Nous remercions Mme Fan Shen pour son soutien à l&#39;élevage du poisson zèbre. Ceci est soutenu par les financements de Dr Bing Hu (Académie chinoise des sciences, &quot;Bai Ren Ji Hua&quot;, NSFC 30870833 et le Programme national des sciences de base de la Chine, 2009CB941302). Toutes les expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par l&#39;Université des Sciences et Technologies de Chine (USTC) Ressources Animales Center et l&#39;Université des soins aux animaux et le Comité d&#39;utilisation.

Partie I: Construire l&#39;appareil OKR Détails sont montrés dans la vidéo de démonstration. Partie II: OKR des adultes Zebrafish Si votre laboratoire a construit un appareil OKR, la clé de l&#39;adulte poisson zèbre OKR est de tenir le poisson. Dans cette partie, nous allons vous montrer étape par étape. Notre appareil compose des pièces suivantes semblable à d&#39;autres cités. La chambre de poissons circulaire est 7,0 cm de diamètre extérieur, 6,5 cm de diamètre intérieur et 7,0 cm de haut. Le tambour autour de la chambre de poisson est de 8,5 cm de diamètre sur 7,5 cm de diamètre intérieur et 7,0 cm de haut. Il pourrait tourner dans le sens anti-horaire à la vitesse de 20 ~ 70 rotations par minute (rpm). Onze cycles de rayures noires et blanches sont tracées sur un papier calque qui ont adhéré à l&#39;extérieur du tambour. Le système d&#39;éclairage est composé de deux tubes de lumière cercle dans 21 cm de diamètre. <img src="/files/ftp_upload/1742/1742fig1.jpg" alt="Figure 1" /> Figure 1. Le modèle de l&#39;éponge. La falaise fait dans ce milieu est de s&#39;assurer que le champ visuel n&#39;était pas protégé. <ol><li> Une éponge comme indiqué dans la Fig.1 composée par deux parties. La partie gauche est la longueur de 3 cm x 3 cm x largeur de 2 cm de haut. La partie droite est la longueur de 3 cm x largeur x 3 cm 3 cm de haut. </li><li> Mettez cette éponge dans le fond de la chambre de poisson et versez l&#39;eau d&#39;élevage dans environ 2 cm de profondeur. Bulles d&#39;air qui se cachent dans l&#39;éponge doit être chassés. </li><li> Insérez une épingle à l&#39;avant de la partie droite de l&#39;éponge, d&#39;environ 2 mm derrière la falaise. </li><li> Anesthetize une zèbre adulte dans le MS-222. </li><li> Mettez le poisson zèbre sur l&#39;éponge avec jusqu&#39;à dorsale et permettent d&#39;un côté de sa nageoire pectorale avant la première épingle. </li><li> La seconde broche est insérée derrière la nageoire pectorale d&#39;autres. Il pourrait éviter le poisson-zèbre le corps diminue en coopérant avec la première épingle (voir aussi la fig. 2). </li><li> Pince sa queue avec une artère pince et fixer la pince artère dans un morceau de caoutchouc (pâte à modeler) qui est collée sur la chambre de poisson. </li><li> La broche troisième et quatrième est planté dans le site près du cloaque pores corps du poisson. Il pourrait empêcher le poisson-zèbre à l&#39;onde de sa queue intensément. </li><li> Le cinquième est situé broches entre la première et la troisième broche puis l&#39;épingle sixième est de l&#39;autre côté d&#39;elle. Ils pourraient empêcher le poisson-zèbre de secouer son corps. </li><li> Les septième et huitième broches est inséré dans les sites à proximité de la branchie de garder la tête de l&#39;orientation. </li><li> Verser l&#39;eau d&#39;élevage dans la chambre de poissons. </li><li> Après le poisson zèbre se réveille, laissez-le s&#39;adapter pendant 5 ~ 10 minutes. </li><li> Allumer la lumière de cet appareil et laissez le poisson-zèbre adapter ce type d&#39;éclairage pendant 2 minutes. </li><li> Allumez le moteur pour entraîner le réseau et permettent d&#39;adapter le poisson zèbre pendant 1 minute. </li><li> Allumez la caméra et d&#39;enregistrer le mouvement des yeux. </li><li> Analyser les 6 angles de vue et les numéros de saccade sur votre ordinateur personnel (Un exemple représentatif de l&#39;œil les angles de l&#39;analyse est montré dans Fig.2). </li></ol><img src="/files/ftp_upload/1742/1742fig2.jpg" alt="Figure 2" /> Figure 2. L&#39;angle entre l&#39;axe des yeux et l&#39;axe du corps. a) Lorsque la direction du réseau est-à-nez temporelle, nous avons défini l&#39;angle au devant du corps du poisson est un angle négatif et l&#39;angle au corps de poisson est un angle positif, et vice versa (pour revue voir aussi brève Réf. 6). Numéros de cette figure représentent la séquence des épingles. b) par exemple l&#39;analyse d&#39;un OKR poissons dans le groupe normal. Un film d&#39;une seconde a été transféré à une série de cadres et les angles des yeux de chaque cadre a été mesurée avec Image-Pro Plus logiciels. Chaque point dans ce chiffre représente chaque image, nous avons mesuré. Notes: <ol class="lalpha"><li> Toutes les broches sont simplement utilisés pour empêcher le poisson-zèbre de secouer son corps, trop serré sera nocif pour les poissons. </li><li> Pince artère pourrait endommager la queue comme sa surface de contact est faible. Une fois la queue est libre, il est très difficile de fixer ce poisson. Enveloppez un bandage autour de la queue est recommandée dans certaines conditions. </li><li> Les septième et huitième broches qui sont plantés dans le site près des branchies ne doit pas être à l&#39;abri de leur vue de caillebotis. </li></ol> Partie III. OKR des larves de poisson zèbre 1. Méthylcellulose Les larves de poisson zèbre peut être immobilisé dans une solution de méthylcellulose à 6% tandis garder vivant pendant une longue période. Préparer une solution de méthylcellulose comme suit. <ol><li> Faire bouillir 200 ml ddH 2 O dans un four à micro-ondes pendant environ 3 minutes au plus haut niveau. </li><li> Transfert de l&#39;eau chaude pour une plaque de cuisson en remuant et mélangez vigoureusement. </li><li> Ajouter 12 g de méthylcellulose dans l&#39;eau chaude progressivement. </li><li> Gardez en remuant pendant 3 minutes après la méthylcellulose est bien dispersé. </li><li> Refroidir les solutions avec de l&#39;eau glacée jusqu&#39;à ce qu&#39;elle devienne claire, puis le placer à 4 ° C. </li></ol><pclass = &quot;jove_step&quot;&gt; 2. Les larves de poisson zèbre <ol><li> Deux paires mâle et une femelle adulte du poisson zèbre dans un réservoir d&#39;accouplement à 17:00 après la tétée. Le lendemain matin, les transférer à un réservoir d&#39;accouplement frais et collecter des embryons après 1 heure. </li><li> Les œufs sont maintenues dans un milieu d&#39;embryon dans un incubateur à 28,5 ± 0,5 ° C avec la lumière: le cycle sombre de 14h10. </li><li> Les oeufs seront éclos à 48 heures après fécondation (HPF) et l&#39;OKR sera testé à 72 HPF ou plus tard. Dans cette vidéo, les larves sont à 5 DPF. </li></ol> 3. Procédure d&#39;OKR <ol><li> Mettez l&#39;appareil sous le stéréoscope équipé d&#39;une caméra CCD et allumer la lumière de cet appareil. </li><li> Ajouter 20 ml méthycellulose 6% dans un plat de 55 mm de Pétri. </li><li> Placez larves plusieurs fois de suite au centre de ce plat. </li><li> Mettez une bulle d&#39;air sur un volume de 1 pl dans le champ de la caméra, il pourrait être aussi un miroir pour refléter l&#39;orientation de la grille. </li><li> Allumez le moteur et enregistrer les mouvements de leurs yeux. </li></ol> Partie IV. OMR des adultes Zebrafish Adulte zèbre OMR pourrait également être réalisées avec cet appareil, il suffit de placer une colonne dans la chambre de poisson pour prévenir les poissons nageant dans le centre (montré dans Fig.3a, la vitesse de rotation est de 36 min, noir-blanc cycles sont 11). <ol><li> Mettez le poisson dans cette chambre de poisson. </li><li> Support d&#39;une colonne de 2 cm de diamètre au centre de cette chambre de poisson. </li><li> Adapter une minute à la fois dans le reste de râpage et caillebotis de rotation. </li><li> Enregistrement du mouvement du poisson zèbre. </li><li> Analyser le taux de coïncidence de temps sur votre ordinateur personnel (un exemple est montré dans Fig.3b, la vitesse de rotation est de 36 min, noir-blanc du cycle est de 11) </li></ol><img src="/files/ftp_upload/1742/1742fig3.jpg" alt="Figure 3" /> Figure 3. OMR du poisson zèbre adulte. (A) Une colonne blanche plastiques a été placé au centre de la chambre de poisson pour prévenir les poissons qui nagent à travers elle. (B) Le taux de coïncidence de poisson zèbre adulte après la grille. Le taux de coïncidence était défini comme la proportion du temps caillebotis suivantes pour la durée d&#39;enregistrement totale. La valeur de 50% indique que le poisson nage au hasard alors que 100% indique la grille suivante poissons très bien. Les deux nerfs optiques ont été écrasés avec une pince bijoux environ 10 secondes. Le comportement de l&#39;OMR est apparu comme une nage aléatoire après écrasement.

<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>

Selon la définition de l&#39;OKR, c&#39;est un phénomène que les animaux déplacer leurs yeux pour poursuivre une grille tournante spontanément. Si ce comportement a été menée pendant une longue période (par exemple, plus de 6 heures), les muscles de l&#39;oeil peut-être fatigué et le OKR sera atténuée. Adulte zèbre OMR a été complètement décrite par les autres 7. Dans cette vidéo, nous venons de vous montrer ce comportement avec notre appareil. Une fois les nerfs optiques sont écrasés, ils ne pouvaient pas suivre cette grille très bien. Nous avons effectué de test de comportement chez les adultes OKR, le poisson-zèbre larvaires et les adultes OMR avec nos personnalisé simple construit un appareil pour évaluer la fonction visuelle du poisson zèbre. Ce test OKR du poisson zèbre adulte dans notre expérience pourrait garder assez forts dans un délai de 4 heures sans lui nuire. Cela pourrait ouvrir OKR test dans l&#39;étude des poissons adultes fonctions plus efficacement.

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using the optokinetic response to study visual function of zebrafish

La réponse optocinétique (OKR) est un comportement que l&#39;animal vibre ses yeux de suivre une grille tournante autour d&#39;elle. Il a été largement utilisé pour évaluer les fonctions visuelles des larves de poisson zèbre 1-5. Néanmoins, le protocole standard pour les larves de poissons ne sont pas encore facilement applicables dans zabrafish adulte. Ici, nous introduisons la façon de mesurer l&#39;OKR du poisson zèbre adulte avec nos personnalisé simple construit un appareil utilisant un nouveau protocole qui est établi dans notre laboratoire. Tant notre appareil et l&#39;étape par étape la procédure d&#39;OKR chez le poisson zèbre adulte sont illustrées dans cette vidéo. En outre, les mesures de l&#39;OKR larvaire, ainsi que la réponse optomoteur (OMR) test de poisson zèbre adulte, il est aussi démontré dans cette vidéo. Ce test OKR du poisson zèbre adulte dans notre expérience peut durer jusqu&#39;à 4 heures. Ces tests OKR appliquée dans les poissons adultes bénéficieront d&#39;une enquête de la fonction visuelle plus efficace lorsque le système de vision adulte de poisson est manipulé. Su-Zou et Qi Wu Yin ont contribué également à ce document.

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

La réponse optocinétique a été largement utilisé pour évaluer les fonctions visuelles du poisson zèbre larvaire. Néanmoins, le protocole standard pour les larves de poissons ne sont pas encore facilement applicables chez les adultes<sup&gt; 1-5</sup&gt;. Ici, nous introduisons la façon de mesurer l&#39;OKR du poisson zèbre adulte en utilisant un nouveau protocole qui est établi dans notre laboratoire.

En utilisant la réponse optocinétique pour étudier la fonction visuelle du poisson zèbre

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

Nanyang Technological University

Research

JoVE Journal

Biology

18.3K vues.  University of Science and Technology of China (USTC). La réponse optocinétique a été largement utilisé pour évaluer les fonctions visuelles du poisson zèbre larvaire. Néanmoins, le protocole standard pour les larves de poissons ne sont pas encore facilement applicables chez les adultes 1-5. Ici, nous introduisons la façon de mesurer l'OKR du poisson zèbre adulte en utilisant un nouveau protocole qui est établi dans notre laboratoire.

Vidéo: 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.

Test sensibilité visuelle à la vitesse et la direction du mouvement chez les lézards

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

Recherche

Biologie

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.

Computer-Generated Stimuli modèle 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.

Microinjection d&#39;embryons de poissons zèbres pour analyser la fonction génique

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.

Méthodes pour l&#39;étude de l&#39;Barbel Zebrafish maxillaire

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.

Salut-C: une méthode d&#39;étude de l&#39;architecture tridimensionnelle des génomes.

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 &#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.

Quantification des γH2AX Foci en réponse aux radiations ionisantes

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.

Laser Ablation de l&#39;pronéphros Zebrafish d&#39;étude rénale régénération épithéliale

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.

Mesure de la force lors de la contraction d&#39;évaluer la fonction musculaire en poisson zèbre

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.

Haute Résolution Mont entier<em&gt; In Situ</em&gt; Hybridation dans les embryons de poisson zèbre pour étudier l&#39;expression des gènes et la fonction

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.

Analyse de néphron Composition et fonction du rein poisson zèbre adulte

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