Gracias a Marie Birne, Tsiola Areti, Estévez Jaymie y Arelys Uribe. Este trabajo fue apoyado en parte por la Fundación de Investigación de CUNY y Howard Hughes Medical Institute Programa de Verano de becas para estudiantes de pregrado (SPUR) y NIH SR3 41702-00-01. La investigación también fue apoyada por el Queens College y algunos de los experimentos se realizaron en el equipo del Fondo para el núcleo de la Imagen, Biología Celular y Molecular de la Universidad de Queens.

Pez cebra se plantearon a través de protocolos estándar de 13 en las instalaciones de animales Queens College a 28 ° C. Los distintos pares de pescado o apareamientos grupo debe ser preformado y los embriones resultantes limpian y se almacenan en una incubadora de 28 ° C. Salud de los peces deben ser examinados todos los días. En la fecundación después de 5 días (DPF), el pescado debe ser separada en los tanques, con una densidad de peces 10-15 y entró en la guardería instalación de pescado. Para que el protocolo que aquí se presenta el pez cebra se recogieron en larva a través de las etapas adultas: 15, 30, 45, 60, 90, 180 DPF. 1. Pez cebra colección tema y la fijación <ol><li> Retire el pescado del tanque de hábitat por medio de net y el lugar en el 0,2% tricaína para anestesiar. </li><li> Coloque el pescado anestesiado en agua helada durante 15 minutos para practicar la eutanasia. </li><li> Hacer paraformaldehido fresco 4% (PFA) en 1x de tampón fosfato salino (PBS). </li><li> Incubar peces recogidos en PFA al 4% durante 1-2 horas a temperatura ambiente durante la noche a 4 ° C en una placa de Petri envuelto en parafilm. Esto mantiene el piso de peces y más tarde hace una disección mucho más fácil. </li><li> Después de la fijación, lavar dos veces en PBS con Tween (PBT). El pescado puede ser tener en PBT a 4 ° C durante un máximo de 10 días. </li></ol> 2. Medición de los peces y la disección <ol><li> Coloque el pescado en una sola media placa de Petri llena de PBS y lo pondré sobre su lado derecho. </li><li> Medir la longitud de los peces del hocico a la base de la cola (el pedúnculo caudal), sin incluir las aletas de la cola, utilizando un calibrador digital, mm (Figura 1). Esta es la longitud estándar (SL). </li><li> Cada pez se le puede asignar un número / combinación de letras para permitir que un pez y el corazón como consecuencia disecados para realizar un seguimiento para su posterior análisis. Datos, incluyendo el número de acciones de los padres, edad en la fijación, las mediciones de SL, se introdujeron en una hoja de cálculo y organizada sobre la base de estas etiquetas. </li><li> Antes de la disección, la etiqueta tiras de tubos PCR con número de seguimiento asignado y se llenan de PBT u otra solución apropiada para su posterior análisis. </li><li> Orientar a los peces ventral hacia arriba en la placa de Petri, mientras que la estabilización del cuerpo con pinzas de sujeción de la cabeza entre los ojos y las agallas (Figura 2). </li></ol> Los peces más pequeños (menores de 12 mm SL): <ol start="4"><li> a) El uso de pinzas cortantes, o una micro-aguja en un soporte de pasador, quitar los músculos pectorales y las aletas del cuerpo para revelar el corazón. El constante movimiento de la pinza durante la disección puede causar corrientes en el PBT que se mueven los peces. Esto es particularmente problemático para los peces más pequeños, por lo tanto una micro-aguja se puede utilizar para eliminar los músculos pectorales y las aletas y abrir la cavidad del cuerpo. </li><li> a) utilizar con cuidado las pinzas para sacar el corazón de la cavidad de debajo de la aurícula. Alternativamente, en algunos peces que puede ser capaz de extraer el corazón, tirando suavemente hacia fuera con bulbo y el resto del corazón seguirá. Tenga cuidado con la ubicación de la aurícula si se utiliza esta técnica, ya que pueden dañarse fácilmente. Si hay tejido extra que viene con el corazón, que está muy bien y pueden ser eliminados después. </li><li> a) Si el pescado contiene un marcador fluorescente cardiacas como Tg (myl7: GFP) twu34, usar un ámbito de fluorescentes para la disección y verificar la extracción del corazón por el florecimiento (Figura 3). </li><li> a) Una vez que el corazón se retira de la cavidad, utilice las pinzas para mantener el corazón y una micro-aguja para extraer adicional no tejido cardíaco y la pared epicárdica de la parte exterior del corazón. </li></ol> Los peces más grandes (más de 12 mm SL): <ol start="4"><li> b) Utilización de la primavera maneja micro-tijeras, hacen tres incisiones de menos de 1mm de profundidad: 1 - corte transversal a través de los dos branquias - corte transversal en el vientre anterior 3 - corte sagital en el lado ventral de conectar los dos cortes (Figura 4). </li><li> b) El uso de pinzas afiladas eliminar los músculos pectorales y las aletas del cuerpo para abrir la cavidad del cuerpo y revelar el tejido plateado del pericardio (Figura 5). Eliminar este tejido de pericardio y el corazón se vuelve visible (Figura 5). </li><li> b) Utilice las tijeras para cortar micro-la arteria conectada al bulbo arterioso, que se encuentra superior a la del corazón. Una vez que esta arteria se corta, se puso las puntas de unas pinzas en el atrio, utilice las pinzas para sacar el corazón de la cavidad. Si hay tejido extra que viene con el corazón, que está muy bien y se puede quitar más tarde. </li><li> b) Una vez que el corazón se retira de la cavidad, use las dos pinzas para mantener el corazón y retirar el tejido extra, o un par de pinzas para mantener el corazón y una micro-aguja para extraer extra, no de tejido cardíaco. El revestimiento epicárdica, evidente por su pigmentación negro, puede ser removido de la parte exterior del corazón, si lo desea, raspando suavemente en el tejido. </li></ol> 3. Fotografía de los corazones <ol><li> Prepare una 23g / L de agar nutritivo plato de Petri y la cubierta de agar solidificado con PBS. </li><li> Usando las pinzas, hacer un pozo en el agar que permite que el corazón descanse en la orientación deseada para la fotografía. </li><li> Remove el centro del tubo de PCR mediante la celebración de la punta del bulbo con las pinzas con firmeza. Coloque el corazón en el plato de agar. </li><li> Orientar el corazón en agar para fotografiar. La muestra se puede girar después de cada imagen para todas las orientaciones posibles (Figura 6). </li><li> Utilice el tiempo de exposición aérea ligeros y cortos para fotografiar el corazón. Haga los ajustes de calidad de la luz con la exposición como sea necesario para obtener la mejor fotografía. </li></ol> 4. Los resultados representativos: Un ejemplo de un corazón bien diseccionado limpia se muestra en múltiples orientaciones en la Figura 6. Cada centro tendrá algunas diferencias en la morfología general, pero el corazón debe ser completa, que contiene un ventrículo intacto, el atrio y bulbo arterioso. Algunos ejemplos de esta variación en la morfología general se muestra en la Figura 7. El negro manchado tejido epicárdico puede arrancar del corazón. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Figura 1"/> Figura 1. Medir el pescado desde el hocico hasta el pedúnculo caudal con una pinza digitales mm. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Figura 2"/> Figura 2. Sostenga el pescado con una pinza entre los ojos y agallas. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Figura 3"/> Figura 3 cardiaco específico marcador fluorescente transgénicos como Tg (myl7: GFP). Twu34, se muestra aquí, puede ayudar en la visualización de los corazones disecados de los peces más pequeños. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Figura 4"/> Figura 4 Tres incisiones para eliminar los músculos pectorales y la piel: 1 - corte transversal a través de las branquias 2 - corte transversal en el vientre anterior 3 - corte sagital en el lado ventral de conectar los dos cortes.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Figura 5"/> . Cartas de la figura 5 Negro etiqueta de las cámaras del corazón: bulbo arterioso (bis), en el ventrículo (V), y el atrio (a). A-La cavidad del cuerpo superior está abierta para mostrar el tejido plateado del pericardio. B-Después de retirar el pericardio, el corazón es visible y fácil de retirar. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Figura 6"/> Orientaciones Figura 6. Diferentes del corazón se muestran. Letras en negro etiqueta de las cámaras del corazón: bulbo arterioso (bis), en el ventrículo (V), y el atrio (a). Flechas grises indican la ubicación y orientación del corazón, en referencia a su posición normal en el cuerpo: anterior (A), posterior (P), dorsal (D), ventral (V), izquierda (L) y derecha (R). Barra de escala indica 400μm. A-ventral vista del corazón B-vista dorsal del corazón C-vista lateral del lado izquierdo D-vista lateral del lado derecho <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Figura 7"/> Figura 7. Morfología de los corazones de diferentes tamaños se muestra. Letras en negro etiqueta de las cámaras del corazón: bulbo arterioso (bis), en el ventrículo (V), y el atrio (a). Barra de escala se indica 400 micras. AC-Corazones forma de pez con menos de 12 mm SL DF-Corazones con forma de pez SL mayor que 12 mm

<ol>
	<li>Ahmad, N., Long, S., Rebagliati, 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+southpaw+joins+the+roster:+the+role+of+the+zebrafish+nodal-related+gene+southpaw+in+cardiac+LR+asymmetry.">A southpaw joins the roster: the role of the zebrafish nodal-related gene southpaw in cardiac LR asymmetry.</a> Trends Cardiovasc Med. 14, 43-49 (2004).</li><li>Armstrong, E. J., Bischoff, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heart+valve+development:+endothelial+cell+signaling+and+differentiation.">Heart valve development: endothelial cell signaling and differentiation.</a> Circ Res. 95, 459-470 (2004).</li><li>Bakkers, J., Verhoeven, M. C., Abdelilah-Seyfried, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shaping+the+zebrafish+heart:+from+left-right+axis+specification+to+epithelial+tissue+morphogenesis.">Shaping the zebrafish heart: from left-right axis specification to epithelial tissue morphogenesis.</a> Dev Biol. 330, 213-220 (2009).</li><li>Glickman, N. S., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+development+in+zebrafish:+coordination+of+form+and+function.">Cardiac development in zebrafish: coordination of form and function.</a> Semin Cell Dev Biol. 13, 507-513 (2002).</li><li>Heicklen-Klein, A., McReynolds, L. J., Evans, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Using+the+zebrafish+model+to+study+GATA+transcription+factors.">Using the zebrafish model to study GATA transcription factors.</a> Semin Cell Dev Biol. 16, 95-106 (2005).</li><li>Schoenebeck, J. J., Yelon, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Illuminating+cardiac+development:+Advances+in+imaging+add+new+dimensions+to+the+utility+of+zebrafish+genetics.">Illuminating cardiac development: Advances in imaging add new dimensions to the utility of zebrafish genetics.</a> Semin Cell Dev Biol. 18, 27-35 (2007).</li><li>Beis, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genetic+and+cellular+analyses+of+zebrafish+atrioventricular+cushion+and+valve+development.">Genetic and cellular analyses of zebrafish atrioventricular cushion and valve development.</a> Development. 132, 4193-4204 (2005).</li><li>Lepilina, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+dynamic+epicardial+injury+response+supports+progenitor+cell+activity+during+zebrafish+heart+regeneration.">A dynamic epicardial injury response supports progenitor cell activity during zebrafish heart regeneration.</a> Cell. 127, 607-619 (2006).</li><li>Wills, A. A., Holdway, J. E., Major, R. J., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulated+addition+of+new+myocardial+and+epicardial+cells+fosters+homeostatic+cardiac+growth+and+maintenance+in+adult+zebrafish.">Regulated addition of new myocardial and epicardial cells fosters homeostatic cardiac growth and maintenance in adult zebrafish.</a> Development. 135, 183-192 (2008).</li><li>Parichy, D. M., Elizondo, M. R., Mills, M. G., Gordon, T. N., Engeszer, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Normal+table+of+postembryonic+zebrafish+development:+staging+by+externally+visible+anatomy+of+the+living+fish.">Normal table of postembryonic zebrafish development: staging by externally visible anatomy of the living fish.</a> Dev Dyn. 238, 2975-3015 (2009).</li><li>Huang, C. J., Tu, C. T., Hsiao, C. D., Hsieh, F. J., Tsai, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Germ-line+transmission+of+a+myocardium-specific+GFP+transgene+reveals+critical+regulatory+elements+in+the+cardiac+myosin+light+chain+2+promoter+of+zebrafish.">Germ-line transmission of a myocardium-specific GFP transgene reveals critical regulatory elements in the cardiac myosin light chain 2 promoter of zebrafish.</a> Dev Dyn. 228, 30-40 (2003).</li><li>Westerfield, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Zebrafish+Book,+A+guide+for+the+laboratory+use+of+zebrafish+(Danio+rerio).">The Zebrafish Book, A guide for the laboratory use of zebrafish (Danio rerio).</a> , (1995).</li></ol>

No hay conflictos de interés declarado.

Estos métodos para la disección del corazón del pez cebra de post-embrionario de peces permite la extirpación completa de un corazón en buen estado. Si bien esta técnica es sencilla y ofrece guías para los cortes externos que evitar que se dañe el corazón, una mano firme es esencial para los peces más pequeños. Un paso clave en la disección es identificar el pericardio plateado como el corazón está directamente dentro de este saco. Visualizar el pericardio proporciona un marcad...

<table border="1"><tbody><tr><th> Material </th><th> Empresa </th><th> Número de catálogo </th><th> Comentarios (opcional) </th></tr><tr><td> Paraformaldehído </td><td> EMD </td><td> PX0055 </td><td> .04g/mL En PBS </td></tr><tr><td> Pinzas # 55 </td><td> FST </td><td> 11295-51 </td><td></td></tr><tr><td> Micro-tijeras </td><td> FST </td><td> 15005-08 </td><td></td></tr><tr><td> Pin titular </td><td> FST </td><td> 26016-12 </td><td></td></tr><tr><td> Micro-agujas </td><td> FST </td><td> 10130-10 </td><td></td></tr><tr><td> Agar nutritivo </td><td> BBL </td><td> 11472 </td><td> 23 g / L en agua destilada H 2 O </td></tr><tr><td> Placas de Petri </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR tiras de 8 tubos </td><td> EE.UU. Científico </td><td> 1402-2500 </td><td></td></tr><tr><td> Calibre digital </td><td> Fisher Scientific </td><td> 14-648-17 </td><td></td></tr></tbody></table>

heart dissection in larval, juvenile and adult zebrafish, danio rerio

Pez cebra se han convertido en un organismo modelo beneficioso y práctico para el estudio del desarrollo embrionario del corazón (ver comentarios de los últimos 1-6), sin embargo, el trabajo de examen de post-embrionario a través del desarrollo adulto cardiaca se ha limitado 7.10. Examinar el cambio de morfología del corazón de maduración y el envejecimiento se ven limitadas por la falta de técnicas disponibles para la puesta en escena y el aislamiento de los corazones juveniles y adultos. Con el fin de analizar el desarrollo del corazón durante la vida útil del pescado, que diseccionan el corazón del pez cebra en varias etapas y una fotografía para su posterior análisis 11. Las características morfológicas del corazón puede ser cuantificado y corazones individuales pueden ser analizados por una serie de métodos estándar. Pez cebra crecen a tasas variables y la maduración se correlaciona mejor con el tamaño de los peces de la edad, por lo tanto, posterior a la fijación, longitud del pez fotografía y medida como un indicador de la maduración de los peces. Este protocolo se explican dos técnicas distintas, el tamaño de la disección dependientes de pez cebra, que van desde 3,5 mm de longitud estándar larvas (SL) con el corazón de la longitud del ventrículo 100μm (VL), a los adultos, con el SL de 30 mm y 1 mm de VL o más grande. Las larvas de peces y adultos tienen el cuerpo muy diferente y la morfología de órganos. Las larvas no sólo son significativamente más pequeños, tienen menos pigmento y cada órgano es visualmente muy difíciles de identificar. Por esta razón, utilizamos distintas técnicas de disección. Se utilizó antes de la disección de los procedimientos de fijación, como hemos descubierto que el corazón disecado directamente después de la eutanasia tienen una morfología más variable, con el balón muy flojo y como aurículas en comparación con el corazón eliminado después de la fijación. Los peces fijo antes de la disección, mantener vivo en la morfología y la posición de la cámara (datos no mostrados). Además, para fines de demostración, nos aprovechamos del corazón (miocardio) específicos GFP transgénicos Tg (myl7: GFP) twu34 (12), que nos permite visualizar todo el corazón y es particularmente útil en las primeras etapas de desarrollo, cuando el corazón morfología es menos clara de los tejidos circundantes. La disección del corazón hace un mayor análisis de la biología celular y molecular que subyacen al desarrollo del corazón y la maduración utilizando hibridación in situ, inmunohistoquímica, la extracción de RNA o otros métodos analíticos más fácil en el post-embrionario del pez cebra. Este protocolo proporciona una técnica valiosa para el estudio de la maduración de desarrollo cardíaco y el envejecimiento.

Watch this Scientific Journal Video about Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio at JoVE.com

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Un método claro y estandarizado para la disección y el aislamiento del corazón del pez cebra en varias etapas de desarrollo se describen. Técnicas de anotación y cuantificación también se discuten.

La disección del corazón en las larvas, juveniles y adultos de pez cebra, Danio rerio</em

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work ...

heart-dissection-in-larval-juvenile-and-adult-zebrafish-danio-rerio

Research

JoVE Journal

Biology

Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

21.4K vistas.  Queens College, City University of New York. Un método claro y estandarizado para la disección y el aislamiento del corazón del pez cebra en varias etapas de desarrollo se describen. Técnicas de anotación y cuantificación también se discuten.

Video: Heart Dissection in Larval, Juvenile and Adult Zebrafish, Danio rerio

Dissection of Larval CNS in Drosophila Melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to examine the expression of various novel and marker proteins.  Staining of whole larvae is impractical because the tough cuticle prevents antibodies from penetrating inside the body cavity.  In order to stain these tissues it is necessary to dissect the animal prior to fixing and staining.  In this article we demonstrate how to dissect Drosophila larvae without damaging the CNS.  Begin by tearing the larva in half with a pair of fine forceps, and then turn the cuticle "inside-out" to expose the CNS.  If the dissection is performed carefully the CNS will remain attached to the cuticle.  We usually keep the CNS attached to the cuticle throughout the fixation and staining steps, and only completely remove the CNS from the cuticle just prior to mounting the samples on glass slides.  We also show some representative images of a larval CNS stained with Eve, a transcription factor expressed in a subset of neurons in the CNS.  The article concludes with a discussion of some of the practical uses of this technique and the potential difficulties that may arise.

In this article we demonstrate how to dissect the central nervous system from third instar Drosophila larvae.

La disección del sistema nervioso central de larvas de Drosophila melanogaster

The central nervous system (CNS) of Drosophila larvae is complex and poorly understood.  One way to investigate the CNS is to use immunohistochemistry to ...

Investigación

Biología

Drosophila Larval NMJ Dissection

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use of the Drosophila motor system is due to its high accessibility. It can be analyzed with single-cell resolution. There are 30 muscles per hemisegment whose arrangement within the peripheral body wall are known. A total of 35 motor neurons attach to these muscles in a pattern that has high fidelity. Using molecular biology and genetics, one can create transgenic animals or mutants. Then, one can study the developmental consequences on the morphology and function of the NMJ. In order to access the NMJ for study, it is necessary to carefully dissect each larva. In this article we demonstrate how to properly dissect Drosophila larvae for study of the NMJ by removing all internal organs while leaving the body wall intact. This technique is suitable to prepare larvae for imaging, immunohistochemistry, or electrophysiology.

This protocol demonstrates how to dissect Drosophila larvae in preparation for immunohistochemistry and/or imaging of the neuromuscular junction.

Drosophila larval NMJ Disección

The Drosophila neuromuscular junction (NMJ) is an established model system used for the study of synaptic development and plasticity. The widespread use ...

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish (Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss.
Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.

Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio

The inner ear sensory epithelium of adult zebrafish is a good model system for understanding the mechanisms of hair cell regeneration in adult vertebrates. This protocol demonstrates the fine dissection of the epithelia, through which we can get tissue samples for studying the regenerative events at cellular and subcellular levels.

La disección fina y gruesa de los epitelios sensoriales del oído interno en adultos de pez cebra ( Danio rerio)

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the ...

Dissection of Organs from the Adult Zebrafish

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition.  Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling.  In addition, the bipotential gonad primordium develops into either testis or ovary.  This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques.  This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

This protocol describes a procedure for identifying and dissecting organs from the adult zebrafish.

La disección de los órganos del pez cebra adultos

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease.  Although experimental ...

Dissection and Staining of Drosophila Larval Ovaries

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells form, and how they interact with their environment is therefore crucial for understanding development, homeostasis and disease. The ovary of the fruit fly Drosophila melanogaster has served as an influential model for the interaction of germ line stem cells (GSCs) with their somatic support cells (niche) 1, 2. The known location of the niche and the GSCs, coupled to the ability to genetically manipulate them, has allowed researchers to elucidate a variety of interactions between stem cells and their niches 3-12.
Despite the wealth of information about mechanisms controlling GSC maintenance and differentiation, relatively little is known about how GSCs and their somatic niches form during development. About 18 somatic niches, whose cellular components include terminal filament and cap cells (Figure 1), form during the third larval instar 13-17. GSCs originate from primordial germ cells (PGCs). PGCs proliferate at early larval stages, but following the formation of the niche a subgroup of PGCs becomes GSCs 7, 16, 18, 19. Together, the somatic niche cells and the GSCs make a functional unit that produces eggs throughout the lifetime of the organism.
Many questions regarding the formation of the GSC unit remain unanswered. Processes such as coordination between precursor cells for niches and stem cell precursors, or the generation of asymmetry within PGCs as they become GSCs, can best be studied in the larva. However, a methodical study of larval ovary development is physically challenging. First, larval ovaries are small. Even at late larval stages they are only 100&mu;m across. In addition, the ovaries are transparent and are embedded in a white fat body. Here we describe a step-by-step protocol for isolating ovaries from late third instar (LL3) Drosophila larvae, followed by staining with fluorescent antibodies. We offer some technical solutions to problems such as locating the ovaries, staining and washing tissues that do not sink, and making sure that antibodies penetrate into the tissue. This protocol can be applied to earlier larval stages and to larval testes as well.

Dissection and Staining of Drosophila Larval Ovaries

How niches and stem cells form during development is an important question with practical implications. In the Drosophila ovary, germ line stem cells and their somatic niches form during larval development. This video demonstrates how to dissect, stain and mount female gonads from late third instar (LL3) Drosophila larvae.

La disección y la tinción de Drosophila Larvas Ovarios

Many organs depend on stem cells for their development during embryogenesis and for maintenance or repair during adult life. Understanding how stem cells ...

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 ...

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal neurons. In contrast, the adult zebrafish retina possesses the robust ability to spontaneously regenerate any neuronal class that is lost in a variety of different retinal damage models, including retinal puncture, chemical ablation, concentrated high temperature, and intense light treatment 1-8. Our lab extensively characterized regeneration of photoreceptors following constant intense light treatment and inner retinal neurons after intravitreal ouabain injection 2, 5, 9. In all cases, resident M&uuml;ller glia re-enter the cell cycle to produce neuronal progenitors, which continue to proliferate and migrate to the proper retinal layer, where they differentiate into the deficient neurons. 
	We characterized five different stages during regeneration of the light-damaged retina that were highlighted by specific cellular responses. We identified several differentially expressed genes at each stage of retinal regeneration by mRNA microarray analysis 10. Many of these genes are also critical for ocular development. To test the role of each candidate gene/protein during retinal regeneration, we needed to develop a method to conditionally limit the expression of a candidate protein only at times during regeneration of the adult retina. 
	Morpholino oligos are widely used to study loss of function of specific proteins during the development of zebrafish, Xenopus, chick, mouse, and tumors in human xenografts 11-14. These modified oligos basepair with complementary RNA sequence to either block the splicing or translation of the target RNA. Morpholinos are stable in the cell and can eliminate or "knockdown" protein expression for three to five days 12. 
	Here, we describe a method to efficiently knockdown target protein expression in the adult zebrafish retina. This method employs lissamine-tagged antisense morpholinos that are injected into the vitreous of the adult zebrafish eye. Using electrode forceps, the morpholino is then electroporated into all the cell types of the dorsal and central retina. Lissamine provides the charge on the morpholino for electroporation and can be visualized to assess the presence of the morpholino in the retinal cells. 
	Conditional knockdown in the retina can be used to examine the role of specific proteins at different times during regeneration. Additionally, this approach can be used to study the role of specific proteins in the undamaged retina, in such processes as visual transduction and visual processing in second order neurons.

In vivo Electroporation of Morpholinos into the Adult Zebrafish Retina

A method to conditionally knockdown a target protein&#x2019;s expression in the adult zebrafish retina is described, which involves intravitreally injecting antisense morpholinos and electroporating them into the retina. The resulting protein is knocked down for several days, which allows testing the protein&#x2019;s role in the regenerating or intact retina.

En electroporación in vivo de Morpholinos en la retina de pez cebra adultos

Many devastating inherited eye diseases result in progressive and irreversible blindness because humans cannot regenerate dying or diseased retinal ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

Cuidado y mantenimiento regulares de un pez cebra (<em&gt; Danio rerio</em&gt;) Laboratorio: Introducción

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

En Vivo Modelado del Genoma Humano Morbid usando Danio rerio

Here,  we present methods for the development of assays to query potentially clinically  significant nonsynonymous changes using in  vivo complementation ...

Gavaging Adult Zebrafish

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or agents in solutions for scientific research. Current methods for administering compounds orally to adult zebrafish are inaccurate due to variability in voluntary consumption by the fish. A gavage procedure was developed to deliver precise quantities of infectious agents to zebrafish for study in biomedical research. Adult zebrafish over 6 months of age were anesthetized with 150 mg/L of buffered MS-222 and gavaged with 5 &mu;l of solution using flexible catheter implantation tubing attached to a cut 22-G needle tip. The flexible tubing was lowered into the oral cavity of the zebrafish until the tip of the tubing extended past the gills (approximately 1 cm). The solution was then injected slowly into the intestinal tract. This method was effective 88% of the time, with fish recovering uneventfully. This procedure is also efficient as one person can gavage 20-30 fish in one hour. This method can be used to precisely administer agents for infectious diseases studies, or studies of other compounds in adult zebrafish.

The increasing use of zebrafish as an animal model requires the development of effective methods for the delivery of known quantities of compounds and solutions. The gavage procedure described below allows for the oral delivery of precise volumes of solution reliably, safely and efficiently to adult zebrafish.

Gavage adultos de pez cebra

The zebrafish has become an important in vivo model in biomedical research. Effective methods must be developed and utilized to deliver compounds or ...

La disección del corazón en las larvas, juveniles y adultos de pez cebra, Danio rerio

Resumen

Explorar más videos

Capítulos en este video