The authors thank Gilberto Morales for fish care and maintenance, and Drs. Claudia Rivera and Hector Malag&#242;n (IFC-UNAM Animal Facility) for their help in rabbit immunization. This work was supported by grants from CONACYT 131226 and PAPIIT-DGAPA-UNAM IN204916.

Todos os experimentos realizados em animais foram aprovados pelo Comitê de Laboratório Animal Care e Use da Universidade Nacional Autônoma do México (UNAM), sob o número CICUAL-Protocol: FLC40-14. (CICUAL: &quot;Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de Fisiologia Celular, Universidad Nacional Autónoma de México&quot;). 1. Preparação do extracto do embrião Proteína <ol><li> Colete 100 - 200 embriões para cada condição para comparar (morphants vs. tipo selvagem) no estágio desejado, por exemplo 72 horas após a fertilização (hpf). </li><li> Embriões lugar em placas de Petri contendo água de peixe (ver Tabela 1) e dechorionate manualmente embriões usando uma pinça de ponta fina. Evitar o uso de pronase durante este passo (ou qualquer outra protease ao longo do protocolo), uma vez que podem digerir o receptor alvo. </li><li> embriões lugar em um tubo de microcentrífuga de 1,5 ml e lavar embriões twgelo com uma solução de tampão de fosfato de 1x (PBS) (ver Tabela 1). </li><li> Adicionar 500 ul de tampão deyolk (ver Tabela 1). </li><li> Liberar mais de gema de cuidado pipetando para cima e para baixo (cerca de 30 - 40 vezes) os embriões na solução. Para 72 hpf e 48 embriões HPF usar azul e amarelo pontas de pipeta, respectivamente. pontas amarelas pode precisar de ser ligeiramente cortada para evitar a destruição de embriões. O lançamento gema de sucesso pode ser verificado observando-se os embriões sob o microscópio. </li><li> Recolhe embriões por centrifugação a 600 xg durante 15 seg. </li><li> Elimine o sobrenadante cuidadosamente, usando uma pipeta. </li><li> Embriões lavar duas vezes com 500 ul de tampão de lavagem (ver Tabela 1) por vortex suavemente na velocidade mais baixa. </li><li> Recolhe embriões por centrifugação a 600 xg durante 15 seg. </li><li> A partir daqui, o processo continua a 4 ° C. </li><li> Elimine o sobrenadante cuidadosamente, usando uma pipeta. </li><li> Ressuspender embriões em 350 ul de tampão de lise (ver Tabela 1) e homogeneizar utilizando um pilão de plástico. </li><li> Incubar embriões lisadas 30 min, com agitação num agitador tubo de ensaio a 4 ° C. </li><li> Centrifugar embriões lisadas a 11000 xg durante 15 minutos a fim de remover os detritos insolúveis. </li><li> Transferência afastada sobrenadante utilizando uma pipeta para um tubo fresco. </li><li> Determinar a proteína total pelo ensaio de proteína de Bradford 4, ou outro procedimento adequado. Se ensaio de Bradford é utilizado, realizar a curva de calibração na presença de concentrações equivalentes dos detergentes presentes no tampão de lise, uma vez que causam uma subestimação da proteína padrão 4. </li></ol> 2. Detecção da proteína endógena do Receptor <ol><li> Affinity Labeling e Immunoprecipitation (todos estes passos, a 4 ° C) <ol><li> Coloque 400-500 ug de proteína total de embriões num tubo de microcentrífuga de 1,5 ml e diluir até 1 ug / uL com tampão 1 (ver Tabela 1). Nota: A fim de obter 500 mg de proteína embrião total, cerca de 100-200 embriões devem ser inicialmente processado. 72 hpf embriões rotineiramente obter ~ 5 ^ g de proteína total de embrião, o qual é suficiente para a detecção betaglicano. </li><li> Adicionar um volume suficiente de estoque de TGF-β2 marcado para atingir uma concentração final de 150 pM e incubar com agitação em um tubo de ensaio balancim 2 h a 4 ° C. TGF-β2 deve ser marcado antes AFLIP é iniciado, com 125 I pelo método da cloramina T como descrito por Cheifetz et ai. 5. CUIDADO: O uso de blindagem para minimizar a exposição ao manusear 125 I ligando marcado. </li><li> Adicionar um volume suficiente de anticorpo não diluído contra o receptor de interesse, a fim de chegar a uma diluição de 1: 100 e continuar a incubação durante mais 2 horas a 4 ° C (de incubação pode ser O / N). Esta é a diluição óptima de anti-soro # 31, utilizado neste estudo e descrito noutro local 3, mas, dependendo da qualidade do umtibody, uma diluição maior ou menor pode ser a ideal. </li><li> Adicionar 50 ul de pérolas de ligação de imunoglobulina adequada (por exemplo, a proteína G-Sepharose &#39;, que foi previamente equilibrada em TNTE e ressuspensas 1: 5 do seu volume original em TNTE) e incubar durante 50 minutos com agitação num agitador tubo de ensaio a 4 ° C. </li><li> Recuperar os grânulos por microcentrifugação a 11000 xg durante 20 seg. </li><li> Elimine o sobrenadante em um recipiente de lixo radioactivo apropriado. </li><li> Lavam-se as esferas de IP-se três vezes com 1 ml de tampão de lavagem de IP (ver Tabela 1), por agitação em vórtice e microcentrifugação a 11000 xg durante 20 seg de cada vez. </li><li> Ressuspender IP-grânulos em 250 ul de tampão de lavagem IP. </li><li> Adicionar 1,5 uL de suberato de dissuccinimidilo (DSS, dissolvido em DMSO a 10 mg / ml). Preparar a solução de DSS imediatamente antes da sua utilização neste passo. </li><li> Incubar durante 15 min a 4 ° C com agitação. </li><li> A fim de parar a reacção de reticulação, adicionar 500 ul Otampão de lavagem f IP, suplementado com suficiente de Tris-Cl pH 7,4 estoque para atingir 25 mM de Tris-Cl. Os grupos amino livres em Tris capturar DSS que não reagiu. </li><li> Centrifuga-se a 11000 xg durante 20 seg para recolher IP-grânulos e descartar o sobrenadante. </li><li> Ressuspender IP-esferas em 30 ul de tampão redutor de Laemmli. </li><li> Ferver as amostras 5 min a 94 ° C. </li><li> Opcionalmente, analisar amostras em um contador gama usando o protocolo do fabricante. </li></ol></li><li> Análise de amostras <ol><li> amostras submetido a uma desnaturação SDS-PAGE. Use poliacrilamida na percentagem adequada para a massa do receptor e executar gel sob procedimento padrão. </li><li> Mergulhar em gel de solução fixadora (ver Tabela 1) durante 30 min à TA. </li><li> Lavar o gel com água destilada durante 15 min. </li><li> Coloque gel em papel de filtro previamente hidratado e cubra com filme de envolvimento Saran. </li><li> gel seco a 80 ° C durante 1 h. </li><li> Expor gel em um scre phosphorimager brancoen à TA S / N. </li><li> Digitalizar tela exposta usando um phosphorimager usando o protocolo do fabricante. </li></ol></li></ol>

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Biochem. 72, 248-254 (1976).</li><li>Cheifetz, 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=Distinct+transforming+growth+factor-b+receptor+subsets+as+determinants+of+cellular+responsiveness+to+three+TGF-b+isoforms.">Distinct transforming growth factor-b receptor subsets as determinants of cellular responsiveness to three TGF-b isoforms.</a> J. Biol. Chem. 265, 20533-20538 (1990).</li><li>L&#243;pez-Casillas, F., Wrana, J. L., Massagu&#233;, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Betaglycan+presents+ligand+to+the+TGF&#946;+signaling+receptor.">Betaglycan presents ligand to the TGF&#946; signaling receptor.</a> Cell. 73 (7), 1435-1444 (1993).</li><li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> PNAS USA. 76 (9), 4350-4354 (1979).</li><li>Maccari, F., Volpi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+and+specific+recognition+of+glycosaminoglycans+by+antibodies+after+their+separation+by+agarose+gel+electrophoresis+and+blotting+on+cetylpyridinium+chloride-treated+nitrocellulose+membranes.">Direct and specific recognition of glycosaminoglycans by antibodies after their separation by agarose gel electrophoresis and blotting on cetylpyridinium chloride-treated nitrocellulose membranes.</a> Electrophoresis. 24 (9), 1347-1352 (2003).</li><li>Volpi, N., Maccari, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosaminoglycan+blotting+and+detection+after+electrophoresis+separation.">Glycosaminoglycan blotting and detection after electrophoresis separation.</a> Methods Mol Biol (Clifton, N.J). 1312, 119-127 (2015).</li><li>Guesdon, J. 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Biochem. 165 (2), 448-455 (1987).</li><li>Das, 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=Specific+radiolabeling+of+a+cell+surface+receptor+for+epidermal+growth+factor.">Specific radiolabeling of a cell surface receptor for epidermal growth factor.</a> PNAS USA. 74 (7), 2790-2794 (1977).</li><li>Massague, J., Guillette, B., Czech, M., Morgan, C., Bradshaw, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+nerve+growth+factor+receptor+protein+in+sympathetic+ganglia+membranes+by+affinity+labeling.">Identification of a nerve growth factor receptor protein in sympathetic ganglia membranes by affinity labeling.</a> J. Biol. Chem. 256 (18), 9419-9424 (1981).</li><li>Massague, J., Pilch, P. F., Czech, M. 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The authors have nothing to disclose.

O uso de transferências de Western com um anticorpo específico contra uma proteína de interesse é uma ferramenta valiosa para estudar a sua expressão 7 durante a embriogénese. No entanto, immunoblotting de proteínas altamente glicosiladas não tem sido muito bem sucedida, devido à sua transferência ineficiente e de ligação fraca para nitrocelulose ou PVDF membranas 8,9. Proteoglicanos são um bom exemplo desta lacuna, por causa de suas cadeias de glicosaminog...

Specific detection of proteins expressed during embryonic development is required to validate expression profiles obtained by measuring their cognate mRNAs with RT-PCR or in situ hybridization (ISH). This is commonly achieved by a western blot of embryo extracts followed by detection with specific antibodies. However, this approach is hard to apply to proteins that are in very low abundance, or that have properties that hamper their quantitative transfer during their blotting. Betaglycan, also known as the type III transforming growth factor &#946; (TGF-&#946;) receptor (TGFBR3), is an example of these difficulties. TGFBR3 is a part time membrane proteoglycan that binds TGF-&#946; through its core protein1, with notably higher affinity for the isoform TGF-&#946;2, a property that distinguishes it from any other TGF-&#946; binding protein2. TGFBR3 in the zebrafish is expressed from 8 hpf on, reaching a maximum by 72 hpf, as detected by RT-PCR of its mRNA3. 
However, despite the availability of a very specific antibody3, every attempt to detect its translated product by western blot proved unsuccessful. Reckoning that TGFBR3's proteoglycan nature, as well as putative low abundance may be accountable for this failure, a detection method, AFLIP, which takes advantage of TGFBR3 high affinity for TGF-&#946;2 was devised. In this method a protein extract from pooled embryos is allowed to specifically bind 125I-labeled TGF-&#946;2 and the receptor-ligand complexes are purified by immunoprecipitation and cross-linked before separation by SDS-PAGE. The migration patterns observed by autoradiography of the gels revealed the presence and nature of the labeled receptor species. This approach combines the ligand specificity of affinity labeling with immunoprecipitation by specific antibodies, increasing detection range, avoiding the inefficient transfer blotting of TGFBR3. Due to its inherent properties, the AFLIP assay is not a quantitative assay but can be used to confidently gauge relative experimental differences in the analyzed receptor.

<table><tbody><tr><td>Disuccinimidyl suberate (DSS)</td><td>ThermoFisher Scientific</td><td>21555</td><td></td></tr><tr><td>Protein G Sepharose 4 Fast Flow</td><td>GE Healthcare Life Sciences</td><td>17-0618-01</td><td></td></tr><tr><td>Gel Dryer Model 583&amp;nbsp;</td><td>BIO-RAD</td><td>1651745</td><td></td></tr><tr><td>Typhoon 9400</td><td>GE Healthcare Life Sciences</td><td>63-0055-78</td><td></td></tr><tr><td>Cobra II Auto gamma counter</td><td>Packard</td><td></td><td></td></tr><tr><td>Exposure Cassette</td><td>Molecular Dynamics</td><td>63-0035-44</td><td></td></tr><tr><td>NaCl</td><td>J.T. Baker</td><td>3624</td><td></td></tr><tr><td>KCl</td><td>J.T. Baker</td><td>3040</td><td></td></tr><tr><td>Na&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3828</td><td></td></tr><tr><td>K&lt;sub&gt;2&lt;/sub&gt;HPO&lt;sub&gt;4&lt;/sub&gt;</td><td>J.T. Baker</td><td>3246</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;OH</td><td>J.T. Baker</td><td>9070</td><td></td></tr><tr><td>CH&lt;sub&gt;3&lt;/sub&gt;COOH</td><td>J.T. Baker</td><td>9508</td><td></td></tr><tr><td>HCHO</td><td>J.T. Baker</td><td>2106</td><td></td></tr><tr><td>SDS</td><td>Sigma-Aldrich</td><td>L4509</td><td></td></tr><tr><td>EDTA</td><td>Sigma-Aldrich</td><td>ED</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>T9284</td><td></td></tr><tr><td>CaCl&lt;sub&gt;2&lt;/sub&gt;</td><td>Sigma-Aldrich</td><td>C3306</td><td></td></tr><tr><td>NaHCO&lt;sub&gt;3&lt;/sub&gt;</td><td>Fisher Scientific</td><td>S233</td><td></td></tr><tr><td>PMSF</td><td>Sigma-Aldrich</td><td>P7626</td><td></td></tr><tr><td>Crystal Sea Marine Mix</td><td>Marine Enterprises International</td><td>http://www.meisalt.com/Crystal-Sea-Marinemix</td><td></td></tr></tbody></table>

affinity labeling detection of endogenous receptors from zebrafish embryos

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish embryos has been implemented. This technique takes advantage of the selectivity and sensitivity conferred by affinity labeling of a given receptor by its ligand with the specificity of the immunoprecipitation. We have used AFLIP to detect the type III TGF-&#946; receptor (TGFBR3), also know as betaglycan, during early zebrafish development. AFLIP was instrumental in validating the efficacy of a TGFBR3 morphant zebrafish phenotype. In the first step, embryo protein extracts are prepared and used to generate 125I-TGF-&#946;2-TGFBR3 complexes that are purified by immunoprecipitation. Later, these complexes are covalently cross-linked and revealed using SDS-PAGE separation and autoradiography detection. This technique requires the availability of a labeled ligand for, and a specific antibody against, the receptor to be detected, and shall be easily adapted to identify any growth factor or cytokine receptor that meets these requirements.

A Figura 1 mostra um resultado representativo obtido com AFLIP. Os sinais nas faixas 1 vêm do 125I-ligando ligado de forma covalente, quer a proteína de peixe-zebra betaglicano núcleo (núcleo BG, abaixo da kDa marcador 150) ou o núcleo BG que foi processado para a sua forma de proteoglicanos por ligação de glicosaminoglicanos (GAG, esfregaço variando de 170 kDa para a parte superior do gel). Este padrão de migração, uma proteína núcleo afiado mais...

Watch this Scientific Journal Video about Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos at JoVE.com

Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Afinidade Detecção de Etiquetagem endógena Receptores de embriões Zebrafish

By combining the powers of Affinity Labeling and Immunoprecipitation (AFLIP), a technique for the detection of low abundance receptors in zebrafish ...

affinity-labeling-detection-endogenous-receptors-from-zebrafish

Research

JoVE Journal

Developmental Biology

5.6K visualizações.  Universidad Nacional Autónoma de México. A novel technique for the detection of low abundance endogenous receptors present in zebrafish embryos is described. We have named it AFLIP because it consists of affinity labeling of the receptor by its ligand linked to immunoprecipitation.

Vídeo: Affinity Labeling Detection of Endogenous Receptors from Zebrafish Embryos

Labeling and Imaging Cells in the Zebrafish Hindbrain

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish embryos, injection of plasmid DNA results in mosaic expression, allowing for the visualization of single cells or small clusters of cells 1 . We describe how injection of plasmid DNA encoding membrane-targeted Green Fluorescent Protein (mGFP) under the control of a ubiquitous promoter can be used for imaging cells undergoing neurulation. Central to this protocol is the methodology for imaging labeled cells at high resolution in sections and also in real time. This protocol entails the injection of mGFP DNA into young zebrafish embryos. Embryos are then processed for vibratome sectioning, antibody labeling and imaging with a confocal microscope. Alternatively, live embryos expressing mGFP can be imaged using time-lapse confocal microscopy. We have previously used this straightforward approach to analyze the cellular behaviors that drive neural tube formation in the hindbrain region of zebrafish embryos 2. The fixed preparations allowed for unprecedented visualization of cell shapes and organization in the neural tube while live imaging complemented this approach enabling a better understanding of the cellular dynamics that take place during neurulation.

Key to understanding the morphogenetic processes that shape the early embryo is the ability to image cells at high resolution. We describe here a technique for labeling single cells or small clusters of cells in whole zebrafish embryos with membrane-targeted Green Fluorescent Protein.

Rotulagem e Células de imagem no rombencéfalo Zebrafish

Key to understanding the morphogenetic processes that shape the early vertebrate embryo is the ability to image cells at high resolution. In zebrafish ...

Neurociência

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 ...

Biologia do Desenvolvimento

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualização de células Multiciliated no Zebrafish através de um protocolo combinado de toda montagem fluorescente In Situ da hibridação e imunofluorescência

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

<meta charset="utf8"></head><body>Marcação de proteína fluorescente em larvas de peixe-zebra usando CRISPR/Cas9 Knock-In

Fluorescence Labeling to Visualize Low-Expressed Proteins in Zebrafish

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in vivo imaging due to its transparency during the early developmental stage. Here, we provide a detailed protocol for performing high-efficiency fluorescence gene KI, rapid screening for KI founders, and low-abundance protein tracing in zebrafish larvae, which will lay a critical foundation for subsequent physio-pathological studies in zebrafish. The current protocol includes complete steps for the sgRNA design for the gene of interest, sgRNA in vitro transcription, Cas9 mRNA in vitro transcription, in vivo sgRNA screen for the one with the highest efficiency, donor plasmid design and construction, microinjection in zebrafish larvae, KI founder screen and zebrafish live imaging. Critical steps, troubleshooting tips, quality control methods, and advantages and applications of this protocol are included and discussed. This protocol assures quick and accurate results at a low cost and has been validated by multiple trials.

<meta charset="utf8"></head><body>Marcação de fluorescência para visualizar proteínas de baixa expressão em peixe-zebra

Here, we present a protocol to label proteins with a fluorescence protein tag in zebrafish larvae, a newly developed and modified in vivo system especially useful for visualizing low-abundance proteins in zebrafish.

Marcação de fluorescência para visualizar proteínas de baixa expressão em peixe-zebra

CRISPR/Cas9-mediated knock-in (KI) technology allows for easier fluorescent-protein tagging in zebrafish (Danio rerio), a preferred model organism for in ...

Genética

Antibody Uptake Assay for Tracking Notch/Delta Endocytosis During the Asymmetric Division of Zebrafish Radial Glia Progenitors

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing organs of both invertebrates and vertebrates, asymmetrically dividing progenitors generates a Notchhi self-renewing and a Notchlo differentiating daughter. In the embryonic zebrafish brain, radial glia progenitors (RGPs)-the principal vertebrate neural stem cells-mostly undergo ACD to give birth to one RGP and one differentiating neuron. The optical clarity and easy accessibility of zebrafish embryos make them ideal for in vivo time-lapse imaging to directly visualize how and when the asymmetry of Notch signaling is established during ACD. Recent studies have shown that dynamic endocytosis of the Notch ligand DeltaD plays a crucial role in cell fate determination during ACD, and the process is regulated by the evolutionarily conserved polarity regulator Par-3 (also known as Pard3) and the dynein motor complex. To visualize the in vivo trafficking patterns of Notch signaling endosomes in mitotic RGPs, we have developed this antibody uptake assay. Using the assay, we have uncovered the dynamicity of DeltaD-containing endosomes during RGP division.

This work develops an antibody uptake assay for imaging intra-lineage Notch/DeltaD signaling in dividing radial glia progenitors of the embryonic zebrafish brain.

Ensaio de Captação de Anticorpos para Rastreamento de Endocitose Notch/Delta Durante a Divisão Assimétrica de Progenitores da Glia Radial de Zebrafish

Asymmetric cell division (ACD), which produces two daughter cells of different fates, is fundamental for generating cellular diversity. In the developing ...

Lineage Labeling of Zebrafish Cells with Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated precursors. Researchers have employed various methods of lineage labeling, such as DiI labeling1 and pressure injection of traceable enzymes2 to ascertain cell fate at later stages of development in model systems. The first fate maps in zebrafish (Danio rerio) were assembled by iontophoretic injection of fluorescent dyes, such as rhodamine dextran, into single cells in discrete regions of the embryo and tracing the labeled cell's fate over time3-5. While effective, these methods are technically demanding and require specialized equipment not commonly found in zebrafish labs. Recently, photoconvertable fluorescent proteins, such as Eos and Kaede, which irreversibly switch from green to red fluorescence when exposed to ultraviolet light, are seeing increased use in zebrafish6-8. The optical clarity of the zebrafish embryo and the relative ease of transgenesis have made these particularily attractive tools for lineage labeling and to observe the migration of cells in vivo7. Despite their utility, these proteins have some disadvantages compared to dye-mediated lineage labeling methods. The most crucial is the difficulty we have found in obtaining high 3-D resolution during photoconversion of these proteins. In this light, perhaps the best combination of resolution and ease of use for lineage labeling in zebrafish makes use of caged fluorescein dextran, a fluorescent dye that is bound to a quenching group that masks its fluorescence9. The dye can then be "uncaged" (released from the quenching group) within a specific cell using UV light from a laser or mercury lamp, allowing visualization of its fluorescence or immunodetection. Unlike iontophoretic methods, caged fluorescein can be injected with standard injection apparatuses and uncaged with an epifluorescence microscope equipped with a pinhole10. In addition, antibodies against fluorescein detect only the uncaged form, and the epitope survives fixation well11. Finally, caged fluorescein can be activated with very high 3-D resolution, especially if two-photon microscopy is employed 12,13. This protocol describes a method of lineage labeling by caged fluorescein and laser uncaging. Subsequenctly, uncaged fluorescein is detected simultaneously with other epitopes such as GFP by labeling with antibodies.

This protocol delineates a way to label and trace the fate of small groups of cells zebrafish embryos using UV-uncaging of caged fluorescein, followed by whole mount immunolabeling to amplify the signal from the uncaged fluorescein.

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

A central problem in developmental biology is to deduce the origin of the myriad cell types present in vertebrates as they arise from undifferentiated ...

Biologia

Semi-automated Imaging of Tissue-specific Fluorescence in Zebrafish Embryos

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are frequently used to identify chemicals that modulate gene expression. Chemical screens that assay fluorescence in live zebrafish often rely on expensive, specialized equipment for high content screening. We describe a procedure using a standard epifluorescence microscope with a motorized stage to automatically image zebrafish embryos and detect tissue-specific fluorescence. Using transgenic zebrafish that report estrogen receptor activity via expression of GFP, we developed a semi-automated procedure to screen for estrogen receptor ligands that activate the reporter in a tissue-specific manner. In this video we describe procedures for arraying zebrafish embryos at 24-48 hours post fertilization (hpf) in a 96-well plate and adding small molecules that bind estrogen receptors. At 72-96 hpf, images of each well from the entire plate are automatically collected and manually inspected for tissue-specific fluorescence. This protocol demonstrates the ability to detect estrogens that activate receptors in heart valves but not in liver.

Described here is a protocol for semi-automated imaging of tissue-specific fluorescence in zebrafish embryos.

Imagem semi-automatizada de Fluorescência de tecidos específicos em embriões Zebrafish

Zebrafish embryos are a powerful tool for large-scale screening of small molecules. Transgenic zebrafish that express fluorescent reporter proteins are ...

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Monte Preparação Plano de observação e análise de amostras de embriões Zebrafish manchado por inteiro Monte<em&gt; Em situ</em&gt; Hibridização

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...

Afinidade Detecção de Etiquetagem endógena Receptores de embriões Zebrafish

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Ensaio de Captação de Anticorpos para Rastreamento de Endocitose Notch/Delta Durante a Divisão Assimétrica de Progenitores da Glia Radial de Zebrafish

Rotulagem de linhagem de células Zebrafish com Laser Uncagable Fluorescein Dextran

Imagem semi-automatizada de Fluorescência de tecidos específicos em embriões Zebrafish

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