Agradecemos a la Sra. Fan Shen por su apoyo a la crianza de pez cebra. Esto es apoyado por las financiaciones al Dr. Hu Bing (Academia China de Ciencias, &quot;Bai Ren Ji Hua&quot;, NSFC 30.870.833 y el Programa Nacional de Ciencia Básica de China, 2009CB941302). Todos los experimentos con animales se realizaron de conformidad con las directrices y normas establecidas por la Universidad de Ciencia y Tecnología de China (USTC) Animal del Centro de Recursos y la Universidad de Cuidado de Animales y el empleo.

Parte I: Construir el aparato okr Los detalles se muestran en el vídeo de demostración. Parte II: okr de adultos de pez cebra Si su laboratorio ha construido un aparato okr, la clave de los adultos de pez cebra okr es mantener a los peces. En esta parte, vamos a mostrar paso a paso. Nuestro aparato compone las siguientes partes similar a los otros mencionados. La cámara circular de pescado es de 7,0 cm de diámetro exterior, 6,5 cm de diámetro interior y 7,0 cm de alto. El tambor alrededor de la cámara de pescado es de 8,5 cm de diámetro a cabo, 7,5 cm de diámetro interior y 7,0 cm de alto. Podría girar en sentido horario y antihorario con la velocidad de 20 ~ 70 rotaciones por minuto (rpm). Once ciclos de rayas blanco y negro se dibuja en un papel los trazos que se adhirió en el exterior del tambor. El sistema de iluminación se compone de dos tubos de un círculo de luz de diámetro de 21 cm. <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="Figura 1" /> Figura 1. El modelo de la esponja. El acantilado hecho en este medio es para asegurarse de que el campo visual no se estaba protegido. <ol><li> Una esponja como se muestra en la Fig. 1 compuesto por dos partes. La parte izquierda es la longitud de 3 cm de ancho x 3 cm de alto x 2 cm. La parte derecha es la longitud de 3 cm de ancho x 3 cm de alto x 3 cm. </li><li> Añadir este esponja en la parte inferior de la cámara de pescado y vierta el agua en la crianza de unos 2 cm de profundidad. Burbuja de aire que se esconden en la esponja debe ser expulsado. </li><li> Insertar un pin en la parte delantera de la parte derecha de la esponja, de unos 2 mm por detrás del acantilado. </li><li> Anestesiar un pez cebra adulto en el MS-222. </li><li> Poner el pez cebra en la esponja con un máximo de dorsales y permitir que una parte de su aleta pectoral antes de que el primer pin. </li><li> El segundo pasador se inserta detrás de la aleta pectoral otros. Se podría evitar el cuerpo del pez cebra reducción mediante la cooperación con el primer pasador (ver también en la fig. 2). </li><li> Abrazadera de la cola con una pinza de la arteria y fijar la pinza de la arteria en un trozo de goma (arcilla) que se adhiere a la cámara de los peces. </li><li> El pasador de tercer y cuarto se planta en el sitio cerca de la cloaca de los poros del cuerpo de los peces. Podría prevenir el pez cebra para saludar la cola intensamente. </li><li> El pin quinta está situada entre el primer y el tercer pin y luego el pin sexto en el otro lado de la misma. Que podrían prevenir el pez cebra de la sacudida de su cuerpo. </li><li> El pasador de séptimo y octavo se inserta en los sitios cerca de las agallas para mantener la orientación de la cabeza. </li><li> Vierta el agua en la cámara de cría de peces. </li><li> Después de que el pez cebra se despierta, vamos a adaptarlo a 5 ~ 10 minutos. </li><li> Encender la luz de este aparato y dejar que el pez cebra adaptar este tipo de iluminación durante 2 minutos. </li><li> Encienda el motor para impulsar la rejilla y permiten adaptar el pez cebra durante 1 minuto. </li><li> Encienda la cámara y registrar el movimiento de los ojos. </li><li> Analizar los 6 ángulos de los ojos y los números sacada en su ordenador personal (un ejemplo representativo del análisis de los ángulos del ojo se muestra en la Fig. 2). </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="Figura 2" /> Figura 2. El ángulo entre el eje óptico y el eje del cuerpo. a) Cuando la dirección de la rejilla es nasal a temporal, que define el ángulo en el frente del cuerpo de los peces es un ángulo negativo y el ángulo en el cuerpo de los peces es un ángulo positivo, a la inversa (para una revisión breve también ver ref. 6). Los números en esta cifra representa la secuencia de los pines. b) okr ejemplo, el análisis de un pez en el grupo normal. Una película de un segundo fue trasladado a una serie de cuadros y los ángulos de los ojos de cada cuadro se midió con Image-Pro Plus. Cada punto en que esta cifra representa cada cuadro se midió. Notas: <ol class="lalpha"><li> Todos los pines son utilizados para prevenir el pez cebra de la sacudida de su cuerpo, demasiado apretado será perjudicial para los peces. </li><li> Abrazadera puede dañar la arteria de la cola como su área de contacto es pequeño. Una vez que la cola es libre, s muy difícil de solucionar este pescado. Envuélvalo con una venda alrededor de la cola se recomienda en algunas condiciones. </li><li> El pasador de séptimo y octavo que se plantan en el sitio cerca de enmalle no debe ser refugio de su vista de la rejilla. </li></ol> Parte III. Okr de las larvas de pez cebra 1. Metilcelulosa Las larvas de pez cebra puede ser inmovilizado en una solución de metilcelulosa al 6%, mientras que mantener vivo durante mucho tiempo. Prepare una solución de metilcelulosa de la siguiente manera. <ol><li> Hervir 200 ml ddH2O en el microondas unos 3 minutos a alto nivel. </li><li> Transferir el agua caliente a una placa de agitación y se remueve vigorosamente. </li><li> Agregar 12 g de metilcelulosa en el agua caliente poco a poco. </li><li> Mantener la agitación durante 3 minutos después de la metilcelulosa es muy dispersa. </li><li> Enfriar la solución con agua helada hasta que se aclare, y luego se coloca a 4 ° C. </li></ol><pclass = &quot;jove_step&quot;&gt; 2. Las larvas de pez cebra <ol><li> Dos pares macho y una hembra de pez cebra adulto en un tanque de acoplamiento a las 17:00 horas después de comer. A la mañana siguiente transferirlos a un depósito de apareamiento fresca y recoger los embriones después de 1 hora. </li><li> Los huevos se mantienen en un medio de embriones en un 28,5 ± 0,5 ° C incubadora con la luz: oscuridad ciclo de 14:10. </li><li> Los huevos serán incubados durante 48 horas después de la fecundación (HPF) y el okr se pondrá a prueba a las 72 HPF o más adelante. En este video, las larvas se encuentran en 5 fdd. </li></ol> 3. Procedimiento de okr <ol><li> Poner el aparato en el estereoscopio equipado con cámara CCD y encender la luz de este aparato. </li><li> Añadir 20 ml Metilcelulosa 6% en un plato de petri de 55 mm. </li><li> Lugar larvas de varios en una fila en el centro de este plato. </li><li> Ponga una burbuja de aire sobre un volumen de 1 l en el campo de la cámara, podría ser como un espejo para reflejar la dirección de la rejilla. </li><li> Encienda el motor y registrar el movimiento de sus ojos. </li></ol> Parte IV. OMR de adultos de pez cebra Pez cebra adulto OMR también podría llevarse a cabo con este aparato, basta con colocar una columna en la cámara de peces para la prevención de los peces que nadan a través del centro (mostrado en Fig.3a, velocidad de rotación es de 36 rpm, blanco y negro son los ciclos de 11). <ol><li> Ponga un pez en esta cámara de pescado. </li><li> Pie de una columna de 2 cm de diámetro en el centro de esta cámara de peces. </li><li> Adaptarse durante un minuto, tanto en todavía rejilla y la reja de rotación. </li><li> Registrar el movimiento del pez cebra. </li><li> Analizar el tipo de coincidencia de la hora de su ordenador personal (un ejemplo se muestra en Fig.3b, velocidad de rotación es de 36 rpm, negro-blanco del ciclo es de 11) </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="Figura 3" /> Figura 3. OMR de adultos de pez cebra. (A) Una columna de plástico blanco se colocó en el centro de la cámara de pescado para la prevención de los peces que nadan a través de ella. (B) El tipo de coincidencia de los adultos de pez cebra después de la rejilla. La tasa de coincidencia se definió como la proporción de la rejilla de tiempo después de que el tiempo de grabación total. El valor del 50% indica que el pez está nadando al azar mientras que el 100% indica la rejilla siguientes peces muy bien. Ambos nervios ópticos fueron aplastados con una pinza de la joyería de unos 10 segundos. El comportamiento de los OMR apareció como una piscina al azar después de aplastar.

<ol>
	<li>Beck, J., Gilland, E., Tank, D., Baker, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+the+Ontogeny+of+Optokinetic+and+Vestibuloocular+Behaviors+in+Zebrafish+Medaka,+and+Goldfish.">Quantifying the Ontogeny of Optokinetic and Vestibuloocular Behaviors in Zebrafish Medaka, and Goldfish.</a> J Neurophysiol. 92, 3546-3561 (2004).</li><li>Brockerhoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measuring+the+optokinetic+response+of+zebrafish+larvae.">Measuring the optokinetic response of zebrafish larvae.</a> Nature Protocols. 1, 2448-2451 (2006).</li><li>Emran, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=OFF+ganglion+cells+cannot+drive+the+optokinetic+reflex+in+zebrafish.">OFF ganglion cells cannot drive the optokinetic reflex in zebrafish.</a> Proc Natl Acad Sci U S A. 104, 19126-19131 (2007).</li><li>Rinner, O., Rick, J., Neuhauss, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contrast+Sensitivity,+Spatial+and+Temporal+Tuning+of+the+Larval+Zebrafish+Optokinetic+Response.">Contrast Sensitivity, Spatial and Temporal Tuning of the Larval Zebrafish Optokinetic Response.</a> Investigative Ophthalmology and Visual Science. 46, 137-142 (2005).</li><li>Roeser, T., Baier, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Visuomotor+Behaviors+in+Larval+Zebrafish+after+GFP-Guided+Laser+Ablation+of+the+Optic+Tectum.">Visuomotor Behaviors in Larval Zebrafish after GFP-Guided Laser Ablation of the Optic Tectum.</a> The Journal of Neuroscience. 23, 3726-3734 (2003).</li><li>Easter, S., Nicola, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Development+of+Vision+in+the+Zebrafish+(Danio+rerio).">The Development of Vision in the Zebrafish (Danio rerio).</a> Developmental Biology. 180, 646-663 (1996).</li><li>Maaswinkel, H., Li, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatio-temporal+frequency+characteristics+of+the+optomotor+response+in+zebrafish.">Spatio-temporal frequency characteristics of the optomotor response in zebrafish.</a> Vision Research. 43, 21-30 (2003).</li></ol>

De acuerdo con la definición de okr, es un fenómeno que los animales se mueven sus ojos para seguir una rejilla de rotación de manera espontánea. Si este comportamiento se ha realizado durante mucho tiempo (por ejemplo, por encima de 6 horas), los músculos del ojo puede estar cansado y el okr será atenuada. Pez cebra adulto OMR ha sido descrita completamente por otros 7. En este vídeo, acaba de mostrar este comportamiento con nuestro aparato. Una vez que los nervios óptico...

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using the optokinetic response to study visual function of zebrafish

Respuesta optocinético (okr) es un comportamiento que un animal hace vibrar sus ojos para seguir una rejilla girando alrededor de ella. Ha sido ampliamente utilizada para evaluar las funciones visuales de las larvas de pez cebra 1-5. Sin embargo, el protocolo estándar para las larvas de peces aún no es fácilmente aplicable en zabrafish adultos. Aquí, presentamos la forma de medir la okr de adultos de pez cebra con nuestro simple costumbre-construido aparatos que utilizan un nuevo protocolo que se establece en nuestro laboratorio. Tanto nuestro equipo y paso a paso el procedimiento de okr en adultos de pez cebra se muestran en este video. Además, las mediciones de la okr larvas, así como la respuesta optomotor (OMR) la prueba de adultos de pez cebra, también se demuestra en este vídeo. Este ensayo okr de pez cebra adulto en nuestro experimento puede durar hasta 4 horas. Prueba okr como aplicada en peces adultos se beneficiarán con la investigación de la función visual más eficientemente cuando los peces adultos sistema de visión se manipula. Su-Qi Zou y Wu Yin contribuyeron igualmente a este trabajo.

Watch this Scientific Journal Video about Using the optokinetic response to study visual function of zebrafish at JoVE.com

Using the optokinetic response to study visual function of zebrafish

Optocinético respuesta ha sido ampliamente utilizada para evaluar las funciones visuales de larvas de pez cebra. Sin embargo, el protocolo estándar para las larvas de peces aún no es fácilmente aplicable en los adultos<sup&gt; 1.5</sup&gt;. Aquí, presentamos la forma de medir la okr de adultos de pez cebra mediante un nuevo protocolo que se establece en nuestro laboratorio.

Usando la respuesta de optocinético para estudiar la función visual del pez cebra

Optokinetic response (OKR) is a behavior that an animal vibrates its eyes to follow a rotating grating around it. It has been widely used to assess the ...

using-the-optokinetic-response-to-study-visual-function-of-zebrafish

Research

JoVE Journal

Biology

18.2K vistas.  University of Science and Technology of China (USTC). Optocinético respuesta ha sido ampliamente utilizada para evaluar las funciones visuales de larvas de pez cebra. Sin embargo, el protocolo estándar para las larvas de peces aún no es fácilmente aplicable en los adultos 1.5. Aquí, presentamos la forma de medir la okr de adultos de pez cebra mediante un nuevo protocolo que se establece en nuestro laboratorio.

Video: Using the optokinetic response to study visual function of zebrafish

Testing Visual Sensitivity to the Speed and Direction of Motion in Lizards

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of the visual system provide more empirical evidence for functional applications. Investigation into the sensory system provides information about the sensory capacity, learning and memory ability, and establishes known baseline behaviour in which to gauge deviations (Burghardt, 1977). However, unlike mammalian or avian systems, testing for learning and memory in a reptile species is difficult. Furthermore, using an operant paradigm as a psychophysical measure of sensory ability is likewise as difficult. Historically, reptilian species have responded poorly to conditioning trials because of issues related to motivation, physiology, metabolism, and basic biological characteristics. Here, I demonstrate an operant paradigm used a novel model lizard species, the Jacky dragon (Amphibolurus muricatus) and describe how to test peripheral sensitivity to salient speed and motion characteristics. This method uses an innovative approach to assessing learning and sensory capacity in lizards. I employ the use of random-dot kinematograms (RDKs) to measure sensitivity to speed, and manipulate the level of signal strength by changing the proportion of dots moving in a coherent direction. RDKs do not represent a biologically meaningful stimulus, engages the visual system, and is a classic psychophysical tool used to measure sensitivity in humans and other animals. Here, RDKs are displayed to lizards using three video playback systems. Lizards are to select the direction (left or right) in which they perceive dots to be moving. Selection of the appropriate direction is reinforced by biologically important prey stimuli, simulated by computer-animated invertebrates.

Testing visual sensitivity in lizards using an operant conditioning paradigm that employs video playback of random-dot kinematograms and computer-generated invertebrates.

Prueba de sensibilidad visual a la velocidad y dirección del movimiento en lagartos

Testing visual sensitivity in any species provides basic information regarding behaviour, evolution, and ecology. However, testing specific features of ...

Investigación

Biología

Computer-Generated Animal Model Stimuli

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In particular, understanding the constraints on visual signal design for communication has been of great interest. Traditional methods for investigating animal interactions have used basic observational techniques, staged encounters, or physical manipulation of morphology. Less intrusive methods have tried to simulate conspecifics using crude playback tools, such as mirrors, still images, or models. As technology has become more advanced, video playback has emerged as another tool in which to examine visual communication (Rosenthal, 2000). However, to move one step further, the application of computer-animation now allows researchers to specifically isolate critical components necessary to elicit social responses from conspecifics, and manipulate these features to control interactions. Here, I provide detail on how to create an animation using the Jacky dragon as a model, but this process may be adaptable for other species. In building the animation, I elected to use Lightwave  3D to alter object morphology, add texture, install bones, and provide comparable weight shading that prevents exaggerated movement. The animation is then matched to select motor patterns to replicate critical movement features. Finally, the sequence must rendered into an individual clip for presentation. Although there are other adaptable techniques, this particular method had been demonstrated to be effective in eliciting both conspicuous and social responses in staged interactions.

Computer-generated stimuli using the Jacky dragon as a model.

Los estímulos generados por ordenador Modelo animal

Communication between animals is diverse and complex. Animals may communicate using auditory, seismic, chemosensory, electrical, or visual signals. In ...

Microinjection of Zebrafish Embryos to Analyze Gene Function

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic oligonucleotides with antisense complementarity to target RNAs, can be added to the embryo to reduce the expression of a particular gene product.  Conversely, processed mRNA can be added to the embryo to increase levels of a gene product.  The vehicle for adding either mRNA or morpholino to an embryo is microinjection.  Microinjection is efficient and rapid, allowing for the injection of hundreds of embryos per hour.  This video shows all the steps involved in microinjection.  Briefly, eggs are collected immediately after being laid and lined up against a microscope slide in a Petri dish.  Next, a fine-tipped needle loaded with injection material is connected to a microinjector and an air source, and the microinjector controls are adjusted to produce a desirable injection volume.  Finally, the needle is plunged into the embryo's yolk and the morpholino or mRNA is expelled.

This video shows how morpholino or mRNA can be injected into zebrafish embryos at the one-cell stage to decrease or increase the level of specific gene products during subsequent development.

Microinyección de embriones de pez cebra para analizar la función génica

One of the advantages of studying zebrafish is the ease and speed of manipulating protein levels in the embryo.  Morpholinos, which are synthetic ...

Methods for the Study of the Zebrafish Maxillary Barbel

Barbels are skin sensory appendages found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal pair and a longer maxillary pair. Barbel tissue contains cells of ectodermal, mesodermal and neural crest origin, including skin cells, glands, taste buds, melanocytes, circulatory vessels and sensory nerves. Unlike most adult tissue, the maxillary barbel is optically clear, allowing us to visualize the development and maintenance of these tissue types throughout the life cycle. 
This video shows early development of the maxillary barbel (beginning approximately one month post-fertilization) and demonstrates a surgical protocol to induce regeneration in the adult appendage (&gt;3 months post-fertilization). Briefly, the left maxillary barbel of an anesthetized fish is elevated with sterile forceps just distal to the caudal edge of the maxilla. A fine, sterile spring scissors is positioned against the forceps to cut the barbel shaft at this level, establishing an anatomical landmark for the amputation plane. Regenerative growth can be measured with respect to this plane, and in comparison to the contralateral barbel. Barbel tissue regenerates rapidly, reaching maximal regrowth within 2 weeks of injury.
Techniques for analyzing the regenerated barbel include dissecting and embedding matched pairs of barbels (regenerate and control) in the wells of a standard DNA electrophoresis gel. Embedded specimens are conveniently photographed under a stereomicroscope for gross morphology and morphometry, and can be stored for weeks prior to downstream applications such as paraffin histology, cryosectioning, and/or whole mount immunohistochemistry. These methods establish the maxillary barbel as a novel in vivo tissue system for studying the regenerative capacity of multiple cell types within the genetic context of zebrafish.

The zebrafish maxillary barbel is an integumentary sense organ containing ectodermal, mesodermal and neural crest derivatives. Importantly, the adult barbel can regenerate after proximal amputation. This video introduces maxillary barbel development and demonstrates a surgical protocol to induce regeneration, followed by collection, embedding and downstream imaging of barbel specimens.

Métodos para el Estudio de la barbo pez cebra maxilar

Barbels are skin sensory appendages found in fishes, reptiles and amphibians.  The zebrafish, Danio rerio, develops two pairs of barbels- a short nasal ...

Hi-C: A Method to Study the Three-dimensional Architecture of Genomes.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, into close spatial proximity 2-6. Deciphering the relationship between chromosome organization and genome activity will aid in understanding genomic processes, like transcription and replication. However, little is known about how chromosomes fold. Microscopy is unable to distinguish large numbers of loci simultaneously or at high resolution. To date, the detection of chromosomal interactions using chromosome conformation capture (3C) and its subsequent adaptations required the choice of a set of target loci, making genome-wide studies impossible 7-10.
We developed Hi-C, an extension of 3C that is capable of identifying long range interactions in an unbiased, genome-wide fashion. In Hi-C, cells are fixed with formaldehyde, causing interacting loci to be bound to one another by means of covalent DNA-protein cross-links. When the DNA is subsequently fragmented with a restriction enzyme, these loci remain linked. A biotinylated residue is incorporated as the 5' overhangs are filled in. Next, blunt-end ligation is performed under dilute conditions that favor ligation events between cross-linked DNA fragments. This results in a genome-wide library of ligation products, corresponding to pairs of fragments that were originally in close proximity to each other in the nucleus. Each ligation product is marked with biotin at the site of the junction. The library is sheared, and the junctions are pulled-down with streptavidin beads. The purified junctions can subsequently be analyzed using a high-throughput sequencer, resulting in a catalog of interacting fragments.
Direct analysis of the resulting contact matrix reveals numerous features of genomic organization, such as the presence of chromosome territories and the preferential association of small gene-rich chromosomes. Correlation analysis can be applied to the contact matrix, demonstrating that the human genome is segregated into two compartments: a less densely packed compartment containing open, accessible, and active chromatin and a more dense compartment containing closed, inaccessible, and inactive chromatin regions. Finally, ensemble analysis of the contact matrix, coupled with theoretical derivations and computational simulations, revealed that at the megabase scale Hi-C reveals features consistent with a fractal globule conformation.

The Hi-C method allows unbiased, genome-wide identification of chromatin interactions (1). Hi-C couples proximity ligation and massively parallel sequencing. The resulting data can be used to study genomic architecture at multiple scales: initial results identified features such as chromosome territories, segregation of open and closed chromatin, and chromatin structure at the megabase scale.

Hi-C: Un método para estudiar la arquitectura tridimensional de los genomas.

The three-dimensional folding of chromosomes compartmentalizes the genome and and can bring distant functional elements, such as promoters and enhancers, ...

Quantification of &gamma;H2AX Foci in Response to Ionising Radiation

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA lesions with respect to survival and preservation of genomic integrity. An early response to the induction of DSBs is phosphorylation of the H2A histone variant, H2AX, at the serine-139 residue, in the highly conserved C-terminal SQEY motif, forming &gamma;H2AX1. Following induction of DSBs, H2AX is rapidly phosphorylated by the phosphatidyl-inosito 3-kinase (PIKK) family of proteins, ataxia telangiectasia mutated (ATM), DNA-protein kinase catalytic subunit and ATM and RAD3-related (ATR)2. Typically, only a few base-pairs (bp) are implicated in a DSB, however, there is significant signal amplification, given the importance of chromatin modifications in DNA damage signalling and repair. Phosphorylation of H2AX mediated predominantly by ATM spreads to adjacent areas of chromatin, affecting approximately 0.03% of total cellular H2AX per DSB2,3. This corresponds to phosphorylation of approximately 2000 H2AX molecules spanning ~2 Mbp regions of chromatin surrounding the site of the DSB and results in the formation of discrete &gamma;H2AX foci which can be easily visualized and quantitated by immunofluorescence microscopy2. The loss of &gamma;H2AX at DSB reflects repair, however, there is some controversy as to what defines complete repair of DSBs; it has been proposed that rejoining of both strands of DNA is adequate however, it has also been suggested that re-instatement of the original chromatin state of compaction is necessary4-8. The disappearence of &gamma;H2AX involves at least in part, dephosphorylation by phosphatases, phosphatase 2A and phosphatase 4C5,6. Further, removal of &gamma;H2AX by redistribution involving histone exchange with H2A.Z has been implicated7,8. Importantly, the quantitative analysis of &gamma;H2AX foci has led to a wide range of applications in medical and nuclear research. Here, we demonstrate the most commonly used immunofluorescence method for evaluation of initial DNA damage by detection and quantitation of &gamma;H2AX foci in &gamma;-irradiated adherent human keratinocytes9.

Quantification of &amp;#947;H2AX Foci in Response to Ionising Radiation

Quantification of DNA double-strand streaks using &gamma;H2AX formation as a molecular marker has become an invaluable tool in radiation biology. Here we demonstrate the use of an immunofluorescence assay for quantification of &gamma;H2AX foci after exposure of cells to radiation.

La cuantificación de γH2AX focos en respuesta a la radiación ionizante

DNA double-strand breaks (DSBs), which are induced by either endogenous metabolic processes or by exogenous sources, are one of the most critical DNA ...

Laser Ablation of the Zebrafish Pronephros to Study Renal Epithelial Regeneration

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in renal failure1. AKI can be caused by a number of factors including ischemia, drug-based toxicity, or obstructive injury1. This results in an inability to maintain fluid and electrolyte homeostasis. While AKI has been observed for decades, effective clinical therapies have yet to be developed. Intriguingly, some patients with AKI recover renal functions over time, a mysterious phenomenon that has been only rudimentally characterized1,2. Research using mammalian models of AKI has shown that ischemic or nephrotoxin-injured kidneys experience epithelial cell death in nephron tubules1,2, the functional units of the kidney that are made up of a series of specialized regions (segments) of epithelial cell types3. Within nephrons, epithelial cell death is highest in proximal tubule cells. There is evidence that suggests cell destruction is followed by dedifferentiation, proliferation, and migration of surrounding epithelial cells, which can regenerate the nephron entirely1,2. However, there are many unanswered questions about the mechanisms of renal epithelial regeneration, ranging from the signals that modulate these events to reasons for the wide variation of abilities among humans to regenerate injured kidneys.
The larval zebrafish provides an excellent model to study kidney epithelial regeneration as its pronephric kidney is comprised of nephrons that are conserved with higher vertebrates including mammals4,5. The nephrons of zebrafish larvae can be visualized with fluorescence techniques because of the relative transparency of the young zebrafish6. This provides a unique opportunity to image cell and molecular changes in real-time, in contrast to mammalian models where nephrons are inaccessible because the kidneys are structurally complex systems internalized within the animal. Recent studies have employed the aminoglycoside gentamicin as a toxic causative agent for study of AKI and subsequent renal failure: gentamicin and other antibiotics have been shown to cause AKI in humans, and researchers have formulated methods to use this agent to trigger kidney damage in zebrafish7,8. However, the effects of aminoglycoside toxicity in zebrafish larvae are catastrophic and lethal, which presents a difficulty when studying epithelial regeneration and function over time. Our method presents the use of targeted cell ablation as a novel tool for the study of epithelial injury in zebrafish. Laser ablation gives researchers the ability to induce cell death in a limited population of cells. Varying areas of cells can be targeted based on morphological location, function, or even expression of a particular cellular phenotype. Thus, laser ablation will increase the specificity of what researchers can study, and can be a powerful new approach to shed light on the mechanisms of renal epithelial regeneration. This protocol can be broadly applied to target cell populations in other organs in the zebrafish embryo to study injury and regeneration in any number of contexts of interest.

Acute kidney injury (AKI) in humans is a common clinical problem caused by damage to the epithelial cells that comprise kidney nephrons, and AKI is associated with high mortality rates of 50-70%1. Following epithelial cell destruction, nephrons have a limited ability to regenerate, though the mechanisms and limitations that guide this phenomenon remain poorly understood. In this video article, we describe our technique for targeted laser ablation of kidney nephron cells in the zebrafish embryo kidney, or pronephros. Our new method can be used to complement nephrotoxicity-induced models of AKI and gain a high-resolution understanding of the cell and molecular alterations that are associated with epithelial regeneration in the kidney nephron.

La ablación con láser de la pronefros pez cebra para el Estudio de la regeneración epitelial renal

Acute kidney injury (AKI) is characterized by high mortality rates from deterioration of renal function over a period of hours or days that culminates in ...

Force Measurement During Contraction to Assess Muscle Function in Zebrafish Larvae

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional measures of muscle health. In this video article, we demonstrate a method to measure force generation during contraction of zebrafish larval trunk muscle. Force measurements are accomplished by placing an anesthetized larva into a chamber filled with a salt solution. The anterior end of the larva is tied to a force transducer and the posterior end of the larva is tied to a length controller. An isometric twitch contraction is elicited by electric field stimulation and the force response is recorded for analysis. Force generation during contraction provides a measure of overall muscle health and specifically provides a measure of muscle function. Although we describe this technique for use with wild-type larvae, this method can be used with genetically modified larvae or with larvae treated with drugs or toxicants, to characterize muscle disease models and evaluate treatments, or to study muscle development, injury, or chemical toxicity.

Force measurements can be used to demonstrate changes in muscle function due to development, injury, disease, treatment or chemical toxicity. In this video, we demonstrate a method to measure force during a maximal contraction of zebrafish larval trunk muscle.

Medición de fuerza durante la contracción para evaluar la función muscular en larvas de pez cebra

Zebrafish larvae provide models of muscle development, muscle disease and muscle-related chemical toxicity, but related studies often lack functional ...

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression both in terms of tissue distribution and developmental stage. Protocols are described for the use of WISH of zebrafish embryos using antisense RNA probes labeled with digoxigenin. Probes are generated by incorporating digoxigenin-linked nucleotides through in vitro transcription of gene templates that have been cloned and linearized. The chorions of embryos harvested at defined developmental stages are removed before incubation with specific probes. Following a washing procedure to remove excess probe, embryos are incubated with anti-digoxigenin antibody conjugated with alkaline phosphatase. By employing a chromogenic substrate for alkaline phosphatase, specific gene expression can be assessed. Depending on the level of gene expression the entire procedure can be completed within 2-3 days.

High Resolution Whole Mount In Situ Hybridization within Zebrafish Embryos to Study Gene Expression and Function

The zebrafish, a small tropical fish, has become a popular model for studying gene function during vertebrate development and disease. The temporal and spatial expression of target genes can be determined by in situ hybridization. Our improved protocol allows for the detection of low abundant transcripts with low non-specific background signal.

Alta resolución todo el montaje de<em&gt; In Situ</em&gt; La hibridación en embriones de pez cebra para estudiar la expresión génica y la función

This article focuses on whole-mount in situ hybridization (WISH) of zebrafish embryos. The WISH technology facilitates the assessment of gene expression ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Análisis de Composición Nephron y función en el riñón adultos de pez cebra

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...