Agradecemos al Prof. Christoph Englert y el Prof. Didier Stainier para regalar la Tg (wt1b: GFP) LI1 y Tg (kdrl: EGFP) s843 cepas, respectivamente, y el Fondo para el pez cebra Taiwán Core para proporcionar Tg (kdrl: mCherry) CI5. Este estudio fue apoyado por subvenciones del Ministerio de Ciencia y Tecnología (96-2628-B-029-002-MY3, 101-2313-B-029-001, 102-2628-B-029-002-MY3 de Taiwán, 102- 2321-B-400-018).

Ética Declaración: Todos los procedimientos experimentales en el pez cebra fueron aprobados por el Comité de Cuidado y Uso de Animales Institucional de la Universidad de Tunghai (Aprobación del IRB NO 101-12.) Y llevada a cabo de conformidad con las directrices aprobadas. 1. Soluciones de archivo para 3β-HSD actividad enzimática de tinción <ol><li> Preparar trans-dehydroandrosterone [10 mg / ml en sulfóxido de dimetilo (DMSO)]. </li><li> Preparar β-nicotinamida adenina dinucleótido hidrato (1,2 mg / ml en tampón fosfato 0,1 M, pH 7,2). </li><li> Preparar nicotinamida (vitamina B3, 50 mg / ml en H 2 O). </li><li> Preparar azul tetrazolio 4-nitro (50 mg / ml en 70% de dimetilformamida). </li><li> Alícuotas todos estos componentes en tubos de microcentrífuga y almacenar a -20 ° C. NOTA: En nuestra experiencia, los ciclos de congelación y descongelación de las alícuotas de todas las soluciones mencionadas no afectan a la intensidad de la 3β-HSD actividad de las manchas. </li></ol> 2. Worificio de montaje en 3β-HSD actividad de tinción NOTA: La actividad enzimática 3β-HSD es detectable en embriones de pez cebra tan pronto como 28 horas después de la fertilización (hpf) cuando se cultivan a 28,5 ° C, lo cual es consistente con la aparición de la expresión de ARNm de hsd3b1 2,7. El protocolo de todo el montaje en la actividad tinción 3β-HSD en este estudio se basa en un método descrito previamente 8 y estaba decidido a tener éxito en el pez cebra en desarrollo hasta 7 días después de la fecundación 9. Para analizar el microambiente vascular del tejido steroidogenic interrenal, todo el montaje 3β-HSD actividad tinción se debe aplicar a las líneas transgénicas que expresan fluorescencia de endotelio específica, como la Tg (kdrl: EGFP) s843 14 y Tg (kdrl: mCherry) CI5 15 . Para analizar el tejido androgénica interrenal teniendo en cuenta el desarrollo del riñón pez cebra, 3β-HSD actividad de tinción puede ser aplied a la Tg (wt1b: GFP) pescado LI1 16, donde se delinea la formación de los glomérulos y túbulos pronéfricos. <ol><li> Se trata el pez cebra en desarrollo mediante su incubación en 0,03% feniltiourea en agua de huevo (sal de 0,06 mg / L arrecife en agua desionizada), a partir de una etapa entre 12 y 24 hpf, para inhibir la formación de pigmento. </li><li> Fijar embriones dechorionated o larvas en (A) 4% de paraformaldehído (PFA) en solución salina tamponada con fosfato (PBS) suplementado con 0,1% de Tween 20 (PFAT) durante 1 hora a temperatura ambiente (RT) o (B) 2% PFAT durante la noche a 4 ° C o durante 2 horas a 4 ° C, seguido por 4 horas a RT. Precaución: PFA es tóxico por inhalación y por contacto con la piel. Es destructivo para la piel, las mucosas, los ojos y el tracto respiratorio superior. </li><li> Lavar el embriones 4x para 10 min con PBS suplementado con 0,1% de Tween 20 (PBST) a TA. NOTA: Es muy importante no secar las muestras, mientras que el cambio de las soluciones de lavado. </li><li> Recién preparar el stasolución caniza antes de las reacciones (Tabla 1). Preparar, vórtice, y partes de centrifugadoras A y B en tubos separados. NOTA: La adición de todos los componentes directamente en un único tubo provocaría la precipitación severa. </li><li> Añadir la solución de la totalidad de la parte A a la totalidad de la solución de la parte B, mezclar bien para preparar la solución de tinción, e inmediatamente distribuir 1 ml en cada tubo de microcentrífuga que contiene embriones fijos y lavadas. </li><li> Envolver tubos de microcentrífuga con papel de aluminio para protegerlas de la luz y el lugar ya sea en un rotador o una plataforma de agitación durante agitación suave a temperatura ambiente. </li><li> Empíricamente determinar el tiempo de reacción mediante el control de las señales a través de microscopía cromogénicos. precipitaciones ligeras van a desarrollar con el tiempo; sin embargo, que no interfieran con el proceso de reacción. NOTA: Para los embriones fijan en 28 HPF, tinción a 25 ° C durante 4 horas genera una señal clara en el tejido interrenal. </li><li> Detener la reacción lavando 4 veces durante 10 minutos en PBST. yof no se requiere un mayor análisis de inmunohistoquímica (IHC), posterior a fijar los embriones teñidos con 4% PFAT durante 60 minutos a temperatura ambiente. De lo contrario, almacenar los embriones lavados a 4 ° C hasta su uso posterior. </li><li> Para todo el montaje de microscopía, borrar los embriones teñidos, después de la fijada, y se lavó con 50% de glicerol en PBS, orientarlos con el lado dorsal hacia arriba sobre un portaobjetos, y observar el uso de un microscopio vertical bajo iluminación de campo claro. NOTA: Para hacer una imagen de alta resolución de la morfología de los tejidos interrenal, el análisis del embrión teñido con actividad 3β-HSD y deyolked plana de montaje se recomienda. A medida que el tejido androgénica interrenal se coloca justo encima del saco vitelino, un análisis ventral plana de montaje del tejido interrenal permite una observación directa y clara de la morfología interrenal 17. </li></ol> 3. Análisis IHC de 3β-HSD embriones Actividad manchados NOTA: Para analizar el microambiente vascular de la steroidogenic tejido, los embriones de actividad manchados de 3β-HSD con un indicador fluorescente específico del endotelio transgénico se someten a transversal vibratome seccionamiento y posterior análisis IHC 12,18. <ol><li> Posterior a la fijación <ol><li> Post-fijar los embriones teñidos con actividad 3β-HSD y se lavó con 2% PFA suplementado con 1% de Triton X-100 durante 1 hora a 4 ° C y lavar 4x para 10 min en PBS que contenía 1% de Triton X-100 (PBSTx) a TA. </li></ol></li><li> Incorporación <ol><li> Prepare un 4% bajo punto de fusión de agarosa al disolver la agarosa en PBS a 95 ° C, alícuota en tubos de microcentrífuga y se almacena a 4-8 ° C. </li><li> Fundir una parte alícuota del 4% de agarosa de bajo punto de fusión a 70 ° C durante 10 minutos, y luego mantener la alícuota a 47 ° C. Incrustar cada embrión en fundido 4% bajo punto de fusión de agarosa en un casquillo individual de un tubo de microcentrífuga de 1,5 ml que sirve como un molde y se deja enfriar hasta que solidifique. </li></ol></li><li> vibratome Seccionamiento <ol><li> Pegue un trozo de cinta de papel a la p secalatform de la vibratome. </li><li> Retire el bloque de agarosa endurecido del molde por una hoja de afeitar. Aplicar pegamento de la cinta de papel en la plataforma, y ​​fijar el bloque de agarosa en la cinta de papel, con la cara anterior de la muestra hacia arriba. Recorte el bloque de agarosa a una forma trapezoidal del prisma, con aproximadamente 4 mm y 8 mm a cada lado de las facetas superior e inferior, respectivamente. </li><li> Enjuague el bloque de agarosa que contiene la muestra con PBS frío suplementado con 0,5% de Triton X-100 (0,5% PBSTx) mediante el uso de un cuentagotas. </li><li> Cortar el bloque de agarosa en 100 micras secciones de acuerdo con el manual de instrucciones de la vibratome. Constantemente enjuagar el bloque de agarosa para evitar que se seque. A medida que se corta cada rebanada, quitarlo de la vibratome utilizando una aguja de 26 G jeringa, y transferir a una placa que contiene punto frío 0,5% PBSTx (500 l / pocillo). </li><li> Recoger las secciones de tejido de actividad positivos 3ß-HSD marcando con un microscopio de disección, y transferir las secciones por un pipunta pette con su extremo de corte (alrededor de 1 mm de diámetro interior en la apertura) en una placa de 96 pocillos que contenía 0,5% frío PBSTx (150 l / pocillo). Las muestras de tejido generalmente se disocian a partir del bloque de agarosa después de este paso. </li></ol></li><li> Permeabilización y Lavado <ol><li> Lavar las rodajas 6x durante 15 minutos en 0,5% PBSTx a TA, 10 min en PBS, y 4x para 10 min en PBS suplementado con 1% de albúmina de suero bovino / 1% de DMSO / 0,1% de Triton X-100 (PBDTx). </li></ol></li><li> El bloqueo <ol><li> Incubar las rebanadas de suero 1 hr en 2% de ternera fetal (FCS) en PBDTx a TA. </li></ol></li><li> Reacción primaria de anticuerpos <ol><li> Se incuban las rodajas durante 1,5 días en PBDTx que contienen un anticuerpo primario (por ejemplo, policlonal de conejo anti-fibronectina humana y de ratón anti-humano quinasa de adhesión focal) a 4 ° C. </li></ol></li><li> Lavado <ol><li> Lavar las rodajas de 6x para 10 min en PBDTx a TA. </li></ol></li><li> El bloqueo <ol><li> Se incuban las rodajas de 1 hora en 2% de FCS en PBDTX a TA. </li></ol></li><li> Reacción secundaria de anticuerpos <ol><li> Incubar las rebanadas de 1,5 días en PBDTx que contienen un anticuerpo secundario (fluoróforo conjugado de cabra anti-conejo o anti-IgG de ratón) a 4 ° C. </li></ol></li><li> Lavado <ol><li> Lavar las rodajas de 6x para 10 min en PBDTx a TA y 4x para 10 min en PBST a TA. NOTA: Para el análisis microscópico confocal, desactive las muestras con 50% de glicerol en PBS. </li></ol></li></ol> 4. Análisis Confocal <ol><li> Observar todo el embrión de montaje y la sección por un microscopio confocal equipado con 10X / 20X y 0,5 / 0,75 lente objetivo. </li><li> Para la detección simultánea de la fluorescencia y la tinción 3β-HSD, ajuste el modo multipista de la siguiente manera: 488 nm láser de argón y 505 a 530 nm de paso de banda de filtro para la detección de GFP; 543 nm láser helio-neón y 560 a 615 nm de paso de banda de filtro para la detección de mCherry; y transmitida canal de luz para la detección de manchas 3β-HSD.El tamaño del agujero de alfiler es de 1 unidad Airey, y la resolución es de 1.024 x 1.024 píxeles. </li><li> Montar las pilas z (con intervalo de 6 micras) como una proyección en 3D, y combinar la proyección y el campo brillante, utilizando el software incorporado confocal. </li></ol>

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The authors have no competing financial interests to declare.

La intensidad de la señal de la actividad 3β-HSD aumentó durante el curso de la reacción. se detectaron señales claras de la actividad 3β-HSD después de 4 horas de reacción para las etapas de 28 hpf en adelante. Sin embargo, la duración de la reacción requiere la determinación empírica, dependiendo de la finalidad del ensayo. En los casos en que la tinción requiere procesamiento durante la noche, un fondo ligeramente azulado tiende a desarrollarse en las muestras. Este problema se puede superar mediante el ...

La glándula suprarrenal, un componente crucial del eje hipotálamo-pituitario-adrenal, segrega esteroides y coordina la homeostasis de esteroides y la respuesta del cuerpo al estrés. La glándula suprarrenal comprende la corteza exterior, que segrega los esteroides de manera-zona específica, y médula interna, que sintetiza las catecolaminas. La glándula interrenal en teleósteos es la contraparte de la glándula suprarrenal en los mamíferos y se compone de células cromafines interrenal y androgénica, que son equivalentes funcionales de la corteza suprarrenal y la médula, respectivamente 1-3. Estudios llevados a cabo utilizando el modelo de pez cebra han informado de que los dos linajes de células esteroidogénicas y cromafines se forman por los mecanismos moleculares y celulares altamente parecidas a las de los mamíferos 1,2. Por lo tanto, el pez cebra es un modelo potencialmente poderosa para el estudio de enfermedades genéticas, el control neuroendocrino, y la biología de sistemas del eje hipotálamo-pituitario-adrenal (interrenal). <p class = &quot;jove_content&quot;&gt; En la glándula adrenal, 3β-HSD cataliza la conversión de la progesterona a partir de pregnenolona, 17α-hidroxiprogesterona de 17α-hydroxypregnelolone, y la androstenediona de dehidroepiandrosterona 4,5. 3β-HSD es esencial para la biosíntesis de todas las clases de esteroides hormonales, a saber, la progesterona, glucocorticoides, mineralocorticoides, andrógenos y estrógenos. Los dos humana 3β-HSD isoenzimas hsd3b1 y HSD3B2 se expresan diferencialmente 6. Hsd3b1 se expresa en la placenta y en los tejidos periféricos, mientras que HSD3B2 se expresa en la corteza adrenal y las gónadas. Hsd3b1 humana y HSD3B2 son co-orthologs de hsd3b1 pez cebra, que se expresa en el tejido interrenal gónadas y adultos; HSD3B2 pez cebra es un gen expresado maternalmente cuyas transcripciones desaparecer antes de la organogénesis 7. El protocolo de todo el montaje en el ensayo de actividad enzimática 3β-HSD para el pez cebra fue desarrollado modificando el método de Levy,s descrito por Milano et al. , En secciones congeladas de ocho especies de teleósteos 8. Debido a la permeabilidad del tejido y la transparencia óptica del pez cebra en desarrollo, todo el montaje 3β-HSD la histoquímica se puede utilizar con éxito para el embrión del pez cebra fija y larvas y específicamente delinear los tejidos interrenal diferenciadas. Este ensayo sensible y rápido se ha aplicado a diversos mutantes y morphants que demuestran diferentes tipos de dysmorphogenesis interrenal. La actividad 3β-HSD interrenal está ausente en el embrión, donde la especificación del tejido interrenal se interrumpe a través de una caída específica del factor de transcripción ff1b y se disminuyó a medida que la diferenciación interrenal se ve afectada por una caída de la coregulator ff1b Prox1 9,10. En particular, la actividad 3β-HSD se puede detectar en los mutantes con defectos severos en fase inicial, tales como una cabeza de alfiler de ojos y estrabismo, donde el 3β-Hhistoquímica sd delinea cómo la migración de las células se ve afectado interrenal 11. La diferenciación del tejido interrenal no se vea comprometida incluso en ausencia completa de la sangre y la vasculatura. Por lo tanto, cómo las señales derivadas del endotelio forma al órgano interrenal en desarrollo puede ser determinada 12,13. En general, este ensayo histoquímico ha sido utilizado con éxito para el estudio de la especificación, diferenciación y migración de las células esteroidogénicas en el modelo de pez cebra. Por lo tanto, debe ser un una herramienta fiable para todas las pantallas genéticas o químicas dirigidas a trastornos de los órganos suprarrenales y interrenal eficiente y.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Para determinar cómo las codevelops tejido interrenal androgénica, con el glomérulo renal pronephric y su naciente sistema vascular, el ensayo de actividad enzimática 3β-HSD se llevó a cabo en el doble transgénico Tg (wt1b: GFP) LI1; Tg (kdrl: mCherry) CI5 embrión en 34 HPF (Figura 1). En esta etapa, el tejido steroidogenic actividad positiva 3β-HSD está situado justo a la línea media y caudal inmediatamente al glomérulo renal p...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

La glándula interrenal en el pez cebra es la contraparte teleósteos de la glándula adrenal de los mamíferos. Este protocolo presenta cómo realizar la deshidrogenasa 3-β-hidroxiesteroide (Δ 5-4 isomerasa; 3β-HSD) ensayo de actividad enzimática, que detecta las células esteroidogénicas diferenciadas en el pez cebra en desarrollo.

La visualización del Tejido Interrenal androgénica, y su microambiente vascular en el pez cebra

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Research

JoVE Journal

Developmental Biology

6.9K vistas.  Tunghai University.  La glándula interrenal en el pez cebra es la contraparte teleósteos de la glándula adrenal de los mamíferos. Este protocolo presenta cómo realizar la deshidrogenasa 3-β-hidroxiesteroide (Δ 5-4 isomerasa; 3β-HSD) ensayo de actividad enzimática, que detecta las células esteroidogénicas diferenciadas en el pez cebra en desarrollo. 

Video: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Análisis para el Desarrollo de riñón de pez cebra con time-lapse de uso de un microscopio de disección equipado para seccionamiento óptico

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Investigación

Biología del Desarrollo

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Inmunotinción fosfo-epítopos en ciliadas Órganos de todo el montaje embriones de pez cebra

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Química

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

La disección de las larvas de pez cebra de tejidos gonadales

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Investigación sobre el cáncer

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 ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Pez cebra adulto lesión modelos para estudiar los efectos de la prednisolona en regeneración de tejido óseo

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Cuantificación del tamaño del hígado en el pez cebra larvario mediante la microscopía Brightfield

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Dissection of the Adult Zebrafish Kidney

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem cells during normal homeostasis and disease states. The understanding of adult stem cells has broad reaching implications for the future of regenerative medicine1. For example, better knowledge about adult stem cell biology can facilitate the design of therapeutic strategies in which organs are triggered to heal themselves or even the creation of methods for growing organs in vitro that can be transplanted into humans1. The zebrafish has become a powerful animal model for the study of vertebrate cell biology2. There has been extensive documentation and analysis of embryonic development in the zebrafish3. Only recently have scientists sought to document adult anatomy and surgical dissection techniques4, as there has been a progressive movement within the zebrafish community to broaden the applications of this research organism to adult studies. For example, there are expanding interests in using zebrafish to investigate the biology of adult stem cell populations and make sophisticated adult models of diseases such as cancer5. Historically, isolation of the zebrafish adult kidney has been instrumental for studying hematopoiesis, as the kidney is the anatomical location of blood cell production in fish6,7. The kidney is composed of nephron functional units found in arborized arrangements, surrounded by hematopoietic tissue that is dispersed throughout the intervening spaces. The hematopoietic component consists of hematopoietic stem cells (HSCs) and their progeny that inhabit the kidney until they terminally differentiate8. In addition, it is now appreciated that a group of renal stem/progenitor cells (RPCs) also inhabit the zebrafish kidney organ and enable both kidney regeneration and growth, as observed in other fish species9-11. In light of this new discovery, the zebrafish kidney is one organ that houses the location of two exciting opportunities for adult stem cell biology studies. It is clear that many outstanding questions could be well served with this experimental system. To encourage expansion of this field, it is beneficial to document detailed methods of visualizing and then isolating the adult zebrafish kidney organ. This protocol details our procedure for dissection of the adult kidney from both unfixed and fixed animals.
Dissection of the kidney organ can be used to isolate and characterize hematopoietic and renal stem cells and their offspring using established techniques such as histology, fluorescence activated cell sorting (FACS)11,12, expression profiling13,14, and transplantation11,15. 
We hope that dissemination of this protocol will provide researchers with the knowledge to implement broader use of zebrafish studies that ultimately can be translated for human application.

The zebrafish kidney is home to both renal and hematopoietic adult stem/progenitor cells, and represents an outstanding opportunity to study these cell types and their progeny in a vertebrate model organism. Here, we demonstrate a detailed dissection procedure that enables the researcher to identify and surgically remove the adult zebrafish kidney, which can be used for applications such as cell isolation, transplantation, and expression studies of kidney and/or blood cell populations.

La disección del riñón adultos de pez cebra

Researchers working in the burgeoning field of adult stem cell biology seek to understand the signals that regulate the behavior and function of stem ...

Biología

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Estudiar la Diabetes a través de los ojos de un pez: Microdissection, visualización y análisis de la vasculatura de retina de pez cebra de adulto tg(fli:EGFP)

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medicina

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 ...