Agradecemos a Bernard Kulemaka e membros do laboratório de Smith para seus comentários úteis e orientação de reagente, Sam Connell e Brent Redford de 3i para responder perguntas de imagem e Deborah Bang, Karen Heed e Kay Stewart para cuidados de zebrafish. Este trabalho foi financiado pela Universidade de Notre Dame, a Elizabeth e Michael Gallagher Family, Alfred P. Sloan Foundation, centro de investigação de Zebrafish na Universidade de Notre e centro de células-tronco e medicina regenerativa da Universidade de Notre Dame. Todos os estudos animais foram feitos em conformidade com a Universidade de Notre Dame IACUC ao Dr. Cody Smith.

Todos os estudos em animais foram aprovados pela Universidade de Notre Dame institucional Cuidado Animal e Comissão de utilização.
1. preparação da amostra de Zebrafish
<ol><li>Coloque um macho adulto e um adulto feminino Tg (proteína conversível) para a câmara de acasalamento por procedimentos-padrão13. Neste manuscrito, use Tg(sox10:eos) peixe9 por causa do acesso, mas outras linhas transgénicas com proteína de photoconvertible pode ser igualmente utilizado. Configure mais de uma câmara no caso de peixe não fazer lay. Permitir que os peixes que permanecem na câmara durante a noite.</li>
	<li>Na manhã seguinte, colete ovos em caixas de Petri 100 mm. Permitem a ovos amadurecer-se à fertilização de pós 24h (hpf) antes de triagem.</li>
	<li>Se animais acima eram heterozigotos para o transgene, pratos de hpf tela 24 Tg(sox10:eos) + embriões com uma fonte de luz nm 488 e GFP filtram conjuntos em um microscópio de dissecação. Tg(sox10:eos) + embriões de isolar e permitir para amadurecer-se 48 hpf.</li>
	<li>Dechorionate embriões manualmente com uma agulha ou pinça.</li>
	<li>Preparar e 5 mL de solução de ponto de agarose de baixo ponto de fusão de 0,8% no microondas.<ol>
<li>Uma vez que a agarose é frio ao toque, coloque 3-4 anestesiados Tg (sox10:eos) + 48 peixe hpf no centro de um vidro de 10mm-lamela inferior placa de Petri.</li>
		<li>Adicione suficiente agarose para cobrir a superfície da lamela, cerca de 1 mL. Use uma agulha de sonda para organizar peixe em seus lados. Permitir que a agarose solidificar para garantir a montagem de animais. Pode ser necessário para continuamente re-organizar o zebrafish até o ágar solidifies14. Solidificação leva aproximadamente 2 minutos.</li>
	</ol>
</li>
	<li>Depois que a agarose solidificou-se por 2 min, adicione lentamente o meio de embrião contendo 0,02% éster de ácido aminobenzoico (tricaina) para prato até a superfície inferior do ágar e prato está submersa. Isto deve ser de aproximadamente 3mL.</li>
</ol>2. microscópio montagem e pre-conversão imagens
<ol><li>Confocal software aberto e selecionar os windows captura e foco [Figura 1]. Sob a janela de captura, selecione a configuração de conversão específicas do laboratório de imagem sob a captura de configuração drop-down guia [Figura 1]. Aqui, a configuração específica do laboratório é chamada de "peixe Imaging".</li>
	<li>Coloque a amostra no escopo confocal e colocar em foco usando o curso e os botões de ajuste fino.</li>
	<li>Abra a janela de foco e localizar a região desejada de interesse (ou seja, o gânglio da raiz dorsal).</li>
	<li>Selecione o laser c488 sob o menu filtro definido. Definir a exposição a 300 ms, poder para 5 laser e intensificar a 75 [Figura 2].</li>
	<li>Marque a caixa 3D na seção tipo de captura . Na seção de captura 3D , selecione usar atual posição e verificar o intervalo ao redor de corrente. Na mesma seção, defina o intervalo de 35 anos, o número de aviões a 36 e o tamanho da etapa 1. O intervalo pode ser aumentado ou diminuído para acomodar para a profundidade da área de imagem. Para a medula espinhal, um valor de intervalo entre 35-40 pilhas normalmente é suficiente [Figura 5].</li>
	<li>Selecione o local atual [Figura 5].</li>
	<li>Clique em Iniciar na parte inferior da janela de captura para adquirir a imagem.</li>
</ol>3. single-cell Photoconversion
<ol><li>Confocal software aberto e selecionar os windows captura e foco [Figura 1]. Sob a janela de captura, selecione a configuração de conversão específicas do laboratório de imagem sob a captura de configuração drop-down guia [Figura 1]. Aqui, a configuração específica do laboratório é chamada de "Peixe ablate chip completo."</li>
	<li>Selecione o laser c488 e c541 sob o menu filtro definido. Se não utilizar o mesmo software de microscópio, encontre o menu para selecionar diferentes lasers e selecione o 488 nm e 541 lasers de nm. Definir os riscos relativos a 300 ms, poder para 5 laser e intensificar a 75 [Figura 2]. Essas configurações do laser são selecionadas com base na produção suficiente sinal fluorescente sem causar fotobranqueamento ou toxicidade. Se toxicidade ou fotobranqueamento é visualizado, reduza a potência do laser ou exposição.</li>
	<li>Abra a janela de foco e clique na guia photomanipulation na janela de foco. Ajuste os parâmetros do laser em conformidade. Mudar a potência da pilha do laser para 2 e clique em ir. Alterar o tamanho de bloco de Raster para 1 e clique em definir. Altere o tamanho de clique duplo para 4. Altere a linha de laser para v405 [Figura 3].</li>
	<li>Abra as configurações de captura avançada na janela de captura. Selecione a guia photomanipulation e alterar as repetições de clique duplo para 2. Clique Okey [Figura 4].</li>
	<li>Selecione a guia XY na janela de foco. Verifique os parâmetros do laser da etapa 2 na guia photomanipulation [Figura 3, Figura 4]. Definir configurações do laser para photoconvert a célula de interesse sem photoconversion das células circundantes.<ol>
<li>Se houver photoconversion de células adjacentes, reduza a potência do laser. Se photoconversion de células não ocorre, poderes do laser podem ser aumentados. Idealmente, conjunto potência do laser para photoconvert apenas a região de interesse e não áreas circunvizinhas.</li>
	</ol>
</li>
	<li>Marque a caixa de timelapse sob o tipo de captura. Em seguida, clique em Iniciar [figura 6A].</li>
	<li>Uma vez que abre a janela de timelapse ao vivo, selecione a ferramenta círculo na barra superior [Figura 7]</li>
	<li>Desenhe um círculo na região centermost da célula. O círculo desenhado com o botão direito, selecione a região FRAPe aguarde 3 segundos. A área selecionada deve tornar-se redutor [Figura 8]. Clique em parar captura.</li>
	<li>Se mais de um animal de imagem, volte para o guia XY no foco menu e selecione posição 2. Em seguida, repita etapas 4.1-4.6.</li>
	<li>Para converter uma população de células, segui os parâmetros de protocolo e laser para etapas 4.1-4.4 exceto em vez de um círculo para FRAP a região de interesse, use a ferramenta de linha de desenho. Desenhar uma linha na região de interesse e FRAP região usando os mesmos parâmetros listados acima.</li>
</ol>4. post-photoconversion Imaging
<ol><li>Uma vez que todos os pontos são photoconverted. Selecione a pilha de imagem padrão de laboratório específicas configuração descrita na precoversion de imagem passo 3. Isto é encontrado sob a captura de configuração drop-down guia na janela de captura [Figura 5].</li>
	<li>Selecione o laser c488 e definir a exposição a 300ms, poder para 5 laser e intensificar a 75 [Figura 2].</li>
	<li>Selecione o laser c541 encontrado sob o mesmo menu como o laser c488. Definir a exposição de 500 ms, poder para 10 laser e intensificar a 75 [Figura 9].</li>
	<li>Marque a caixa 3D na seção tipo de captura . Na seção de captura 3D , selecione usar atual posição e verificar o intervalo ao redor de corrente. Na mesma seção, defina o intervalo de 35 anos, o número de aviões a 36 e o tamanho da etapa 1. O número de série pode aumentar ou diminuir para acomodar a desejada profundidade de imagem [Figura 5].</li>
	<li>Se houver vários pontos no guia de foco XY, selecione a opção lista multiponto na janela de captura. Se não, selecione o local atual.</li>
	<li>Clique em Iniciar na parte inferior da janela de captura para adquirir a imagem.</li>
</ol>

<ol>
	<li>Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W., Prasher, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Green+fluorescent+protein+as+a+marker+for+gene+expression.">Green fluorescent protein as a marker for gene expression.</a> Science. 263 (5148), 802-805 (1994).</li><li>Livet, J., Weissman, T. 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=Transgenic+strategies+for+combinatorial+expression+of+fluorescent+proteins+in+the+nervous+system.">Transgenic strategies for combinatorial expression of fluorescent proteins in the nervous system.</a> Nature. 450 (7166), 56-62 (2007).</li><li>Goins, W. F., Krisky, D., 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=Herpes+simplex+virus+vectors+for+gene+transfer+to+the+nervous+system.">Herpes simplex virus vectors for gene transfer to the nervous system.</a> J Neurovirol. 3 Suppl 1, S80-S88 (1997).</li><li>Boevink, P., Cruz, S., Hawes, C., Harris, N., Oparka, K. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Virus-mediated+delivery+of+the+green+fluorescent+protein+to+the+endoplasmic+reticulum+of+plant+cells.">Virus-mediated delivery of the green fluorescent protein to the endoplasmic reticulum of plant cells.</a> Plant J. 10 (5), 935-941 (1996).</li><li>Day, R. N., Davidson, M. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+fluorescent+protein+palette:+tools+for+cellular+imaging.">The fluorescent protein palette: tools for cellular imaging.</a> Chem Soc Rev. 38 (10), 2887-2921 (2009).</li><li>Wiedenmann, J., Ivanchenko, S., 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=EosFP,+a+fluorescent+marker+protein+with+UV-inducible+green-to-red+fluorescence+conversion.">EosFP, a fluorescent marker protein with UV-inducible green-to-red fluorescence conversion.</a> Proc Natl Acad Sci U S A. 101 (45), 15905-15910 (2004).</li><li>Maurel, D., Banala, S., Laroche, T., Johnsson, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Photoactivatable+and+Photoconvertible+Fluorescent+Probes+for+Protein+Labeling.">Photoactivatable and Photoconvertible Fluorescent Probes for Protein Labeling.</a> ACS Chem Biol. 5 (5), 507-516 (2010).</li><li>Terskikh, A., Fradkov, 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=&amp;#34;Fluorescent+timer&amp;#34;:+protein+that+changes+color+with+time.">&amp;#34;Fluorescent timer&amp;#34;: protein that changes color with time.</a> Science. 290 (5496), 1585-1588 (2000).</li><li>McGraw, H. F., Snelson, C. D., Prendergast, A., Suli, A., Raible, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postembryonic+neuronal+addition+in+Zebrafish+dorsal+root+ganglia+is+regulated+by+Notch+signaling.">Postembryonic neuronal addition in Zebrafish dorsal root ganglia is regulated by Notch signaling.</a> Neural Dev. 7 (23), (2012).</li><li>Smith, C. J., Morris, A. D., Welsh, T. G., Kucenas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contact-Mediated+Inhibition+Between+Oligodendrocyte+Progenitor+Cells+and+Motor+Exit+Point+Glia+Establishes+the+Spinal+Cord+Transition+Zone.">Contact-Mediated Inhibition Between Oligodendrocyte Progenitor Cells and Motor Exit Point Glia Establishes the Spinal Cord Transition Zone.</a> PLoS biology. 12 (9), e1001961 (2014).</li><li>Ravanelli, A. M., Appel, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motor+neurons+and+oligodendrocytes+arise+from+distinct+cell+lineages+by+progenitor+recruitment.">Motor neurons and oligodendrocytes arise from distinct cell lineages by progenitor recruitment.</a> Genes Dev. 29 (23), 2504-2515 (2015).</li><li>Smith, C. J., Johnson, K., Welsh, T. G., Barresi, M. J. F., Kucenas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radial+glia+inhibit+peripheral+glial+infiltration+into+the+spinal+cord+at+motor+exit+point+transition+zones.">Radial glia inhibit peripheral glial infiltration into the spinal cord at motor exit point transition zones.</a> Glia. , (2016).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., 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=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dynamics. 203 (3), 253-310 (1995).</li><li>Kirby, B. B., Takada, 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=In+vivo+time-lapse+imaging+shows+dynamic+oligodendrocyte+progenitor+behavior+during+zebrafish+development.">In vivo time-lapse imaging shows dynamic oligodendrocyte progenitor behavior during zebrafish development.</a> Nat Neurosci. 9 (12), 1506-1511 (2006).</li><li>Danielian, P. S., Muccino, D., Rowitch, D. H., Michael, S. K., McMahon, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modification+of+gene+activity+in+mouse+embryos+in+utero+by+a+tamoxifen-inducible+form+of+Cre+recombinase.">Modification of gene activity in mouse embryos in utero by a tamoxifen-inducible form of Cre recombinase.</a> Curr Biol. 8 (24), 1323-1326 (1998).</li></ol>

Os autores não têm nada para divulgar.

Em tecidos complexos, distintos tipos de células organizam em domínios específicos. Recentemente, as técnicas têm sido utilizadas para células individuais de etiqueta dentro destes tecidos grandes estruturas1,2,3. Aqui vamos demonstrar duas técnicas que podem ser utilizadas da mesma forma para visualizar tanto interações célula única e interações de população celular dentro de tecidos complexos. A vantagem da téc...

Várias células distintas interagem para construir e manter o complexos tecidos animais e vegetais. Estas células são frequentemente intercalados e difícil de distinguir de vizinhos em um nível de célula única sem microscopia de alta resolução que exigem a fixação do tecido. No entanto, para compreender a forma como estes tecidos, são mantidas e tornar-se doente, tem sido essencial para investigar como single as células dentro do tecido estão interagindo ao longo do tempo. Idealmente, estas experiências exigem a rotulagem de células únicas dentro de um tecido de forma não-invasiva, sem a exigência de fixação. Os cientistas agora desenvolveram inúmeras técnicas para realizar esta tarefa1,2,3,4.
A descoberta e a implementação da proteína medusas verde fluorescente (GFP) foi uma abordagem interessante que permitiu para a rotulagem de células distintas em um ambiente de tecido1. Usando promotores de célula específica, é possível geneticamente, selecionar um subconjunto de células que são rotulados1. Alternativamente, viral induzida expressão de GFP pode ser utilizada para usuário-selecionado expressão de GFP3,4. Embora bastante útil, expressão mediada genética de GFP não permite expressão selecionado pelo usuário dentro de um subconjunto de células no tecido; e a expressão viral de GFP, apesar de vantajoso, pode ser invasiva. Com o advento de técnicas inteligentes como Brainbow expressar proteínas fluorescentes distintas mais escassa dentro de tecidos e derivados GFP, tornou-se possível visualizar células únicas e as interações entre eles em tecido complexo2, 5. no entanto, essas abordagens rotular as células de uma forma aleatória. Se o experimento desejado requer a visualização de uma única célula ou a população de células é definido pelo experimentador, são, portanto, limitados. Com tais experiências, seria vantajoso ter uma proteína fluorescente geneticamente expressa que pode ser manipulada para distinguir, de forma única célula, que de outras células não-fluorescente fluorescentes.
Para atingir esse objetivo e visualizar a biologia celular de células únicas dentro de um tecido vivo complexo, a comunidade científica usa única célula photoconversion de proteínas fluorescentes distintas6,7,8. Usando geneticamente controlado a expressão de uma proteína de photoconvertible (i.e., eos, kaede, etc.) que transita de um verde para vermelho estado fluorescente quando exposto aos raios UV (488 nm) a luz, podemos distinguir uma única célula de sua fluorescente etiquetadas vizinhos de7,6,8. Esta abordagem utiliza um aparelho ligado ao nosso microscópio confocal que pode direcionar a luz de uma pilha de laser para uma região de difração limitada de interesse. Com esta técnica, podemos rotular ou células únicas ou maiores populações em uma maneira definida pelo usuário9,10,11. A técnica é minimamente invasiva em comparação com injeções de célula única de GFP viral. Como uma prova de conceito, mostramos que podemos photoconvert células únicas dentro de um gânglio no sistema nervoso periférico e as maiores populações de photoconvert como células localizadas no lado ventral da medula espinhal9,10, 11,12. Podemos então Visualizar estas photoconverted populações de células 24h mais tarde para ganhar a introspecção em seu movimento e diferenciação durante o desenvolvimento.

<table border="0" cellpadding="0" cellspacing="0" width="1846">
	<tbody>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt;width:320pt" width="427">Tg(sox10:eos) zebrafish animals</td>
			<td style="width:125pt" width="167"></td>
			<td style="width:85pt" width="113"></td>
			<td style="width:854pt" width="1139">Fish were obtained and crossed from the fish facility at the University of Notre Dame</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">100 X 15 mm petri dish</td>
			<td>VWR</td>
			<td>25384-302</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Embryo medium</td>
			<td></td>
			<td></td>
			<td>Embryo medium is made weekly and provided by the fish facility at the University of Notre Dame containing 5L RO water, 30uL methylene blue, and 200mL salt stock.</td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.8% Low Melting Point Agarose</td>
			<td>dot scientific inc</td>
			<td>9012-36-6</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">35 X 10 mm glass-coverslip bottom petri dish</td>
			<td>Ted Pella Inc.</td>
			<td>14021-20</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Needle dissecting probe</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">0.002% 3-aminobenzoic acid ester (Tricaine)</td>
			<td>Fluka analytical</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Fluorescent Dissecting Microscope with GFP filters</td>
			<td>Zeiss Axiozoom</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Confocal microscope with lasers to excite GFP and RFP filter sets</td>
			<td>3i spinning disk confocal</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">UV light source (laser) for photoconversion</td>
			<td>405 nm laser</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Slidebook software</td>
			<td>3i</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21" style="height:15.75pt">
			<td height="21" style="height:15.75pt">Methylene blue</td>
			<td>Kordon</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

single-cell photoconversion in living intact zebrafish

Tecidos animais e vegetais é composto de populações distintas de células. Estas células interagem ao longo do tempo para construir e manter o tecido e podem causar doenças quando interrompido. Os cientistas desenvolveram técnicas inteligentes para investigar características e dinâmica natural dessas células no tecido intacto por expressar proteínas fluorescentes em subconjuntos de células. No entanto, às vezes, experiências exigem mais selecionada visualização de células dentro do tecido, às vezes a forma unicelular ou população de células. Para alcançar este objectivo e Visualizar células únicas dentro de uma população de células, os cientistas têm utilizado a célula única photoconversion de proteínas fluorescentes. Para demonstrar essa técnica, mostramos aqui como direcionar a luz UV para uma célula Eos-expressando um interesse de uma intacta, vivendo zebrafish. Nós então imagem essas células de Eos+ photoconverted 24h depois para determinar como eles mudaram no tecido. Descrevemos duas técnicas: única célula photoconversion e photoconversions das populações de células. Estas técnicas podem ser usadas para visualizar interações célula-célula, célula-destino e diferenciação e migrações de célula, tornando-se uma técnica que é aplicável em numerosas questões biológicas.

Photoconversion de proteínas fluorescentes pode ser usado para rotular células distintas dentro de um tecido de6. Para demonstrar isso, Tg(sox10:eos) peixe9 foram usados para expressar a proteína photoconvertible Eos sob as sequências reguladoras de sox10. Os animais Tg(sox10:eos) em 48 hpf foram montados primeiro e então fotografada para detectar qualquer photoconversion não-específicos que possam ter ocorr...

Watch this Scientific Journal Video about Single-cell Photoconversion in Living Intact Zebrafish at JoVE.com

Single-cell Photoconversion in Living Intact Zebrafish

Aqui, apresentamos um protocolo para mostrar como célula photoconversion é conseguida através da exposição UV para áreas específicas, expressando a proteína fluorescente, Eos, em animais vivos.

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 ...

single-cell-photoconversion-in-living-intact-zebrafish

Research

JoVE Journal

Developmental Biology

5.9K visualizações.  University of Notre Dame. Aqui, apresentamos um protocolo para mostrar como célula photoconversion é conseguida através da exposição UV para áreas específicas, expressando a proteína fluorescente, Eos, em animais vivos.

Vídeo: Single-cell Photoconversion in Living Intact Zebrafish

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological systems. In FDAP experiments, the clearance of proteins within living organisms can be measured. A protein of interest is tagged with a photoconvertible fluorescent protein, expressed in vivo and photoconverted, and the decrease in the photoconverted signal over time is monitored. The data is then fitted with an appropriate clearance model to determine the protein half-life. Importantly, the clearance kinetics of protein populations in different compartments of the organism can be examined separately by applying compartmental masks. This approach has been used to determine the intra- and extracellular half-lives of secreted signaling proteins during zebrafish development. Here, we describe a protocol for FDAP experiments in zebrafish embryos. It should be possible to use FDAP to determine the clearance kinetics of any taggable protein in any optically accessible organism.

Measuring Protein Stability in Living Zebrafish Embryos Using Fluorescence Decay After Photoconversion FDAP

Protein levels in cells and tissues are often tightly regulated by the balance of protein production and clearance. Using Fluorescence Decay After Photoconversion (FDAP), the clearance kinetics of proteins can be experimentally measured in vivo.

Medir a estabilidade da proteína em Viver embriões Zebrafish Usando Fluorescência Decay Após Fotoconversão (FDAP)

Protein stability influences many aspects of biology, and measuring the clearance kinetics of proteins can provide important insights into biological ...

Biologia do Desenvolvimento

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Análise de Zebrafish larvas Músculo Esquelético Integrity com Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

Imaging Subcellular Structures in the Living Zebrafish Embryo

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Imagiologia estruturas celulares no embrião vivo Zebrafish

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such ...

Biosensing Motor Neuron Membrane Potential in Live Zebrafish Embryos

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the cells are located. Specifically, they have been developed to analyze the electrical activity of excitable cells, such as spinal neurons. In such a scenario, it is crucial to preserve the integrity of the spinal cell, but it is also important to preserve the anatomy and physiological shape of the systems involved. Indeed, the comprehension of the manner in which the nervous system-and other complex systems-works must be based on a systemic approach. For this reason, the live zebrafish embryo was chosen as a model system, and the spinal neuron membrane voltage changes were evaluated without interfering with the physiological conditions of the embryos. 
Here, an approach combining the employment of zebrafish embryos with a FRET-based biosensor is described. Zebrafish embryos are characterized by a very simplified nervous system and are particularly suited for imaging applications thanks to their transparency, allowing for the employment of fluorescence-based voltage indicators at the plasma membrane during zebrafish development. The synergy between these two components makes it possible to analyze the electrical activity of the cells in intact living organisms, without perturbing the physiological state. Finally, this non-invasive approach can co-exist with other analyses (e.g., spontaneous movement recordings, as shown here).

Protocols described here allow for the study of the electrical properties of excitable cells in the most non-invasive physiological conditions by employing zebrafish embryos in an in vivo system together with a fluorescence resonance energy transfer (FRET)-based genetically encoded voltage indicator (GEVI) selectively expressed in the cell type of interest.

Potencial de membrana de neurônio motor biossensível em embriões vivos de peixe-zebra

The protocols described here are designed to allow researchers to study cell communication without altering the integrity of the environment in which the ...

Induction of Hypoxia in Living Frog and Zebrafish Embryos

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and zebrafish embryos. Our system comprises a chamber featuring a simple setup that is nevertheless robust to induce and maintain a specific oxygen concentration and temperature in any experimental solution of choice. The presented system is very cost-effective but highly functional, it allows induction and sustainment of hypoxia for direct experiments in vivo and for various time periods up to 48 h.
To monitor and study the effects of hypoxia, we have employed two methods - measurement of levels of hypoxia-inducible factor 1alpha (HIF-1&#945;) in whole embryos or specific tissues and determination of retinal stem cell proliferation by 5-ethynyl-2&#8242;-deoxyuridine (EdU) incorporation into the DNA. HIF-1&#945; levels can serve as a general hypoxia marker in the whole embryo or tissue of choice, here embryonic retina. EdU incorporation into the proliferating cells of embryonic retina is a specific output of hypoxia induction. Thus, we have shown that hypoxic embryonic retinal progenitors decrease proliferation within 1 h of incubation under 5% oxygen of both frog and zebrafish embryos.
Once mastered, our setup can be employed for use with small aquatic model organisms, for direct in vivo experiments, any given time period and under normal, hypoxic or hyperoxic oxygen concentration or under any other given gas mixture.

We introduce a novel hypoxic chamber system for use with aquatic organisms such as frog and zebrafish embryos. Our system is simple, robust, cost-effective and allows the induction and sustainment of hypoxia in vivo and for up to 48 h. We present 2 reproducible methods to monitor the effectiveness of hypoxia.

Indução de hipóxia em sapos vivos e embriões de peixe-zebra

Here, we introduce a novel system for hypoxia induction, which we developed to study the effects of hypoxia in aquatic organisms such as frog and ...

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 ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Análises de célula única Transcriptomic de células endócrinas no pâncreas de rato

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the development of neuronal connectivity. During establishment of the retino-tectal projection, optic axons extend from the eye and navigate through distinct regions of the brain to reach their target tissue, the optic tectum. Once optic axons enter the tectum, they elaborate terminal arbors that function to increase the number of synaptic connections they can make with target interneurons in the tectum. Here, we describe a method to express DNA encoding green fluorescent protein (GFP), and gain- and loss-of-function gene constructs, in optic neurons (retinal ganglion cells) in Xenopus embryos. We explain how to microinject a combined DNA/lipofection reagent into eyebuds of one day old embryos such that exogenous genes are expressed in single or small numbers of optic neurons. By tagging genes with GFP or co-injecting with a GFP plasmid, terminal axonal arbors of individual optic neurons with altered molecular signaling can be imaged directly in brains of intact, living Xenopus tadpoles several days later, and their morphology can be quantified. This protocol allows for determination of cell-autonomous molecular mechanisms that underlie the development of optic axon arborization in vivo.

Microinjection of DNA into Eyebuds in Xenopus laevis Embryos and Imaging of GFP Expressing Optic Axonal Arbors in Intact, Living Xenopus Tadpoles

This protocol aims to demonstrate how to microinject a DNA/DOTAP mixture into eyebuds of one day old Xenopus laevis embryos, and how to image and reconstruct individual green fluorescent protein (GFP) expressing optic axonal arbors in tectal midbrains of intact, living Xenopus tadpoles.

Microinjection do ADN em Eyebuds em embriões do laevis de Xenopus e imagem latente de GFP que expressa Arbors axonal óticos em girinos intact, vivendo de Xenopus

The primary visual projection of tadpoles of the aquatic frog Xenopus laevis serves as an excellent model system for studying mechanisms that regulate the ...

Visualizing the Developing Brain in Living Zebrafish using Brainbow and Time-lapse Confocal Imaging

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in vivo imaging techniques can provide a clear window into these processes in the living organism. For example, dividing cells and their progeny can be followed in real time as the nervous system forms. In recent years, technical advances in multicolor techniques have expanded the types of questions that can be investigated. The multicolor Brainbow approach can be used to not only distinguish among like cells, but also to color-code multiple different clones of related cells that each derive from one progenitor cell. This allows for a multiplex lineage analysis of many different clones and their behaviors simultaneously during development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells over real time within the developing zebrafish nervous system. This is particularly useful for following cellular interactions among like cells, which are difficult to label differentially using traditional promoter-driven colors. Our approach can be used for tracking lineage relationships among multiple different clones simultaneously. The large datasets generated using this technique provide rich information that can be compared quantitatively across genetic or pharmacological manipulations. Ultimately the results generated can help to answer systematic questions about how the nervous system develops.

In vivo imaging is a powerful tool that can be used to investigate the cellular mechanisms underlying nervous system development. Here we describe a technique for using time-lapse confocal microscopy to visualize large numbers of multicolor Brainbow-labeled cells in real time within the developing zebrafish nervous system.

Visualizando o cérebro em desenvolvimento em zebrafish vivo usando Brainbow e Imagem Confocal de lapso de tempo

Development of the vertebrate nervous system requires a precise coordination of complex cellular behaviors and interactions. The use of high resolution in ...

Eye Removal in Living Zebrafish Larvae to Examine Innervation-dependent Growth and Development of the Visual System

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis support continuous growth of the entire visual system, both in the eye and the brain. Coordinated growth between the retinae and the optic tectum ensures accurate retinotopic mapping as new neurons are added in the eyes and brain. To address whether retinal axons provide crucial information for regulating tectal stem and progenitor cell behaviors such as survival, proliferation, and/or differentiation, it is necessary to be able to compare innervated and denervated tectal lobes within the same animal and across animals. 
Surgical removal of one eye from living larval zebrafish followed by observation of the optic tectum achieves this goal. The accompanying video demonstrates how to anesthetize larvae, electrolytically sharpen tungsten needles, and use them to remove one eye. It next shows how to dissect brains from fixed zebrafish larvae. Finally, the video provides an overview of the protocol for immunohistochemistry and a demonstration of how to mount stained embryos in low-melting-point agarose for microscopy.

The article explains how to surgically remove eyes from living zebrafish larvae as the first step toward investigating how retinal input influences optic tectum growth and development. In addition, the article provides information about larval anesthetization, fixation, and brain dissection, followed by immunohistochemistry and confocal imaging.

Remoção de olhos em larvas de zebrafish vivos para examinar o crescimento e desenvolvimento dependentes da inervação do sistema visual

Zebrafish exhibit remarkable life-long growth and regenerative abilities. For example, specialized stem cell niches established during embryogenesis ...