We thank O. Subach for providing the original H2B-PSmOrange plasmid and our fish facility team for fish care. We are grateful to the Nikon Imaging Center at the University of Heidelberg for access to microscopy equipment and analysis software. We acknowledge the support of the Core Facility Live Cell Imaging Mannheim at the CBTM (DFG INST 91027/10-1 FUGG). This work was supported by the Excellenzcluster CellNetworks, EcTop Spatio-temporal coordination of signaling processes (EcTop 2), University of Heidelberg to C.A.B. and the Medical Faculty Mannheim of the University Heidelberg and the DFG (FOR 1036/2, 298/3-1 and 298/6-1) to M.C.

1. H2B-PSmOrange mRNA transcrição in vitro e mRNA Purificação <ol><li> Linearizar o H2B-PSmOrange contendo pCS2 + plasmídeo utilizando a enzima de restrição NotI de acordo com as instruções do fabricante. Nota: Execute as seguintes etapas usando proteções adequadas, tais como luvas e bata de laboratório para evitar a contaminação e degradação do mRNA. </li><li> Purifica-se o ADN linearizado utilizando um kit de purificação ou de fenol-clorofórmio PCR métodos baseados acordo com os protocolos dos fabricantes. </li><li> Use 1 ug de ADN molde linearizados para transcrição sp6-ARNm de acordo com as instruções do fabricante. </li><li> Remover o DNA pela adição de 1,0 U de RNase livre ADNasel durante 10-20 min a 37 ° C. </li><li> Limpar o mRNA usando um RNA limpar kit de acordo com o protocolo do fabricante. </li><li> Para aumentar a pureza e concentração de ARNm, o ARNm de precipitar por adição de 6 ul de acetato de sódio (3,0 M, pH 5,2) e 150 ul de etanol a 96%. Incubar a mixed solução a -20 ° C durante pelo menos 30 minutos e até 24 h. </li><li> Recolhe-se o ARNm por fiação da solução a 18,407 xg durante 45 min a 4 ° C. Descartar o líquido e ressuspender o ARNm em 150 ul de etanol a 70%. Misturar e centrifugar a solução durante mais 15 min a 4 ° C. Descartar o líquido, secar o sedimento durante 5 minutos em gelo e ressuspender o ARNm em 20 ul de ARNase ultrapura água livre. </li><li> Avaliar a concentração e pureza do ARNm por medição fotométrica de uma diluição 1: 100 de ARNm (ARNm 1 ul em 99 ul ultrapura RNase isenta de água). </li><li> Para armazenagem a curto prazo, manter a solução de ARNm a -20 ° C. Para armazenamento a longo prazo, manter o ARNm a -80 ° C. </li></ol> 2. H2B-PSmOrange mRNA microinjeção em embriões Zebrafish <ol><li> Configurar o acasalamento pares na noite antes da injeção usando tg (FOXD3: GFP); tg (FLH: GFP) peixes transgénicos casal (ou qualquer outro peixe transgénico GFP). Deixar o peixe separada por colocação de um espaçadorentre eles para controlar o tempo de acasalamento. </li><li> Remova o espaçador na manhã de injecção e deixar o companheiro de peixe para 20 min. </li><li> Durante o acasalamento tempo, dilui-se o ARNm H2B-PSmOrange até à concentração final de 130 pg / NL (em RNase isenta de água) e a transferência de 6 ul de solução de injecção para um capilar de microinjecção utilizando uma ponta de microloader. Verifique o volume de injeção com uma lâmina de vidro calibrado. Ajuste a pressão de injeção para injetar 2 solução mRNA nl (260 pg). </li><li> Recolher os ovos em uma placa de petri de plástico esterilizado contendo Embryo 1x médio (E3). </li><li> Transferir 20 a 30 embriões de um prato de injecção, utilizando uma pipeta de pasteur de plástico. </li><li> Injectar 2 nl de ARNm contendo uma solução para a célula ou apenas abaixo da célula na gema no estádio de uma célula-11. </li><li> Transferência de embriões injetado em uma-placa de petri contendo meio 1x E3, retire os embriões que se desenvolveram normalmente não fertilizados ou não e cultivá-las a 28 ° C. Nota: A proteção Luz of os embriões não é necessário durante todo o experimento. </li><li> Elevar embriões de peixe-zebra em meio 1x E3 e adicionar PTU 0,2 mM (1-fenil-2 tioureia) após a gastrulação para inibir a pigmentação. Mudar o meio duas vezes por dia para minimizar o perigo de contaminações bacterianas. </li></ol> 3. Embryo Embedding <ol><li> Seleccione a GFP (verde) e PSmOrange (laranja / vermelho) embriões co-expressando usando um microscópio binocular equipado com uma lâmpada fluorescente e filtros de emissão adequados. Transferir os embriões positivos em uma placa de petri estéril contendo meio 1x E3. </li><li> Embriões Dechorionate sob um microscópio estereoscópico com a pinça 12. </li><li> Transferência de um embrião em um tubo de 1,5 ml contendo 1,0 ml de pré-aquecido 1,0% de agarose de baixa fusão (LMA) preparadas em água ultrapura, utilizando uma pipeta de corte de ponta (P200). Nota: Aquecer o LMA a 80 ° C e arrefecer durante 3-5 minutos à temperatura ambiente antes de incorporar o embrião. </li><li> Transferir o embrião em 150 ul LMA numacompartimentado lamela e ajustar a orientação do embrião, por exemplo, o lado dorsal para baixo na experiência descrita para o laser confocal de varredura microscópio invertido A1R + (ou qualquer outro microscópio adequado), utilizando uma ponta de plástico fino. Quando o LMA é polimerizado, encher a câmara com água de peixe contendo PTU 0,2 mM e 0,02% de acetato de 3-aminobenzoato metanossulfonato (tricaina) para a anestesia. </li><li> Antes de imagem da amostra, esperar 15 min para determinar o efeito de tricaina. A anestesia impede movimentos embrião, que pode ser monitorado usando um estereomicroscópio equipado com iluminação de campo claro. Nota: As câmaras podem ser reutilizado duas vezes. </li></ol> 4. PSmOrange fotoconversão <ol><li> Colocar a amostra sob o microscópio confocal e digitalizar a amostra para identificar a área para photoconvert usando 488 nm e 561 nm lasers para geração de imagens GFP e PSmOrange respectivamente. Nota: Use o confocal de varredura a laser do microscópio invertido A1R + no sta invertidoge TiEcontrolled pelo software de imagem de microscópio e equipado com 488 nm, 561 nm e 640 nm lasers para fotoconversão e imagem. No entanto, a aplicação, certamente, não está restringido a esta microscópio, enquanto as linhas de laser para a conversão e de imagem estão disponíveis. <ol><li> Coloque o objectivo de ar 20X no lugar (NA: 0,75; WD: 1,0 mm; FOV: 0,64 x 0,64 mm). </li><li> Use as seguintes configurações microscópio para detectar a proteína PSmOrange antes fotoconversão: 561 nm laser, 0,74 mW medido no plano de foco acima do objetivo. Nota: Isto corresponde a 40% neste sistema (de medida no plano do foco é a única forma de determinar a potência efectiva). Ajustar a potência de laser de acordo com as necessidades experimentais como a eficiência de injecções H2B-PSmOrange pode variar. </li><li> Adicionar fatores de zoom para destacar a área de interesse. ampliações reduzir o tempo de aquisição de imagem e, portanto, fototoxicidade. </li></ol></li><li> Adquirir uma z-stack que cubra a estruturas de interesse utilizando 488 nm e 561 nm lasers no modo sequencial (modo de linha 1-&gt; 4). Fixar o z-passo entre 1,0 e 2,0 mm. média linha ea frequência de scaneamento pode ser usado para otimizar a qualidade da imagem. </li><li> Digitalizar a amostra e selecione a região de interesse ferramenta (ROI) para realçar a área para photoconvert. Fixar o ROI como área de estimulação. Selecione o módulo Foto Activation / Branqueamento, ativar o laser de 488 nm, marcando a respectiva caixa de e definir o laser de 488 nm a 80% (potência do laser 1 mW medida no objetivo). Defina a velocidade de digitalização para 0,5 seg / Frame. </li><li> Abra o utilitário fotoconversão (ND Stimulation) e especifique as configurações fotoconversão. <ol><li> Clique no comando &quot;Adicionar&quot; para especificar o protocolo de fotoconversão. </li><li> Ajuste &quot;Fase&quot; 1 no menu &quot;Aquisição / Estimulação&quot; a &quot;Aquisição&quot; e indicar o número de imagens a serem adquiridos antes fotoconversão no menu &quot;Loops&quot;. </li><li> Ajuste &quot;Fase&quot; 2 para &quot;Stimulation &quot;e digite o número de eventos de estimulação a ser realizada. </li><li> Ajuste &quot;Fase&quot; 3 como descrito para &quot;Fase&quot; 1. Opcionalmente, insira uma espera &quot;Phase&quot; depois de &quot;Phase&quot; 2 digitando o tempo de espera antes da última rodada de aquisição de imagem. </li><li> Uma vez que todos os parâmetros são definidos, aplicar a definição estimulação e executar o fotoconversão. Nota: A potência do laser, a frequência de digitalização e número de iterações para cada rodada de fotoconversão pode variar e ser diferente de embrião para embrião. Uma configuração para começar com é mostrado na Tabela 1. </li></ol></li><li> Adquirir uma z-stack final usando 488 nm, 561 nm e 640 nm lasers de aplicar as configurações como acima. Definir o laser de 640 nm para visualizar a PSmOrange convertido utilizando alta potência do laser (até 4,5 mW). </li></ol> 5. Desmontar Embrião de LMA <ol><li> Remover o embrião contendo câmara do microscópio. Remova cuidadosamente o embrião do u agarosecantar fórceps. </li><li> Transferência do embrião para um novo plástico esterilizado placa de petri ou em um de 6 poços de placa esterilizada contendo 1x E3 e UTP 0,2 mM. Incubar o embrião a 28 ° C até à fase de desenvolvimento desejado. </li></ol> 6. Analisando o Fate of Photoconverted PSmOrange proteína células que expressam <ol><li> Re-inserir o embrião como descrito nos pontos 3.3 e 3.4. </li><li> Colocar a amostra sob o microscópio confocal e digitalizar a amostra para identificar as células photoconverted (640 nm) na estrutura transgênica GFP de interesse (488 nm). </li><li> Adquirir uma z-stack cobrindo as estruturas de interesse utilizando 488 nm, 561 nm e 640 nm lasers no modo sequencial (modo de linha 1-&gt; 4). Fixar o z-passo entre 1,0 e 2,0 mm. média linha ea frequência de scaneamento pode ser usado para otimizar a qualidade da imagem. </li><li> Use um dos seguintes métodos para identificar células, que GFP co-express e a proteína photoconverted. <ol><li> personalizado feito Imagem automática FijiJ 3 Macro <ol><li> Convolve a pilha original usando o plugin &quot;GaussianBlur&quot; (→ Processo → Filtros → GaussianBlur) e subtrair as pilhas lisas do original usando o plugin &quot;Calculadora de Imagens&quot; (→ Processo → Calculadora de Imagem). Usar este passo de visualizar as estruturas de interesse e reduzir o tempo de processamento para a análise. </li><li> Aplicar limites específicos para os canais verde e vermelho-distante, a fim de destacar as células photoconverted (→ Imagem → Ajuste → Threshold). Use o &quot;Analisar Particles&quot; ferramenta para detectar as áreas de limiar com um ambiente adequado (→ Analisar → Analisar Partículas). </li><li> Abra o plugin &quot;Calculadora Imagem&quot; e exibir os ROIs sobrepostos no canal verde e vermelho-distante em amarelo (→ Processo → Calculadora de Imagem). </li></ol></li><li> software de imagem de microscópio para avaliação de dados 3D <ol><li> Mostrar a pilha no3D utilizando a opção view show de volume (→ 3D Menu de visualização → Mostrar Volume View). Use a interface gráfica para selecionar os canais verde, vermelho e far-vermelhos, ajustar o brilho eo contraste e recortar a pilha 3D para destacar a área photoconverted (Figura 1). Nota: métodos similares para analisar os dados estão disponíveis na maior parte do software comum para análise de imagem. </li></ol></li></ol></li></ol>

<ol>
	<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. (4350), (2012).</li><li>Subach, O. 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=A+photoswitchable+orange-to-far-red+fluorescent+protein,+PSmOrange.">A photoswitchable orange-to-far-red fluorescent protein, PSmOrange.</a> Nature Methods. 8, 771-777 (2011).</li><li>Beretta, C. A., Dross, N., Bankhead, P., Carl, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+ventral+habenulae+of+zebrafish+develop+in+prosomere+2+dependent+on+Tcf7l2+function.">The ventral habenulae of zebrafish develop in prosomere 2 dependent on Tcf7l2 function.</a> Neural Development. 8, 19 (2013).</li><li>Ando, R., Hama, H., Yamamoto-Hino, M., Mizuno, H., Miyawaki, A. <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, 12651-12656 (2002).</li><li>Habuchi, S., Tsutsui, H., Kochaniak, A. B., Miyawaki, A., van Oijen, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mKikGR,+a+monomeric+photoswitchable+fluorescent+protein.">mKikGR, a monomeric photoswitchable fluorescent protein.</a> PLoS One. 3, e3944 (2008).</li><li>McKinney, S. A., Murphy, C. S., Hazelwood, K. L., Davidson, M. W., Looger, L. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+bright+and+photostable+photoconvertible+fluorescent+protein.">A bright and photostable photoconvertible fluorescent protein.</a> Nature Methods. 6, 131-133 (2009).</li><li>Chudakov, D. M., Lukyanov, S., Lukyanov, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tracking+intracellular+protein+movements+using+photoswitchable+fluorescent+proteins+PS-CFP2+and+Dendra2.">Tracking intracellular protein movements using photoswitchable fluorescent proteins PS-CFP2 and Dendra2.</a> Nature Protocols. 2, 2024-2032 (2007).</li><li>Subach, F. V., 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=Photoactivatable+mCherry+for+high-resolution+two-color+fluorescence+microscopy.">Photoactivatable mCherry for high-resolution two-color fluorescence microscopy.</a> Nature Methods. 6, 153-159 (2009).</li><li>Patterson, G. H., Lippincott-Schwartz, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+photoactivatable+GFP+for+selective+photolabeling+of+proteins+and+cells.">A photoactivatable GFP for selective photolabeling of proteins and cells.</a> Science. 297, 1873-1877 (2002).</li><li>Subach, F. V., Patterson, G. H., Renz, M., Lippincott-Schwartz, J., Verkhusha, V. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bright+monomeric+photoactivatable+red+fluorescent+protein+for+two-color+super-resolution+sptPALM+of+live+cells.">Bright monomeric photoactivatable red fluorescent protein for two-color super-resolution sptPALM of live cells.</a> J Am Chem Soc. 132, 6481-6491 (2010).</li><li>Rosen, J. N., Sweeney, M. F., Mably, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microinjection+of+zebrafish+embryos+to+analyze+gene+function.">Microinjection of zebrafish embryos to analyze gene function.</a> J Vis Exp. (25), e1115 (2009).</li><li>Concha, M. L., 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=Local+tissue+interactions+across+the+dorsal+midline+of+the+forebrain+establish+CNS+laterality.">Local tissue interactions across the dorsal midline of the forebrain establish CNS laterality.</a> Neuron. 39, 423-438 (2003).</li><li>Auer, T. O., Duroure, K., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CRISPR/Cas9-mediated+conversion+of+eGFP-+into+Gal4-transgenic+lines+in+zebrafish.">CRISPR/Cas9-mediated conversion of eGFP- into Gal4-transgenic lines in zebrafish.</a> Nature Protocols. 9, 2823-2840 (2014).</li><li>Auer, T. O., Duroure, K., De Cian, A., Concordet, J. P., Del Bene, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highly+efficient+CRISPR/Cas9-mediated+knock-in+in+zebrafish+by+homology-independent+DNA+repair.">Highly efficient CRISPR/Cas9-mediated knock-in in zebrafish by homology-independent DNA repair.</a> Genome Research. 24, 142-153 (2014).</li><li>Keller, P. J., Schmidt, A. D., Wittbrodt, J., Stelzer, E. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+zebrafish+early+embryonic+development+by+scanned+light+sheet+microscopy.">Reconstruction of zebrafish early embryonic development by scanned light sheet microscopy.</a> Science. 322, 1065-1069 (2008).</li></ol>

The authors have nothing to disclose.

embriões transgénicos portadores de repórteres fluorescentes têm ajudado fundamentalmente para entender o desenvolvimento embrionário. No entanto, existe ainda a necessidade essencial de promotores de facilitar a visualização específica de estruturas particulares. Na sua ausência, os pesquisadores dependem de técnicas como a fotoconversão de proteínas fluorescentes para saber mais sobre a origem eo desenvolvimento de sua estrutura de interesse. Esta por sua vez é um pré-requisito fundamental para identific...

Exponentially improving imaging techniques allow following developmental processes over time periods of up to about four consecutive days3. In zebrafish and many other animal model systems, specific cells, tissues, axonal or vascular structures are marked by transgenic green or sometimes red or yellow fluorescent proteins to facilitate visualization. However, in most transgenic lines the transgene is not specifically expressed in the cells of interest but also additional structures, which hinders the precise tracking of for instance single cells or groups of cells.
Fluorescent photoconvertible proteins are well suited for cell tracking during embryonic development. The prerequisites for the application of such proteins are a long-lived nature, a well-separated emission range upon conversion and bright fluorescence. Available photoconvertible proteins comprise those that change their emission range upon conversion such as Kaede4, KiGR5, mEos26, PS-CFP2 or Dendra27 and others which are only fluorescent when photoactivated (PAmCherry8, PAGFP9 or PATagRFP10). Their applications to track cells in existing FP-transgenic animals are however limited as they often involve a green fluorescent state or do not fulfill all of the above criteria. Only recently, Subach and colleagues reported the PSmOrange protein, which changes its emission from orange/red to far red upon photoconversion and was successfully applied in cells in culture and cultured cells injected into mice2.
To investigate the protein's suitability for cell tracking in a living embryo, we generated an expression construct for the microinjection of nuclear-tagged H2B-PSmOrange into zebrafish embryos. We find that the protein fulfills all prerequisites for successful cell tracking in GFP transgenic backgrounds during the first 4 (and possibly more) days of zebrafish embryonic development. During this time, most of the cell migratory events are completed in fish making the PSmOrange system an excellent addition to the zebrafish toolkit.

<table><tbody><tr><td>PCR Purification Kit</td><td>Qiagen</td><td>28104</td><td></td></tr><tr><td>mMESSAGE mMACHINE SP6 Transcription kit</td><td>Ambion</td><td>AM1340</td><td></td></tr><tr><td>RNeasy MiniElute Cleanup kit</td><td>Qiagen</td><td>74204</td><td></td></tr><tr><td>Plastic Pasteur</td><td>alpha laboratories</td><td>LW4000</td><td></td></tr><tr><td>Original H2B-PSmOrange Plasmid</td><td>Addgene</td><td>31920</td><td>The plasmid described in the paper is available in the Carl lab</td></tr><tr><td>FemtoJet Microinjector</td><td>Eppendorf</td><td>5247 000.013</td><td></td></tr><tr><td>Forceps (5 Inox)</td><td>NeoLab</td><td>2-1633</td><td></td></tr><tr><td>Lab-Tek II Chambered #1.5 German Coverglass System&amp;nbsp;</td><td>Nunc</td><td>155382</td><td></td></tr><tr><td>Nikon A1R+</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>Nikon PLAN Apo &amp;lambda; 20X air objective&amp;nbsp;</td><td>Nikon GmbH Germany</td><td>No Number</td><td></td></tr><tr><td>NIS Elements AR Software (v. 4.30.02)</td><td>Nikon GmbH Germany/Laboratory Imaging</td><td>No Number</td><td></td></tr></tbody></table>

tracking cells in gfp-transgenic zebrafish using the photoconvertible psmorange system

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing developmental processes as they happen in the living animal. Cells of interest can be labeled by using a tissue specific promoter to drive the expression of a fluorescent protein (FP) for the generation of transgenic lines. Using fluorescent photoconvertible proteins for this purpose additionally allows to precisely follow defined structures within the expression domain. Illuminating the protein in the region of interest, changes its emission spectrum and highlights a particular cell or cell cluster leaving other transgenic cells in their original color. A major limitation is the lack of known promoters for a large number of tissues in the zebrafish. Conversely, gene- and enhancer trap screens have generated enormous transgenic resources discretely labeling literally all embryonic structures mostly with GFP or to a lesser extend red or yellow FPs. An approach to follow defined structures in such transgenic backgrounds would be to additionally introduce a ubiquitous photoconvertible protein, which could be converted in the cell(s) of interest. However, the photoconvertible proteins available involve a green and/or less frequently a red emission state1 and can therefore often not be used to track cells in the FP-background of existing transgenic lines. To circumvent this problem, we have established the PSmOrange system for the zebrafish2,3. Simple microinjection of synthetic mRNA encoding a nuclear form of this protein labels all cell nuclei with orange/red fluorescence. Upon targeted photoconversion of the protein, it switches its emission spectrum to far red. The quantum efficiency and stability of the protein makes PSmOrange a superb cell-tracking tool for zebrafish and possibly other teleost species.

A Figura 1 ilustra um exemplo do sistema PSmOrange fotoconversão. O complexo pineal é uma estrutura conservada no diencéfalo dorsal vertebrado. Como em muitos outros vertebrados, esse complexo é constituído por o órgão pineal no centro do diencéfalo e as células parapineal do lado esquerdo. Experimentos uncaging elegantes, mas demoradas mostraram que as células parapineal originam na parte anterior do órgão pineal 13. Em tg (FOXD3: GFP); tg (FLH: G...

Watch this Scientific Journal Video about Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System at JoVE.com

Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Acompanhando as células GFP em peixes-zebra transgénicos Usando o Sistema Photoconvertible PSmOrange

The rapid development of transparent zebrafish embryos (Danio rerio) in combination with fluorescent labelings of cells and tissues allows visualizing ...

tracking-cells-gfp-transgenic-zebrafish-using-photoconvertible

Research

JoVE Journal

Developmental Biology

8.5K visualizações.  Heidelberg University. We established the photoconvertible PSmOrange system as a powerful, straight-forward and cost inexpensive tool for in vivo cell tracking in GFP transgenic backgrounds. This protocol describes its application in the zebrafish model system.

Vídeo: Tracking Cells in GFP-transgenic Zebrafish Using the Photoconvertible PSmOrange System

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Photoconversion de célula única no Zebrafish intacta vida

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Biologia do Desenvolvimento

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Seguindo os movimentos de tecido endocárdico através de célula Photoconversion no embrião Zebrafish

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

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 ...

Biologia

Photoconversion of Purified Fluorescent Proteins and Dual-probe Optical Highlighting in Live Cells

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of fluorescence can be changed by exposure to light of a specific wavelength. Optical highlighting allows noninvasive marking of a subpopulation of fluorescent molecules, and is therefore ideal for tracking single cells or organelles.
Critical parameters for efficient photoconversion are the intensity and the exposure time of the photoconversion light. If the intensity is too low, photoconversion will be slow or not occur at all. On the other hand, too much intensity or too long exposure can photobleach the protein and thereby reduce the efficiency of photoconversion.
This protocol describes a general approach how to set up a confocal laser scanning microscope for pc-FP photoconversion applications. First, we describe a procedure for preparing purified protein droplet samples. This sample format is very convenient for studying the photophysical behavior of fluorescent proteins under the microscope. Second, we will use the protein droplet sample to show how to configure the microscope for photoconversion. And finally, we will show how to perform optical highlighting in live cells, including dual-probe optical highlighting with mOrange2 and Dronpa.

This protocol describes a general approach to perform photoconversion of fluorescent proteins on a confocal laser scanning microscope. We describe procedures for the photoconversion of puried protein samples, as well as for dual-probe optical highlighting in live cells with mOrange2 and Dronpa.

Fotoconversão de proteínas purificadas fluorescentes e Dual-sonda Destacando Optical em células vivas

Photoconvertible fluorescent proteins (pc-FPs) are a class of fluorescent proteins with "optical highlighter" capability, meaning that the color of ...

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 ...

Single Cell Fate Mapping in Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, lymphatic, and blood vessel lineages arise in developing embryos requires fate mapping and lineage tracing of undifferentiated precursor cells. Recently, photoactivatable proteins which include: Eos1, 2, PAmCherry3, Kaede4-7, pKindling8, and KikGR9, 10 have received wide interest as cell tracing probes. The fluorescence spectrum of these photosensitive proteins can be easily converted with UV excitation, allowing a population of cells to be distinguished from adjacent ones. However, the photoefficiency of the activated protein may limit long-term cell tracking11. As an alternative to photoactivatable proteins, caged fluorescein-dextran has been widely used in embryo model systems7, 12-14. Traditionally, to uncage fluorescein-dextran, UV excitation from a fluorescence lamp house or a single photon UV laser has been used; however, such sources limit the spatial resolution of photoactivation. Here we report a protocol to fate map, lineage trace, and detect single labeled cells. Single cells in embryos injected with caged fluorescein-dextran are photoactivated with near-infrared laser pulses produced from a titanium sapphire femtosecond laser. This laser is customary in all two-photon confocal microscopes such as the LSM 510 META NLO microscope used in this paper. Since biological tissue is transparent to near-infrared irradiation15, the laser pulses can be focused deep within the embryo without uncaging cells above or below the selected focal plane. Therefore, non-linear two-photon absorption is induced only at the geometric focus to uncage fluorescein-dextran in a single cell. To detect the cell containing uncaged fluorescein-dextran, we describe a simple immunohistochemistry protocol16 to rapidly visualize the activated cell. The activation and detection protocol presented in this paper is versatile and can be applied to any model system. 
Note: The reagents used in this protocol can be found in the table appended at the end of the article.

A method is described to photoactivate single cells containing a caged fluorescent protein using two-photon absorption from a Ti:Sapphire femtosecond laser oscillator. To fate map the photoactivated cell, immunohistochemistry is used. This technique can be applied to any cell type.

Mapeamento Destino única célula em Zebrafish

The ability to differentially label single cells has important implications in developmental biology. For instance, determining how hematopoietic, ...

Cell Tracking Using Photoconvertible Proteins During Zebrafish Development

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of genetically-encoded fluorescent proteins has proven a valuable strategy for elucidating dynamic morphogenetic processes as they occur in the intact organism. During the past decade, the development of photoactivatable and photoconvertible fluorescent proteins has opened the possibility to investigate the fate of discrete subpopulations of tagged proteins1. Unlike photoactivatable proteins, photoconvertible fluorescent proteins (PCFPs) are readily tracked and imaged in their native emission state prior to photoconversion, making it easier to identify and select regions by optical inspection. PCFPs, such as Kaede2, KikGR3, Dendra4 and EosFP5, can be shifted from green to red upon exposure to UV or blue light due to a His-Tyr-Gly tripeptide sequence which forms a green chromophore that can be photoconverted to a red one by a light-catalyzed &beta;-elimination and subsequent extension of a &pi;-conjugated system3. PCFPs and their monomeric variants are useful tools for tracking cells6-10 and studying protein dynamics11-14, respectively. During recent years, PCFPs have been expressed in different animal model, such as zebrafish6, chicken7,8 and mouse9,10 for cell fate tracking. Here we report a protocol for cell-specific photoconversion of PCFPs in the living zebrafish embryo and further tracking of photoconverted proteins at later developmental stages. This methodology allows studying, in a tissue-specific manner, cell biological events underlying morphogenesis in the zebrafish animal model.

Here, we present a method for the photoactivated switch of photoconvertible fluorescent proteins (PCFPs) in the living zebrafish embryo and further tracking of photoconverted protein at specific time points during development. This methodology allows monitoring of cell biological events underlying different developmental processes in a live vertebrate organism.

Rastreamento de celular utilizando proteínas Photoconvertible Durante o Desenvolvimento Zebrafish

Embryogenesis is a dynamic process that is best studied by using techniques that allow the documentation of developmental changes in vivo. The use of ...

Generation of Dispersed Presomitic Mesoderm Cell Cultures for Imaging of the Zebrafish Segmentation Clock in Single Cells

Segmentation is a periodic and sequential morphogenetic process in vertebrates. This rhythmic formation of blocks of tissue called somites along the body axis is evidence of a genetic oscillator patterning the developing embryo. In zebrafish, the intracellular clock driving segmentation is comprised of members of the Her/Hes transcription factor family organized into negative feedback loops. We have recently generated transgenic fluorescent reporter lines for the cyclic gene her1 that recapitulate the spatio-temporal pattern of oscillations in the presomitic mesoderm (PSM). Using these lines, we developed an in vitro culture system that allows real-time analysis of segmentation clock oscillations within single, isolated PSM cells. By removing PSM tissue from transgenic embryos and then dispersing cells from oscillating regions onto glass-bottom dishes, we generated cultures suitable for time-lapse imaging of fluorescence signal from individual clock cells. This approach provides an experimental and conceptual framework for direct manipulation of the segmentation clock with unprecedented single-cell resolution, allowing its cell-autonomous and tissue-level properties to be distinguished and dissected.

Somitogenesis is a rhythmic developmental process that spatially patterns the body axis of vertebrate embryos. Previously, we developed transgenic zebrafish lines that use fluorescent reporters to observe the cyclic genes that drive this process. Here, we culture dispersed cells from these lines and image their oscillations over time in vitro.

Geração de Disperso Presomitic mesoderme culturas de células for Imaging da Segmentação Relógio Zebrafish em células individuais

Segmentation is a  periodic and sequential morphogenetic process in vertebrates. This rhythmic  formation of blocks of tissue called somites along the ...

ROS Live Cell Imaging During Neuronal Development

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the different ROS, hydrogen peroxide (H2O2) is best characterized with respect to roles in cellular signaling. H2O2 has been implicated during the development in several species. For example, a transient increase in H2O2 has been detected in zebrafish embryos during the first days following fertilization. Furthermore, depleting an important cellular H2O2 source, NADPH oxidase (NOX), impairs nervous system development such as the differentiation, axonal growth, and guidance of retinal ganglion cells (RGCs) both in&#160;vivo and in vitro. Here, we describe a method for imaging intracellular H2O2 levels in cultured zebrafish neurons and whole larvae during development using the genetically encoded H2O2-specific biosensor, roGFP2-Orp1. This probe can be transiently or stably expressed in zebrafish larvae. Furthermore, the ratiometric readout diminishes the probability of detecting artifacts due to differential gene expression or volume effects. First, we demonstrate how to isolate and culture RGCs derived from zebrafish embryos that transiently express roGFP2-Orp1. Then, we use whole larvae to monitor H2O2 levels at the tissue level. The sensor has been validated by the addition of H2O2. Additionally, this methodology could be used to measure H2O2 levels in specific cell types and tissues by generating transgenic animals with tissue-specific biosensor expression. As zebrafish facilitate genetic and developmental manipulations, the approach demonstrated here could serve as a pipeline to test the role of H2O2 during neuronal and general embryonic development in vertebrates.

This protocol describes the use of a genetically encoded hydrogen peroxide (H2O2)-biosensor in cultured zebrafish neurons and larvae for assessing the physiological signaling roles of H2O2 during nervous system development. It can be applied to different cell types and modified with experimental treatments to study reactive oxygen species (ROS) in general development.

Imagem de células vivas ROS durante o desenvolvimento neuronal

Reactive oxygen species (ROS) are well-established signaling molecules, which are important in normal development, homeostasis, and physiology. Among the ...

Neurociência

Protein Phase Separation Assay: An Optogenetic Method for Mutant RNA-Binding Protein Phase Separation in Spinal Motor Neurons of Zebrafish Larvae

Source: Asakawa, K. et al. <a href="https://www.jove.com/v/62932">Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae.</a> J. Vis. Exp. (2022)
In this video, we describe a phase separation assay, wherein intracellular proteins comprising intrinsically disordered regions fused to a photosensitive oligomerization domain are induced via blue-light exposure to self-associate into membrane-less liquid-like condensates.

Ensaio de Separação de Fase de Proteína: Um Método Optogenético para Separação de Fase de Proteína de Ligação a RNA Mutante em Neurônios Motores Espinhais de Larvas de Peixe-Zebra

Source: Asakawa, K. et al. Optogenetic Phase Transition of TDP-43 in Spinal Motor Neurons of Zebrafish Larvae. J. Vis. Exp. (2022)
In this video, we ...