Financiamento foi fornecido pela National Institutes of Health (NIH: K01-DK087814-01A1 para D.L.S.), o programa de Universidade de estado de Califórnia para Educação & pesquisa em biotecnologia (CSUPERB: controle Molecular do nicho Hematopoietic vertebrados para D.L.S.) e da Secretaria de pós-graduação na Universidade do estado da Califórnia Chico (a A.C.B).

Conselho Consultivo institucional Cuidado Animal e Comissão de utilização (IACUC) Universidade Estadual da Califórnia, Chico, aprovou todos os métodos descritos abaixo.
1. bleach 48 hpf Zebrafish embriões
<ol><li>Com 15 cm (w) x 15 cm (l) x recipientes plásticos de 7cm (h) criar 3 estações de lavagem: 1) com 1 mL de água sanitária de 5% em 1000 mL de meio de embrião (E3; ver tabela 1), 2) com E3 estéril e 3) com E3 estéril. Certifique-se de que cada contêiner tem 500 mL de solução a submergir os embriões em. Nota: Para cada condição testada, pelo menos 10 embriões serão necessários. Este procedimento de lavagem pode acomodar até 200 embriões.</li>
	<li>Com uma pipeta de transferência, colocar os embriões em um coador de chá e mergulhe os ovos na estação de lavagem #1 por 5 min. Retire o coador de chá (com embriões dentro) e coloque no estação de lavagem #2 por 5 min; Repita para a estação de lavagem #3 para um final 5 min.</li>
	<li>Após a última lavagem, enxaguamento embriões de coador de chá, transformando-o de cabeça para baixo sobre um limpa de Petri de 10 cm e enxaguando suavemente embriões fora o coador de chá com E3 estéril e uma pipeta de transferência.</li>
</ol>2. Dechorionate embriões
<ol><li>Com uma pipeta de transferência, remover e descartar E3 tanto quanto possível da caixa de Petri e adicionar 500 μL de dechorionation protease (10 mg/mL) de embriões. Incube a temperatura ambiente por 5 min. Bata levemente o lado da caixa de Petri para remover completamente o chorions.</li>
	<li>Com uma pipeta sorológica, adicione 20 mL de E3 estéril para diluir a protease. Permitir que embriões resolver e remover o E3 com uma pipeta de transferência. Repita essa etapa de lavagem 3 vezes para remover todos os vestígios de protease a dechorionation.</li>
</ol>3. preparação de amostras de embrião
<ol><li>Usando uma pipeta P1000, coloque 10 embriões em um tubo de microcentrifugadora único estéril 1,5 mL.</li>
	<li>Retire E3 com uma pipeta e descarte. Cuidado: Pipetar com cuidado como embriões podem ser facilmente deslocados e descartados durante as etapas de lavagem a seguir.</li>
</ol>4. preparação de embriões para dissociação
<ol><li>Transferir as amostras para fluxo laminar hood e lavar os embriões, adicionando 1 mL estéril E3. Permitir que os embriões resolver a parte inferior do tubo e remover o sobrenadante com uma pipeta P1000. Repita para um total de 3 lavagens.</li>
	<li>Após a última lavagem, remova a E3 e descarte. Adicione 1 mL de 10mm ditiotreitol (DTT) na E3 para remover o revestimento de muco (e qualquer esporos, leveduras ou bactérias que podem ser presos nele) cercando o zebrafish embrionário. Colocar o tubo microcentrifuga horizontalmente e incubar a temperatura ambiente na capa de fluxo laminar para 25 min.</li>
</ol>5. o embrião dissociação
<ol><li>Lave a amostra 3 vezes com 1 mL de estéril DPBS (com Ca2 + e Mg2 +). Após a última lavagem, adicione 500 μL de DPBS (com Ca2 + e Mg2 +) e adicione 5 μL de protease de dissociação de 5 mg/mL (26 U/mL). Atenção: Esta enzima precisa de Ca2 + e Mg2 +, para garantir que o DPBS contém esses íons.</li>
	<li>Incubar as amostras a 37 ° C em um agitador orbital horizontal a 180 rpm para amostras de 60 min. lugar do capuz de fluxo laminar e triture os embriões com uma P1000 até amostras são totalmente dissociadas. Atenção: Verifique embriões periodicamente para determinar quando eles se tornam dissociados. A solução de embrião deve ter alguma quantidade de tecido presente; Não deve ser completamente homogêneo. É possível digerir excesso, que destruirá as HSPCs (ver suplementar Figura 1).</li>
</ol>6. preparação dos embriões dissociadas
<ol><li>Embriões de pipeta 10 dissociada para o reservatório superior de um poliestireno 5ml redondo tubo inferior com uma tampa de filtro de célula de 35 μm.</li>
	<li>Com uma pipeta, enxaguar o tubo de Petri com solução de transferência para poliestireno tubo de 5 mL contendo e PBS estéril (com nenhum Ca2 + ou Mg2 +) filtrados células da etapa 6.1. Repita até 4 mL de líquido está presente no tubo de poliestireno de 5 mL. Nota: PBS, DPBS ou outras soluções isotônicas em buffer podem ser utilizadas nesta fase, desde que não contenham Ca2 + ou Mg2 +
</li>
	<li>Centrifugar tubos a 4 ° C e 300 x g por 5 min para a amostra homogeneizada célula de Pelotas. Com uma pipeta, remover e descartar o sobrenadante do tubo de fundo redondo. Tome cuidado para não perturbar as células peletizadas na parte inferior do tubo. Ressuspender as células em 100 µ l de PBS.</li>
</ol>7. preparação de metilcelulose
<ol><li>Gerar 1 x solução completa metilcelulose (tabela 1) e adicionar 2,5 mL de tubos estéreis fundo redondo 14 mL com 16G agulhas e seringas de 3 mL. Use um tubo para cada condição testada.</li>
	<li>
Adicione citocinas, moléculas pequenas ou outros agentes para ser investigado para cada amostra.
	<ol>
<li>Para diferenciação mieloide, adicione 1% carpa soro e 0,3 mg/mL zebrafish recombinante granulócitos da colônia fator (Gcsf)34 para meio de metilcelulose. Ver Svoboda et al 37 para uma descrição completa sobre como gerar citocinas de soro e recombinação de carpa.</li>
		<li>Para diferenciação eritroide, adicione 1% carpa soro e 0,1 mg/mL zebrafish recombinante eritropoietina (Epo)38 a médio de metilcelulose.</li>
		<li>Para examinar os progenitores multilineage, adicione Epo e Gcsf. Cuidado: Citocinas e aditivos devem não total superior a 10% do volume total, como o meio não será suficientemente viscoso para discernir as colônias individuais.</li>
	</ol>
</li>
</ol>8. adição de embriões dissociadas de metilcelulose
<ol><li>Utilizando uma pipeta, adicione 100 μL de dissociada 10 embriões para a superfície da metilcelulose preparado com soro recombinante de citocinas e carpas. Tampa da tampa e vórtice suavemente para homogeneizar completamente a amostra. Cada tubo agora deve conter o tecido dissociado de 10 embriões.</li>
	<li>Usando 3 mL seringas e agulhas de 16G, alíquota de 1ml da amostra em separado 35 mm 2 placas de Petri. Certifique-se que a amostra é totalmente dispersos por todo o prato de Petri. Repita para cada amostra.</li>
	<li>Coloque os pratos de Petri 35mm com células em metilcelulose em uma placa de Petri de 15 cm. Para cada prato 15cm, adicione uma placa de Petri 35mm com 5 mL de água estéril para umidificar as amostras. Incube a 32 ° C e 5% de CO2 por 7-10 dias.</li>
</ol>9. visualização de HSPC-derivado unidades formadoras (CFUs)
<ol><li>Após 7 dias de incubação, coloca amostras em um microscópio invertido em 40-100 X para visualização e enumeração. Neste ponto, colônias individuais podem ser isoladas com uma pipeta e submetidas a análise de coloração e/ou gene.</li>
</ol>

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J., 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=Hematopoietic+stem+cell+arrival+triggers+dynamic+remodeling+of+the+perivascular+niche.">Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.</a> Cell. 160, 241-252 (2015).</li></ol>

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O modelo de sistema de zebrafish tornou-se um modelo eficiente, eficaz e barato para o estudo de hematopoiese vertebrado primitivo e definitivo. Geração de ensaios que são rápidos, baratos e apresentar algumas dificuldades técnicas pode ser utilizada para teste de pequenas moléculas, analisando os embriões mutantes e elucidação moleculares vias importantes para a biologia HSPC. Em vitro chapeamento de HSPCs de zebrafish adulto é um método eficaz para estudar mutagênese e citocinas hematopoiéticos de...

Hematopoiese é o processo de fazer a multidão de células maduras do sangue necessário para a sobrevivência do organismo. É uma chave no desenvolvimento do processo que envolve a diferenciação de células-tronco hematopoiéticas (linfócitos) em uma variedade de tipos de células desenvolvente restrito que compõem o sangue maduro. Estes linfócitos auto devem renovar para que o sistema nunca está esgotado e eles devem persistir de desenvolvimento embrionário precoce até à morte. Nos vertebrados, constante diferenciação e proliferação de células estaminais e progenitoras hematopoiéticas (HSPCs) são necessários para repor adequadamente a maioria das células do sangue que são pós mitóticas e sendo reciclados todos os dias. Linfócitos geram células maduras do sangue por primeira diferenciação em subconjuntos de células progenitoras restrito; comum progenitores linfoides (CLPs)1, que eventualmente produzir células T, B e NK e comum progenitores mieloides (CMPs)2 que geram granulócitos, eritrócitos, macrófagos e megacariócitos. Estes progenitores estão empenhados em gerar linhagens de células específicas e diferenciarem ainda mais em mais células progenitoras desenvolvente restrito como progenitores mieloide eritroide (MEPs) que geram eritrócitos e plaquetas ou granulócitos progenitores do macrófago (GMPs) que geram basófilos, eosinófilos, neutrófilos e macrófagos2. Identificando e isolando esses progenitores permitem a identificação de importantes vias moleculares envolvidos na diferenciação hematopoiética e muitas doenças hematopoiéticas, como a leucemia surgem quando essas células progenitoras não corretamente Diferencie-se.
Ao longo das últimas décadas, o modelo de sistema de peixe-zebra (Danio rerio) tornou-se uma ferramenta chave de pesquisa para estudos hematopoiéticas adultas e embrionárias. Zebrafish são passível de análise genética e são as espécies de vertebrados modelo filogeneticamente menores que têm um sistema vascular semelhante e sistema hematopoiético aos seres humanos. Desenvolvem de embriões de zebrafish ex utero, e dentro de 48 horas pós fertilização (hpf) gerar HSPCs3,4,5,6,7,8. Zebrafish são também altamente fecundo, com fêmeas deitado sobre 100 ovos em uma única embreagem, permitindo a replicação experimental e tamanhos de amostra grande. Zebrafish embriões são opticamente transparentes, permitindo a visualização microscópica do sistema hematopoiético. Várias linhas de transgênicas fluorescentes de zebrafish marcando linfócitos como runx1: EGFP peixe9, cd41: EGFP peixe10, e kdrl:mCherry; CMap: animais de duplo-positivo do GFP3 , permitem a visualização em tempo real de HSC surgimento e expansão na vivo3,4,7,8, 9. Geração rápido tempo e desenvolvimento ex utero do zebrafish conduziu a seu uso em mutagênese estudos11,12,13,14,15 e drogas rastreio de16,17,18,19,20 para compostos que mantêm a promessa terapêutica para distúrbios do sangue humano. Em geral, conservação do sistema hematopoiético, a presença e o desenvolvimento fácil de linhas transgénicas e tempo de regeneração rápida fez o zebrafish um modelo barato, rápido, flexível e ideal para estudos hematopoiéticos.
Numerosos métodos de isolamento e testes de linfócitos foram desenvolvidos em mamíferos sistemas hematopoiéticos. Os investigadores podem utilizar uma combinação de receptores de superfície celular para marcar linfócitos21,22,23,24, bem como explorar a capacidade dos linfócitos de efluxo tintura25,26. Depois que eles são rotulados, fluorescência-ativado da pilha (FACS) de classificação permite que a sua separação física. Provar que é uma célula de um HSC requer irradiando um animal hospedeiro para destruir HSPCs endógenos, transplantando linfócitos putativos e observando a longo prazo, linhagem multi reconstituição de todos amadurecem tipos de células do sangue. Estes ensaios funcionam bem em ratos, como existem numerosos da pilha-superfície anticorpos contra células hematopoiéticas e linhagens puras do mouse que facilitam a correspondência imune para transplante. No entanto, alguns zebrafish anticorpos de superfície de células hematopoiéticas foram gerados27, dificultando a identificação e isolamento de linfócitos. A maneira mais comum para marcar e isolar linfócitos zebrafish é com animais transgénicos, segundo o qual uma sequência do promotor celular específico está dirigindo expressão de uma proteína fluorescente. Estudos têm visualizado e enumerado de linfócitos na parede ventral da aorta dorsal com microscopia utilizando esta técnica3,4,8,9. Outros laboratórios geraram variedades clonais de zebrafish28,29 e realizaram transplantes bem sucedidos em animais MHC-combinadas30. No entanto, essas técnicas são custo proibitivo para muitos laboratórios são tecnicamente difíceis e são demoradas. Para solucionar esses problemas, laboratórios têm gerado vários ensaios em vitro para testar para a presença, as taxas de proliferação e a capacidade de diferenciação de HSPCs31,32,33, 34,35,36. Estes ensaios mostram proliferação e diferenciação dos HSPCs em vitro31,32,33,34,35,36, o resgate de hematopoiéticas defeitos36e um método eficiente para descobrir e testar citocinas33,34,35. Eles também têm sido utilizados para identificar os genes responsáveis pela HSPC biologia31,32. Neste estudo, tomamos estes ensaios um passo adiante, permitindo a quantificação de HSPCs em um embrião em desenvolvimento do zebrafish. Estes ensaios também podem ser utilizados para quantificar o número de HSPCs em animais mutantes e os animais tratados com drogas hematopoiéticas interrupções. Em essência, estes ensaios são rápidos, presentes alguns desafios técnicos e maneiras baratas de dosar números HSPC, examinar sua proliferação e investigar blocos na diferenciação.

<table><tbody><tr><td>10% Bovine Serum Albumin in Iscove&#039;s MDM</td><td>StemCell Technologies&amp;nbsp;</td><td>9300</td><td></td></tr><tr><td>DPBS (10x) with Calcium&lt;sup&gt;2+&lt;/sup&gt; and Magnesium&lt;sup&gt;2+&lt;/sup&gt;</td><td>Life Technologies</td><td>14080-055</td><td></td></tr><tr><td>HyClone PBS (1x)</td><td>GE Healthcare Life Sciences</td><td>sh30256.01</td><td></td></tr><tr><td>&amp;nbsp;DMEM 500 mL</td><td>Corning CellGrow</td><td>10-017-CV</td><td></td></tr><tr><td>&amp;nbsp;Ham&#039;s F12 500 mL</td><td>Corning CellGrow</td><td>10-080-CV</td><td></td></tr><tr><td>FBS 500 mL</td><td>Gemini Bio-Products</td><td>100-108</td><td></td></tr><tr><td>HEPES 100 mL (1 M)</td><td>Gibco life technologies</td><td>15-630-080</td><td></td></tr><tr><td>Penicillin/streptomycin (5000 U/mL&lt;br/&gt; and 5000 mg/mL) with L-Glutamine (200 mM)</td><td>Corning Mediatech</td><td>30-009-CI</td><td></td></tr><tr><td>Gentamycin Sulfate 10 mL (50 mg/mL)</td><td>Corning Mediatech</td><td>30-005-CR</td><td></td></tr><tr><td>1.5 mL MCF tube</td><td>FisherBrand</td><td>05-408-129</td><td></td></tr><tr><td>3 mL 23gx1 injection needle with Luer lock</td><td>BD Safety Glide</td><td>305905</td><td></td></tr><tr><td>5 mL polystyrene round bottom tube with cell strainer cap</td><td>Corning Falcon</td><td>352235</td><td></td></tr><tr><td>Methocel MC</td><td>Sigma-Aldrich</td><td>64630</td><td></td></tr><tr><td>14 mL Polystyrene round bottom tube</td><td>Corning Falcon</td><td>352057</td><td></td></tr><tr><td>10 mm polystyrene easygrip Petri dish</td><td>Corning Falcon</td><td>351008</td><td></td></tr><tr><td>Librease TM</td><td>Roche Sigma-Aldrich</td><td>5401119001</td><td>dissociation protease</td></tr><tr><td>Pronase</td><td>Roche Sigma-Aldrich</td><td>11459643001</td><td>dechorionation protease</td></tr><tr><td>15 cm Petri dish</td><td>Corning Falcon</td><td>351058</td><td></td></tr><tr><td>AB</td><td>Zebrafish International Resource Center (ZIRC)</td><td>ZL1</td><td>zebrafish strain used</td></tr><tr><td>Dithiolthreitol (DTT)</td><td>Sigma-Aldrich</td><td>646563</td><td></td></tr></tbody></table>

development of an in vitro assay to quantitate hematopoietic stem and progenitor cells (hspcs) in developing zebrafish embryos

Hematopoiese é um processo essencial de celular no qual células estaminais e progenitoras hematopoiéticas (HSPCs) diferenciarem-se na multiplicidade de linhagens diferentes de células que compõem o sangue maduro. Isolamento e identificação destes HSPCs é difícil, porque eles são definidos ex post facto; Eles só podem ser definidos após sua diferenciação em linhagens de células específicas. Ao longo das últimas décadas, o peixe-zebra (Danio rerio) tornou-se um organismo modelo para estudar a hematopoiese. Embriões de zebrafish desenvolvem ex uteroe pela pós-fertilização de 48 h (hpf) geraram definitivo HSPCs. ensaios para avaliar a diferenciação HSPC e capacidades de proliferação foram desenvolvidas, utilizando o transplante e subsequente reconstituição do sistema hematopoiético além de Visualizar linhas transgénicas especializadas com microscopia confocal. No entanto, estes ensaios são custo proibitivo, tecnicamente difícil e demorado para muitos laboratórios. Desenvolvimento de um modelo in vitro para avaliar HSPCs seria custo-eficaz, mais rápido, e presentes menos dificuldades em relação ao anteriormente descritos métodos, permitindo que os laboratórios avaliar rapidamente telas mutagênese e drogas que afetam a biologia HSPC. Este romance em vitro ensaio para avaliar a HSPCs é realizado pelo chapeamento dissociado do zebrafish toda embriões e adicionando fatores exógenos que promovem a proliferação e diferenciação de HSPC. Os embriões são dissociados em células únicas e chapeados com HSPC-apoio fatores de estimulante de colônia que levá-los a gerar colônia formando unidades (CFUs) que surgem a partir de uma célula progenitora único. Estes ensaios devem permitir análise mais cuidadosa das vias moleculares responsáveis pela HSPC proliferação, diferenciação e regulamento, que permitirá que os pesquisadores a entender os fundamentos da hematopoiese vertebrado e sua desregulação durante a doença.

Para avaliar os números HSPC no zebrafish embrionário, 48 embriões hpf foram digeridos, banhados em metilcelulose com fatores de crescimento hematopoiéticos-apoio exógenos e incubados durante 7 dias (Figura 1A). Após 7 dias, unidades formadora de Colônia (CFUs) foram enumerados (Figura 1B) e imagem (Figura 1C). Controlando as diferentes citoci...

Watch this Scientific Journal Video about Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos at JoVE.com

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Aqui, apresentamos um método simples para dosar o tronco e progenitoras células hematopoiéticas (HSPCs) no zebrafish embrionária. HSPCs de zebrafish dissociado são banhados em metilcelulose com fatores de apoio, diferenciando-se em sangue maduro. Isto permite a detecção de sangue defeitos e permite para ser facilmente conduzida de despistagem de drogas.

Desenvolvimento de um ensaio In Vitro para dosar o Progenitor e Hematopoietic Stem Cells (HSPCs) no desenvolvimento de embriões de peixe-zebra

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

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Universidad de Cartagena UDEC

Research

JoVE Journal

Developmental Biology

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

7.9K visualizações.  California State University, Chico. Aqui, apresentamos um método simples para dosar o tronco e progenitoras células hematopoiéticas (HSPCs) no zebrafish embrionária. HSPCs de zebrafish dissociado são banhados em metilcelulose com fatores de apoio, diferenciando-se em sangue maduro. Isto permite a detecção de sangue defeitos e permite para ser facilmente conduzida de despistagem de drogas.

Vídeo: Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Large-scale Zebrafish Embryonic Heart Dissection for Transcriptional Analysis

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

To analyse cardiac gene expression profiles during zebrafish heart development, total RNA has to be extracted from isolated hearts. Here, we present a protocol for collecting functional/beating hearts by rapid manual dissection from zebrafish embryos to obtain cardiac-specific mRNA.

Em larga escala Zebrafish Embryonic Coração de dissecação para a análise transcricional

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the ...

Biologia do Desenvolvimento

Transplantation of Whole Kidney Marrow in Adult Zebrafish

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an organism. The functional definition of a HSC is a transplanted cell that has the ability to reconstitute all the blood lineages of an irradiated recipient long term. This designation was established by decades of seminal work in mammalian systems. Using hematopoietic cell transplantation (HCT) and reverse genetic manipulations in the mouse, the underlying regulatory factors of HSC biology are beginning to be unveiled, but are still largely under-explored. Recently, the zebrafish has emerged as a powerful genetic model to study vertebrate hematopoiesis. Establishing HCT in zebrafish will allow scientists to utilize the large-scale genetic and chemical screening methodologies available in zebrafish to reveal novel mechanisms underlying HSC regulation. In this article, we demonstrate a method to perform HCT in adult zebrafish. We show the dissection and preparation of zebrafish whole kidney marrow, the site of adult hematopoiesis in the zebrafish, and the introduction of these donor cells into the circulation of irradiated recipient fish via intracardiac injection. Additionally, we describe the post-transplant care of fish in an "ICU" to increase their long-term health. In general, gentle care of the fish before, during, and after the transplant is critical to increase the number of fish that will survive more than one month following the procedure, which is essential for assessment of long term (&lt;3 month) engraftment. The experimental data used to establish this protocol will be published elsewhere. The establishment of this protocol will allow for the merger of large-scale zebrafish genetics and transplant biology.

In this article, we demonstrate a method to perform HCT in adult zebrafish.

Transplante de Medula Renal inteiro em Zebrafish Adulto

Hematopoietic stem cells (HSC) are a rare population of pluripotent cells that maintain all the differentiated blood lineages throughout the life of an ...

Biologia

Isolation and Culture of Adult Zebrafish Brain-derived Neurospheres

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration in vertebrates. However, an in-depth analysis of the molecular mechanisms underlying zebrafish adult neurogenesis has been limited due to the lack of a reliable protocol for isolating and culturing neural adult stem/progenitor cells. Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from zebrafish whole brain or from the telencephalon, tectum and cerebellum regions of the adult zebrafish brain. The protocol involves, first the microdissection of zebrafish adult brain, then single cell dissociation and isolation of self-renewing multipotent neural stem/progenitor cells. The entire procedure takes eight days. Additionally, we describe how to manipulate gene expression in zebrafish neurospheres, which will be particularly useful to test the role of specific signaling pathways during adult neural stem/progenitor cell proliferation and differentiation in zebrafish.

Here we provide a reproducible method to examine adult neurogenesis using a neurosphere assay derived from the whole brain or from either the telencephalic, tectal or cerebellar regions of the adult zebrafish brain. Additionally, we describe the procedure to manipulate gene expression in zebrafish neurospheres.

Isolamento e Cultura de neuroesferas Zebrafish Adulto derivado do cérebro

The zebrafish is a highly relevant model organism for understanding the cellular and molecular mechanisms involved in neurogenesis and brain regeneration ...

Isolation and Characterization of Single Cells from Zebrafish Embryos

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Isolamento e caracterização de células individuais de embriões Zebrafish

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been ...

Time-lapse Live Imaging of Clonally Related Neural Progenitor Cells in the Developing Zebrafish Forebrain

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To understand the behavior of neural progenitor cells in the complex in vivo environment, time-lapse live imaging of neural progenitor cells in an intact brain is critically required. In this video, we exploit the unique features of zebrafish embryos to visualize the development of forebrain neural progenitor cells in vivo. We use electroporation to genetically and sparsely label individual neural progenitor cells. Briefly, DNA constructs coding for fluorescent markers were injected into the forebrain ventricle of 22 hours post fertilization (hpf) zebrafish embryos and electric pulses were delivered immediately. Six hours later, the electroporated zebrafish embryos were mounted with low melting point agarose in glass bottom culture dishes. Fluorescently labeled neural progenitor cells were then imaged for 36hours with fixed intervals under a confocal microscope using water dipping objective lens. The present method provides a way to gain insights into the in vivo development of forebrain neural progenitor cells and can be applied to other parts of the central nervous system of the zebrafish embryo.

The present video demonstrates a method which takes advantage of the combination of electroporation and confocal microscopy to perform live imaging on individual neural progenitor cells in the developing zebrafish forebrain. In vivo analysis of the development of forebrain neural progenitor cells at a clonal level can be achieved in this way.

Lapso de tempo de imagens ao vivo de relação clonal células progenitoras neurais no desenvolvimento Zebrafish Encéfalo anterior

Precise patterns of division, migration and differentiation of neural progenitor cells are crucial for proper brain development and function1,2. To ...

Neurociência

Generation of Parabiotic Zebrafish Embryos by Surgical Fusion of Developing Blastulae

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for candidate genes of interest, migratory behaviors of cells, and secreted signals in distinct genetic settings. Because parabiotic animals share a common circulation, any blood or blood-borne factor from one animal will be exchanged with its partner and vice versa. Thus, cells and molecular factors derived from one genetic background can be studied in the context of a second genetic background. Parabiosis of adult mice has been &#160;used extensively to research aging, cancer, diabetes, obesity, and brain development. More recently, parabiosis of zebrafish embryos has been used to study the developmental biology of hematopoiesis. In contrast to mice, the transparent nature of zebrafish embryos permits the direct visualization of cells in the parabiotic context, making it a uniquely powerful method for investigating fundamental cellular and molecular mechanisms. The utility of this technique, however, is limited by a steep learning curve for generating the parabiotic zebrafish embryos. This protocol provides a step-by-step method on how to surgically fuse the blastulae of two zebrafish embryos of different genetic backgrounds to investigate the role of candidate genes of interest. In addition, the parabiotic zebrafish embryos are tolerant to heat shock, making temporal control of gene expression possible. This method does not require a sophisticated set-up and has broad applications for studying cell migration, fate specification, and differentiation in vivo during embryonic development.

This protocol provides step-by-step instruction on how to generate parabiotic zebrafish embryos of different genetic backgrounds. When combined with the unparalleled imaging capabilities of the zebrafish embryo, this method provides a uniquely powerful means to investigate cell-autonomous versus non-cell-autonomous functions for candidate genes of interest.

Geração de parabiótica embriões Zebrafish por Fusion cirúrgica de Desenvolvimento blástulas

Surgical parabiosis of two animals of different genetic backgrounds creates a unique scenario to study cell-intrinsic versus cell-extrinsic roles for ...

Genotyping and Quantification of In Situ Hybridization Staining in Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in developmental biology for decades. Traditionally, most gene expression studies relied on visual evaluation of the ISH signal, a method that is prone to bias, particularly in cases where sample identities are known a priori. We have previously reported on a method to circumvent this bias and provide a more accurate quantification of ISH signals. Here, we present a simple guide to apply this method to quantify the expression levels of genes of interest in ISH-stained embryos and correlate that with their corresponding genotypes. The method is particularly useful to quantify spatially restricted gene expression signals in samples of mixed genotypes and it provides an unbiased and accurate alternative to the traditional visual scoring methods.

Gene editing technologies have enabled researchers to generate zebrafish mutants to investigate gene function with relative ease. Here, we provide a guide to perform parallel embryo genotyping and quantification of in situ hybridization signals in zebrafish. This unbiased approach provides greater accuracy in phenotypical analyses based on in situ hybridization.

Genotipagem e Quantificação da Coloração de Hibridização in Situ em Zebrafish

In situ hybridization (ISH) is an important technique that enables researchers to study mRNA distribution in situ and has been a critical technique in ...

Using Flow Cytometry to Detect and Quantitate Altered Blood Formation in the Developing Zebrafish

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells (HSPCs). This is a critical process that occurs throughout the lifespan of organisms, and disruption of the molecular pathways involved during embryogenesis can have catastrophic long-term consequences. For a multitude of reasons, zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop externally, and by 7 days postfertilization (dpf) have produced most of the subtypes of definitive blood cells that will persist for their lifetime. Assays to assess the number of hematopoietic cells have been developed, mainly utilizing specific histological stains, in situ hybridization techniques, and microscopy of transgenic animals that utilize blood cell-specific promoters driving the expression of fluorescent proteins. However, most staining assays and in situ hybridization techniques do not accurately quantitate the number of blood cells present; only large differences in cell numbers are easily visualized. Utilizing transgenic animals and analyzing individuals with fluorescent or confocal microscopy can be performed, but the quantitation of these assays relies on either counting manually or utilizing expensive imaging software, both of which can make errors. Development of additional methods to assess blood cell numbers would be economical, faster, and could even be automated to quickly assess the effect of CRISPR-mediated genetic modification, morpholino-mediated transcript reduction, and the effect of drug compounds that affect hematopoiesis on a large scale. This novel assay to quantitate blood cells is performed by dissociating whole zebrafish embryos and analyzing the amount of fluorescently labelled blood cells present. These assays should allow elucidation of molecular pathways responsible for blood cell generation, expansion, and regulation during embryogenesis, which will allow researchers to further discover novel factors altered during blood diseases, as well as pathways essential during the evolution of vertebrate hematopoiesis.

This assay is a simple method to quantitate hematopoietic cells in developing embryonic zebrafish. Blood cells from dissociated zebrafish are subjected to flow cytometry analysis. This allows the detection of blood defects in mutant animals and after genetic modification.

Usando citometria de fluxo para detectar e quantificar a formação sanguínea alterada no zebrafish em desenvolvimento

The diversity of cell lineages that comprise mature blood in vertebrate animals arise from the differentiation of hematopoietic stem and progenitor cells ...

Isolation of the Side Population in Myc-induced T-cell Acute Lymphoblastic Leukemia in Zebrafish

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to efflux the DNA-binding dye Hoechst 33342. This "side population" of cells can be identified using flow cytometric methods after the Hoechst 33342 dye is excited by an ultraviolet (UV) laser. The side population of many cell types contains stem- or progenitor-like cells. However, not all cell types have an identifiable side population. Danio rerio, zebrafish, have a robust in vivo model of T-cell acute lymphoblastic leukemia (T-ALL), but whether these zebrafish T-ALLs have a side population is unknown. The method described here outlines how to isolate the side population cells in zebrafish T-ALL. To begin, the T-ALL in zebrafish is generated via the microinjection of tol2 plasmids into one-cell stage embryos. Once the tumors have grown to a stage at which they expand into more than half of the animal's body, the T-ALL cells can be harvested. The cells are then stained with Hoechst 33342 and examined by flow cytometry for side population cells. This method has broad applications in zebrafish T-ALL research. While there are no known cell surface markers in zebrafish that confirm whether these side population cells are cancer stem cell-like, in vivo functional transplantation assays are possible. Furthermore, single-cell transcriptomics could be applied to identify the genetic features of these side population cells.

Here, we describe a technique to isolate the side population cells from a zebrafish model of myc-induced T-cell acute lymphoblastic leukemia (T-ALL). This side population assay is highly sensitive and is described for zebrafish T-ALL, but it may be applicable to other malignant and non-malignant zebrafish cell types.

De células T isolamento da população Side em Myc induzida por leucemia linfoblástica aguda no peixe-zebra

Heterogeneous cell populations, from either healthy or malignant tissues, may contain a population of cells characterized by a differential ability to ...

Imunologia e Infecção

<meta charset="utf8"></head><body>Transplante robusto de células de peixe-zebra para pesquisa de regeneração e desenvolvimento

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

<meta charset="utf8"></head><body>Autor em destaque: Mapeando a conectividade celular no sistema nervoso do peixe-zebra durante o desenvolvimento e regeneração

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Transplante de células de alta resolução em peixe-zebra embrionário e larval

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...