam utilizado marcos brutas, como o olho às células-alvo e têm contado com injeção mRNA Kaede, ensaios fotoconversão ou enjaulado dextrano fluoresceína para fotocon...

Fendas orofaciais representam a deformidade craniofacial mais prevalente, com 1/700-1, 000 entregas afetadas 1. Rompimento de desenvolvimento craniofacial embrionário precoce pode levar à formação de fissura de lábio e palato (FL / P). Enquanto causas para fenda sindrômica foram amplamente demonstrado, as bases genéticas e epigenéticas de formas não sindrômicas de fissuras orofaciais ainda precisa ser descoberto 2-4. A fim de compreender a etiologia e patogénese destas malformações, é necessário para elucidar o desenvolvimento de estruturas craniofaciais numa base celular. Em todas as espécies de vertebrados células da crista neural cranianos (CNCC) migram do tubo neural dorsal para preencher os arcos da faringe, o que contribuirá para a formação das estruturas orofaciais. Rompimento de desenvolvimento da crista neural embrionária precoce pode levar à formação de malformações craniofaciais incluindo CL / P 5-7. Em anúnciodição de semelhanças estruturais entre peixe-zebra e desenvolvimento craniofacial mamíferos (CNCCs residem nas regiões homólogas), a rede reguladora do gene é altamente conservado. Também tem sido mostrado que CNCCs desenvolver da mesma maneira entre as espécies de peixe-zebra e amniote 8, fazendo com que o peixe-zebra de um organismo poderosa para o estudo da base de desenvolvimento e genética de CL / P. Tem muitas vantagens, incluindo o tamanho pequeno, rápido e ex-utero desenvolvimento embrionário, e as taxas de reprodução de altura. Além disso, o embrião é opticamente transparente, tornando-se passível de observação de eventos complexos de desenvolvimento sob o microscópio 9. É um modelo animal ideal para o estudo de migração e diferenciação de células da crista neural cranianos. Expandindo publicado anteriormente de trabalho de 8, 10, 11, o padrão migratório da CNCC foi descrito em detalhes usando as SOX10: Kaede modelo transgênico 5. Kaede é uma proteína de foto-conversível que turns de verde para vermelho após a ativação de fotos e faz com que seja possível traçar CNCCs precisão. Durante esta transformação da estrutura peptídica é clivada, sugerindo que a conversão é estável, o que significa que as células podem ser rastreados para o seu destino final 12. Linhas transgénicas marcadas com kaede sob o controlo transcricional de SOX10 mostrou que o paladar e a placa amniota etmóide de peixe-zebra são formados por fusão de homologamente saliências maxilares bilaterais (MXP) com a proeminência ITontonasal (FNP) e que a costura de fusão em forma de Y é análoga entre espécies. Entre outras aplicações, as SOX10: kaede modelo transgénico de peixe-zebra foi usado para gerar o vídeo de embriões de peixes-zebra em diferentes estágios de desenvolvimento para mostrar a formação de estruturas craniofaciais normais e anormais. Fotoconversão de grupos específicos de células faz com que seja possível acompanhar o seu desenvolvimento. Com este método de abordagem para criar imagens em tempo real de desenvolver craniofacestruturas ial em peixe-zebra é introduzida, tornando mais fácil para demonstrar visualmente esse processo complexo de desenvolvimento. Este protocolo destina-se a partilhar a experiência de gerar esses vídeos usando o desenvolvimento normal da placa etmoidal em SOX10: zebrafish transgênicos Kaede como exemplo. Este protocolo pode ainda ser aplicada a fazer vídeos de lapso de tempo de qualquer estrutura derivadas de células da crista neural cranial em peixe-zebra.

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

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

Vertebrate palatogenesis é um processo de desenvolvimento altamente coreografada e complexo, que envolve a migração de crista neural craniana (CNC) células, convergência e extensão de proeminências faciais e maturação do esqueleto craniofacial. Para estudar a contribuição da crista neural craniana para regiões específicas do paladar zebrafish um SOX10: Kaede linha zebrafish transgênicos foi gerada. SOX10 fornece restrição linhagem da proteína kaede repórter para a crista neural, tornando assim o processo de marcação de uma célula mais precisa do que a tradicional corante ou injecção de ARNm repórter. Kaede é uma proteína de foto-conversível que muda de verde para vermelho após a ativação da foto e faz com que seja possível seguir as células com precisão. Os SOX10: linha transgênica Kaede foi utilizada para realizar a análise de linhagem para delinear as populações de células CNC que dão origem ao maxilar contra elementos mandibulares e ilustram homologia de proeminências faciais para amniotas. Este protocolo descreve o passos para gerar um vídeo time-lapse ao vivo de um SOX10: embrião de peixe-zebra Kaede. Desenvolvimento da placa etmoidal servirá como um exemplo prático. Este protocolo pode ser aplicado para fazer uma gravação confocal de lapso de tempo de qualquer kaede ou semelhante da proteína repórter em peixes-zebra transgénicos photoconvertible. Além disso, ele pode ser usado para capturar não só o normal, mas também o desenvolvimento anormal de estruturas craniofaciais nos mutantes de peixe-zebra.

No SOX10: CNCCs linhagem transgênica Kaede, migratórias e pós migratórias são rotulados fluorescente verde. As células marcadas com CNCC Kaede verde fluorescente recapitular endógeno expressão de mRNA SOX10 5. Entre outras aplicações, este modelo animal foi usado para visualizar melhor o desenvolvimento de estruturas craniofaciais CNCC dependentes. O desenvolvimento normal das estruturas específicas e também o desenvolvimento patológico de malformações craniofaciais...

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

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

Visualização de dados experimental tornou-se um elemento-chave na apresentação de resultados para a comunidade científica. Geração de gravação de lapso de tempo ao vivo de embriões de crescimento contribui para uma melhor apresentação e compreensão dos processos de desenvolvimento complexos. Este protocolo é um guia para marcação celular passo-a-passo através de fotoconversão de proteína Kaede no peixe-zebra.

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, ...

visualization-craniofacial-development-sox10-kaede-transgenic

Research

JoVE Journal

Biology

12.7K visualizações.  Massachusetts General Hospital.  Visualização de dados experimental tornou-se um elemento-chave na apresentação de resultados para a comunidade científica. Geração de gravação de lapso de tempo ao vivo de embriões de crescimento contribui para uma melhor apresentação e compreensão dos processos de desenvolvimento complexos. Este protocolo é um guia para marcação celular passo-a-passo através de fotoconversão de proteína Kaede no peixe-zebra. 

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

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.  ...

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

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

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

Vivem de imagem do cérebro Zebrafish embrionárias por microscopia confocal

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

Time-lapse Imaging of Mitosis After siRNA Transfection

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

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

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 ...

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

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

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

Multicolor imagem em tempo-lapso de Transgênicos Zebrafish: Visualizando Células-Tronco da retina Ativado por Ablação célula-alvo Neuronal

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

Time-lapse Microscopy of Early Embryogenesis in Caenorhabditis elegans

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

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 ...

Electroporation of Craniofacial Mesenchyme

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

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

Eletroporação de Mesênquima Craniofacial

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

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

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

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

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 ...

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

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

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

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

Longo prazo, de alta resolução de imagem Lapse Confocal Tempo de<em&gt; Cotilédone Arabidopsis</em&gt; Epiderme durante a germinação

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

Analyzing Craniofacial Morphogenesis in Zebrafish Using 4D Confocal Microscopy

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

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

Analisando Craniofacial morfogênese em Zebrafish Usando 4D microscopia confocal

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