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 los experimentos llevados a cabo en animales fueron aprobados por el Comité para el Laboratorio de Cuidado y Uso de la Universidad Nacional Autónoma de México (UNAM) Animal, bajo el número CICUAL-Protocolo: FLC40-14. (CICUAL: &quot;Comité Institucional para el Cuidado y Uso de los Animales de Laboratorio del Instituto de Fisiología Celular de la Universidad Nacional Autónoma de México&quot;). 1. Preparación de extracto de proteína de embriones <ol><li> Recoger 100 - 200 embriones para cada condición de comparar (morphants vs tipo salvaje) en la etapa deseada, por ejemplo, 72 h después de la fecundación (HPF). </li><li> Coloque los embriones en placas de Petri que contienen los peces de agua (ver Tabla 1) y dechorionate manualmente embriones utilizando unas pinzas de punta fina. Evitar el uso de pronasa durante este paso (o cualquier otra proteasa durante todo el protocolo), ya que puede digerir el receptor diana. </li><li> Coloque los embriones en un tubo de microcentrífuga de 1,5 ml y se lavan los embriones twhielo con solución tampón de fosfato 1x (PBS) (véase la Tabla 1). </li><li> Añadir 500 l de tampón deyolk (ver Tabla 1). </li><li> Liberar la mayor parte de la yema pipeteando suavemente hacia arriba y hacia abajo (aproximadamente 30 - 40 veces) los embriones en la solución. Para el 72 y 48 HPF embriones HPF utilizar azul y amarillo, puntas de pipeta, respectivamente. puntas amarillas puede ser necesario cortar un poco para evitar la destrucción de embriones. La liberación de yema de éxito se puede verificar mediante la observación de los embriones bajo el microscopio. </li><li> Recoger los embriones por centrifugación a 600 xg durante 15 seg. </li><li> Desechar el sobrenadante con cuidado utilizando una pipeta. </li><li> Lavado de embriones dos veces con 500 l de tampón de lavado (ver Tabla 1) por agitación suave a la velocidad más baja. </li><li> Recoger los embriones por centrifugación a 600 xg durante 15 seg. </li><li> A partir de aquí, continuar con el procedimiento a 4 ° C. </li><li> Desechar el sobrenadante con cuidado utilizando una pipeta. </li><li> embriones resuspender en 350 l de tampón de lisis (véase la Tabla 1) y homogeneizar usando una mano de mortero de plástico. </li><li> Incubar los embriones lisadas 30 min con agitación en un agitador tubo de ensayo a 4 ° C. </li><li> Centrifugar lisaron embriones a 11.000 xg durante 15 min con el fin de eliminar los residuos insolubles. </li><li> Transferencia aclaró sobrenadante usando una pipeta a un tubo nuevo. </li><li> Determinación de proteínas totales mediante el ensayo de proteínas de Bradford 4, u otro procedimiento adecuado. Si se utiliza el ensayo de Bradford, lleve a cabo la curva de calibración en presencia de concentraciones equivalentes de los detergentes presentes en el tampón de lisis, ya que provocan una subestimación de la proteína estándar 4. </li></ol> 2. Detección de proteína endógena Receptor <ol><li> Etiquetado de afinidad e inmunoprecipitación (todos estos pasos a 4 ° C) <ol><li> Coloque 400-500 g de proteína total de embriones en un tubo de microcentrífuga de 1,5 ml y diluir a 1 g / l con tampón 1 (ver Tabla 1). Nota: Con el fin de obtener 500 g de proteína total del embrión, a unos 100 - 200 embriones deben ser procesados ​​inicialmente. 72 HPF embriones producen rutinariamente ~ 5 g de proteína embrión total, que es suficiente para la detección betaglicano. </li><li> Añadir suficiente volumen de TGF-β2 etiqueta de valores para alcanzar una concentración final de 150 pM y se incuba con agitación en un tubo de ensayo basculante 2 horas a 4 ° C. TGF-β2 debe ser etiquetado antes de iniciar AFLIP, con 125 I por el método de la cloramina T como se describe por Cheifetz et al. 5. PRECAUCIÓN: El uso de blindaje para minimizar la exposición durante la manipulación marcado con 125I ligando. </li><li> Añadir suficiente volumen de anticuerpo sin diluir contra el receptor de interés con el fin de llegar a una dilución de 1: 100 y continuar la incubación durante otras 2 horas a 4 ° C (incubación puede ser O / N). Esta es la dilución óptima de antisuero # 31, que se utiliza en este estudio y descrito en otra parte 3, pero dependiendo de la calidad de la unatibody, una dilución mayor o menor puede ser la óptima. </li><li> Añadir 50 l de perlas de unión a inmunoglobulina adecuado (por ejemplo, la proteína G-Sepharose, que se equilibró previamente en RTFP y se resuspendió 1: 5 de su volumen original en RTFP) e incubar durante 50 min con agitación en un agitador tubo de ensayo a 4 ° DO. </li><li> Recuperar las cuentas por microcentrifugación a 11.000 xg durante 20 seg. </li><li> Desechar el sobrenadante en un recipiente de basura radiactiva adecuada. </li><li> Se lavan las IP-cuentas tres veces con 1 ml de tampón de lavado IP (ver Tabla 1), por agitación e microcentrifugación a 11.000 xg durante 20 segundos cada vez. </li><li> Resuspender IP-perlas en 250 l de tampón de lavado IP. </li><li> Añadir 1,5 l de suberato de disuccinimidilo (DSS, se disolvió en DMSO a 10 mg / ml). Preparar la solución de DSS justo antes de su uso en esta etapa. </li><li> Incubar durante 15 min a 4 ° C con agitación. </li><li> Con el fin de detener la reacción de reticulación, añadir 500 l Otampón de lavado f IP, suplementado con suficiente Tris-Cl pH 7,4 de stock para llegar a 25 mM Tris-Cl. Los grupos amino libres en Tris capturan DSS no reaccionado. </li><li> Se centrifuga a 11.000 xg durante 20 segundos para recoger IP-cuentas y desechar el sobrenadante. </li><li> Resuspender IP-perlas en 30 l de tampón reductor Laemmli. </li><li> Hervir las muestras de 5 min a 94 ° C. </li><li> Opcionalmente, el análisis de muestras en un contador gamma utilizando el protocolo del fabricante. </li></ol></li><li> Análisis de las muestras <ol><li> Las muestras sometidas a desnaturalización SDS-PAGE. Utilice poliacrilamida en el porcentaje adecuado para la masa del receptor y ejecutar gel bajo procedimiento estándar. </li><li> Sumergir gel en solución de fijación (véase la Tabla 1) durante 30 min a TA. </li><li> Lavar gel con agua destilada durante 15 min. </li><li> Coloque gel en papel de filtro previamente hidratado y cubrir con papel film película. </li><li> gel seco a 80 ° C durante 1 hr. </li><li> Exponer en gel en una scre PhosphorImager blancoa temperatura ambiente en O / N. </li><li> Analiza pantalla expuesta mediante un PhosphorImager usando el protocolo del fabricante. </li></ol></li></ol>

<ol>
	<li>L&#243;pez-Casillas, F., 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=Structure+and+expression+of+the+membrane+proteoglycan+betaglycan,+a+component+of+the+TGF-&#946;+receptor+system.">Structure and expression of the membrane proteoglycan betaglycan, a component of the TGF-&#946; receptor system.</a> Cell. 67 (4), 785-795 (1991).</li><li>Cheifetz, S., Andres, J. L., Massague, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+transforming+growth+factor-beta+receptor+type+III+is+a+membrane+proteoglycan.+Domain+structure+of+the+receptor.">The transforming growth factor-beta receptor type III is a membrane proteoglycan. Domain structure of the receptor.</a> J. Biol. Chem. 263 (32), 16984-16991 (1988).</li><li>Kamaid, 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=Betaglycan+knock-down+causes+embryonic+angiogenesis+defects+in+zebrafish.">Betaglycan knock-down causes embryonic angiogenesis defects in zebrafish.</a> Genesis. 53, 583-603 (2015).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+Rapid+and+Sensitive+Method+for+the+Quantitation+of+Microgram+Quantities+of+Protein+Utilizing+the+Principle+of+Protein-Dye+Binding.">A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Principle of Protein-Dye Binding.</a> Anal. 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. L., Ternynck, T., Avrameas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+avidin-biotin+interaction+in+immunoenzymatic+techniques.">The use of avidin-biotin interaction in immunoenzymatic techniques.</a> J Histochem Cytochem. 27 (8), 1131-1139 (1979).</li><li>Bratthauer, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+avidin-biotin+complex+(ABC)+method+and+other+avidin-biotin+binding+methods.">The avidin-biotin complex (ABC) method and other avidin-biotin binding methods.</a> Methods Mol Biol (Clifton, N.J). 588, 257-270 (2010).</li><li>Heimer, R., Molinaro, L., Sampson, P. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+by+125I-cationized+cytochrome+c+of+proteoglycans+and+glycosaminoglycans+immobilized+on+unmodified+and+on+positively+charged+Nylon+66.">Detection by 125I-cationized cytochrome c of proteoglycans and glycosaminoglycans immobilized on unmodified and on positively charged Nylon 66.</a> Anal. 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. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+resolution+of+three+major+insulin+receptor+structures+with+unique+subunit+stoichiometries.">Electrophoretic resolution of three major insulin receptor structures with unique subunit stoichiometries.</a> PNAS USA. 77 (12), 7137-7141 (1980).</li><li>Hoosein, N. M., Gurd, R. S. <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+Glucagon+Receptors+in+Rat+Brain.">Identification of Glucagon Receptors in Rat Brain.</a> PNAS USA. 81, 4368-4372 (1984).</li></ol>

The authors have nothing to disclose.

El uso de transferencias de Western con un anticuerpo específico contra una proteína de interés es una herramienta valiosa para estudiar su expresión durante la embriogénesis 7. Sin embargo, la inmunotransferencia de proteínas altamente glicosiladas no ha sido muy exitoso debido a su transferencia ineficiente y débil unión a nitrocelulosa o PVDF membranas 8,9. Los proteoglicanos son un buen ejemplo de este inconveniente, debido a sus cadenas de glicosaminoglican...

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.

La Figura 1 muestra un resultado representativo obtenido con AFLIP. Las señales en el carril 1 provienen de la I-ligando 125 covalentemente ya sea a la proteína de pez cebra betaglicano núcleo (BG núcleo, por debajo del marcador de 150 kDa) o el núcleo de BG que ha sido procesada a su forma de proteoglicanos mediante la unión de los glicosaminoglicanos (GAG, frotis que van desde 170 kDa a la parte superior del gel). Este patrón de migración, una prote?...

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.

Detección de afinidad etiquetado Endógeno receptores de embriones de pez cebra

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 vistas.  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.

Video: 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.

Etiquetado y las Pilas de imágenes en la parte posterior del cerebro de pez cebra

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

Investigación

Neurociencias

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.

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

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

Biología del Desarrollo

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.

Visualización de las células Multiciliated en el pez cebra a través de un protocolo combinado de todo Monte fluorescente In Situ hibridación e inmunofluorescencia

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>Marcaje de proteínas fluorescentes en larvas de pez cebra mediante 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>Marcaje de fluorescencia para visualizar proteínas de baja expresión en el pez cebra

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.

Marcaje de fluorescencia para visualizar proteínas de baja expresión en el pez cebra

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.

Ensayo de captación de anticuerpos para el seguimiento de la endocitosis notch/delta durante la división asimétrica de los progenitores radiales de la glía del pez cebra

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.

Etiquetado linaje de células de pez cebra con láser Uncagable fluoresceína 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 ...

Biología

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.

Imaging semi-automatizado de fluorescencia específica de tejido en embriones de pez cebra

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.

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje<em&gt; In situ</em&gt; La hibridación

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

Detección de afinidad etiquetado Endógeno receptores de embriones de pez cebra

Resumen

Explorar más videos

Capítulos en este video

Etiquetado y las Pilas de imágenes en la parte posterior del cerebro de pez cebra

La formación de imágenes estructuras subcelulares en el embrión vivo de pez cebra

Visualización de las células Multiciliated en el pez cebra a través de un protocolo combinado de todo Monte fluorescente In Situ hibridación e inmunofluorescencia

Marcaje de fluorescencia para visualizar proteínas de baja expresión en el pez cebra

Marcaje de fluorescencia para visualizar proteínas de baja expresión en el pez cebra

Marcaje de fluorescencia para visualizar proteínas de baja expresión en el pez cebra

Ensayo de captación de anticuerpos para el seguimiento de la endocitosis notch/delta durante la división asimétrica de los progenitores radiales de la glía del pez cebra

Etiquetado linaje de células de pez cebra con láser Uncagable fluoresceína Dextran

Imaging semi-automatizado de fluorescencia específica de tejido en embriones de pez cebra

Preparativos Piso en observación y análisis de embrión de pez cebra muestras teñidas por todo el montaje