Merci à Marie Birne, Areti Tsiola, Jaymie Estevez et Arelys Uribe. Ce travail a été soutenu en partie par CUNY Research Foundation et Howard Hughes Medical Institute Programme d&#39;été de subvention pour les étudiants (Spur) et NIH RO3 41702-00-01. La recherche a été également soutenue par Queens College et quelques-unes des expériences ont été effectuées sur l&#39;équipement de la Core Facility for Imaging, biologie cellulaire et moléculaire au Queens College.

Le poisson zèbre ont été soulevées en utilisant des protocoles standard de 13 dans l&#39;installation de Queens College animaux à 28 ° C. Paires de poissons individuels ou de groupe accouplements doivent être préformés et les embryons ainsi obtenus nettoyés et stockés dans un incubateur à 28 ° C. La santé des poissons doivent être examinés quotidiennement. Lors de la fécondation après 5 jours (DPF), les poissons devraient être séparés dans des réservoirs à une densité de 10 à 15 poissons et est entré dans la nurserie de poissons installation. Pour le protocole présenté ici le poisson-zèbre ont été collectées à travers la larve stades adultes: 15, 30, 45, 60, 90, 180 DPF. 1. Collecte de l&#39;objet et la fixation de poisson zèbre <ol><li> Retirer le poisson du réservoir de l&#39;habitat en utilisant net et le lieu de 0,2% tricaïne à anesthésier. </li><li> Placer le poisson dans l&#39;eau glacée anesthésié pendant 15 minutes à euthanasier. </li><li> Faire frais paraformaldéhyde à 4% (PFA) en 1x tampon phosphate salin (PBS). </li><li> Incuber les poissons recueillis dans 4% des PFA pendant 1-2 heures à température ambiante puis une nuit à 4 ° C dans une boîte de Pétri enveloppées dans du parafilm. Cela maintient le plat de poisson et rend la dissection plus tard, beaucoup plus facile. </li><li> Après fixation, rincer deux fois dans du PBS avec du Tween (PBT). Le poisson peut être garder en PBT à 4 ° C pour un maximum de 10 jours. </li></ol> 2. Mesure de poisson et de la dissection <ol><li> Placer le poisson dans la seule moitié boîte de Pétri remplie de PBS frais et déposez-le sur son côté droit. </li><li> Mesurer la longueur des poissons du museau à la base de la queue (le pédoncule caudal), non compris ailerons de queue, en utilisant un appareil photo numérique mm étrier (figure 1). Il s&#39;agit de la longueur standard (SL). </li><li> Chaque poisson peut être attribué un numéro / lettre combinaison afin de permettre un poisson et le cœur résultant disséqués pour être suivis pour une analyse ultérieure. Les données, y compris le numéro de stock parental, l&#39;âge à la fixation, les mesures de SL, ont été saisies dans un tableur et organisée en fonction de ces étiquettes. </li><li> Avant la dissection, bandes étiquette du tube PCR avec numéro de suivi attribué et remplir de PBT ou autre solution appropriée pour une analyse ultérieure. </li><li> Orienter le côté ventral du poisson en boîte de Pétri tout en stabilisant le corps avec des pinces tenant la tête entre les yeux et les branchies (figure 2). </li></ol> Les petits poissons (plus petite que 12mm SL): <ol start="4"><li> a) En utilisant une pince forte, ou une micro-aiguille dans un porte-broches, retirez les muscles pectoraux et les ailettes du corps pour révéler le cœur. Le mouvement constant de la pince lors de la dissection peut causer des courants dans les PBT qui déplacent les poissons. Ceci est particulièrement problématique pour les petits poissons, donc une micro-aiguille peut être utilisé pour enlever les pectoraux et les nageoires et ouvrir la cavité abdominale. </li><li> a) l&#39;utilisation de la pince doucement pour ramasser le cœur hors de la cavité sous l&#39;atrium. Alternativement, dans certains poissons que vous pourrez peut-être retirer le coeur en tirant doucement sur le bulbe et le reste du coeur va suivre. Soyez prudent sur l&#39;emplacement de l&#39;atrium si vous utilisez cette technique car elle peut être facilement endommagé. S&#39;il ya des tissus supplémentaire qui vient avec le cœur, c&#39;est très bien et peut être effacé après. </li><li> a) Si le poisson contient un marqueur fluorescent cardiaques telles que Tg (myl7: GFP) twu34, utilisez la portée fluorescentes pour la dissection et de vérifier l&#39;ablation du cœur par la fluorescence (figure 3). </li><li> a) Une fois que le cœur est enlevé de la cavité, utilisez la pince pour tenir le cœur et une micro-aiguille pour enlever supplémentaires non cardiaque tissus et la doublure épicardiques de l&#39;extérieur du cœur. </li></ol> Les gros poissons (plus de 12mm SL): <ol start="4"><li> b) En utilisant le printemps manipulé des micro-ciseaux, faire trois incisions de moins de 1mm de profondeur: 1 - transversale coupé à travers les 2 branchies - coupe transversale au ventre antérieur 3 - coupe sagittale sur la face ventrale reliant les deux coupes (figure 4). </li><li> b) En utilisant une pince forte retirer les muscles pectoraux et les ailettes du corps pour ouvrir la cavité du corps et de révéler les tissus argentés du péricarde (figure 5A). Retirer ce tissu péricardique et le cœur va devenir visible (figure 5B). </li><li> b) Utiliser les ciseaux pour couper les micro-l&#39;artère reliée au bulbe artériel, situé au cœur supérieure. Une fois que cette artère est coupée, mettre les conseils pince sous l&#39;atrium, utilisez la pince pour ramasser le cœur hors de la cavité. S&#39;il ya des tissus supplémentaire qui vient avec le cœur, c&#39;est très bien et peut être retiré plus tard. </li><li> b) Une fois que le cœur est enlevé de la cavité, utiliser les deux pinces pour tenir le cœur et enlever le tissu supplémentaire, ou une paire de pince pour tenir le cœur et une micro-aiguille pour retirer supplémentaire, non cardiaque tissulaire. La doublure épicardiques, évidente par sa pigmentation noire, peut être enlevé de l&#39;extérieur du coeur si désiré en grattant doucement le tissu. </li></ol> 3. Photographie des cœurs <ol><li> Préparer une 23g / L d&#39;agar nutritif boîte de Pétri et la couverture d&#39;agar solidifié avec du PBS. </li><li> Utilisation de la pince, faire un puits dans la gélose qui permet au cœur de se reposer dans l&#39;orientation désirée pour photographier. </li><li> Remove au coeur du tube de PCR en tenant l&#39;extrémité de la bulbe avec la pince fermement. Placez le coeur dans le plat d&#39;agar. </li><li> Orienter le cœur dans l&#39;agar à photographier. L&#39;échantillon peut être tourné après chaque image pour toutes les orientations possibles (figure 6). </li><li> Utilisez le temps d&#39;exposition généraux légère et courte pour photographier les cœurs. Faire des ajustements pour la qualité de lumière en utilisant l&#39;exposition que nécessaire pour obtenir la meilleure photo. </li></ol> 4. Les résultats représentatifs: Un exemple d&#39;un cœur bien disséqué propre est montré dans de multiples orientations dans la figure 6. Chaque coeur aura quelques différences dans la morphologie générale, mais le cœur doit être complet, contenant un ventricule intact, atrium et artériel bulbe. Quelques exemples de cette variation dans la morphologie globale sont présentés dans la figure 7. Les taches noires épicardiques tissus peuvent également enlever du cœur. <img src="/files/ftp_upload/3165/3165fig1.jpg" alt="Figure 1"/> Figure 1. Mesurer le poisson du museau au pédoncule caudal en utilisant un appareil photo numérique mm étrier. <img src="/files/ftp_upload/3165/3165fig2.jpg" alt="Figure 2"/> Figure 2. Tenez le poisson avec une pince entre les yeux et les branchies. <img src="/files/ftp_upload/3165/3165fig3.jpg" alt="Figure 3"/> Figure 3 Un cardiaques spécifiques marqueur fluorescent transgéniques telles que Tg (myl7: GFP). Twu34, montré ici, peut aider à visualiser les cœurs disséqués par des poissons plus petits. <img src="/files/ftp_upload/3165/3165fig4.jpg" alt="Figure 4"/> Figure 4 trois incisions pour enlever les muscles pectoraux et de la peau: 1 - coupe transversale à travers les branchies 2 - coupe transversale au ventre antérieur 3 - coupe sagittale sur la face ventrale reliant les deux coupes.. <img src="/files/ftp_upload/3165/3165fig5.jpg" alt="Figure 5"/> . Figure 5 lettres noires le label cavités du cœur: bulbe artériel (BA), ventricule (v), et l&#39;atrium (a). A-La cavité supérieure du corps est ouverte pour montrer le tissu argenté du péricarde. B-Après avoir enlevé le péricarde, le cœur est visible et facile à enlever. <img src="/files/ftp_upload/3165/3165fig6.jpg" alt="Figure 6"/> Orientations Figure 6. Différents du cœur sont affichés. Lettres noires le label cavités du cœur: bulbe artériel (BA), ventricule (v), et l&#39;atrium (a). Flèches grises indiquent l&#39;emplacement et l&#39;orientation du cœur en référence à son emplacement normal dans l&#39;organisme: antérieure (A), postérieure (P), dorsale (D), ventrale (V), gauche (L) et droite (R). La barre d&#39;échelle indique 400 microns. A-ventrale vue sur le coeur B-vue dorsale du cœur C-latérale du côté gauche D-latérale du côté droit <img src="/files/ftp_upload/3165/3165fig7.jpg" alt="Figure 7"/> Figure 7. Morphologie des coeurs de différentes tailles est montré. Lettres noires le label cavités du cœur: bulbe artériel (BA), ventricule (v), et l&#39;atrium (a). La barre d&#39;échelle indique 400 um. Poissons sous forme AC-Coeurs avec SL moins 12mm Poissons forment DF-Coeurs avec SL supérieure à 12mm

<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>

Aucun conflit d&#39;intérêt déclaré.

Ces méthodes pour la dissection du cœur du poisson zèbre post-embryonnaire des poissons permet l&#39;élimination complète d&#39;un cœur en bon état. Bien que cette technique est simple et fournit des guides pour des coupes externes qui permettra d&#39;éviter d&#39;endommager le cœur, une main ferme est essentiel pour les petits poissons. Une étape clé lors de la dissection est d&#39;identifier le péricarde argentée comme le cœur est directement dans ce sac. Visualiser le péric...

<table border="1"><tbody><tr><th> Matériau </th><th> Société </th><th> Numéro de catalogue </th><th> Commentaires (optionnel) </th></tr><tr><td> Paraformaldéhyde </td><td> EMD </td><td> PX0055 </td><td> .04g/mL En PBS </td></tr><tr><td> Pince # 55 </td><td> TSF </td><td> 11295-51 </td><td></td></tr><tr><td> Micro-ciseaux </td><td> TSF </td><td> 15005-08 </td><td></td></tr><tr><td> Détenteur de Pin </td><td> TSF </td><td> 26016-12 </td><td></td></tr><tr><td> Micro-aiguilles </td><td> TSF </td><td> 10130-10 </td><td></td></tr><tr><td> Nutrient Agar </td><td> BBL </td><td> 11472 </td><td> 23g / L dans H 2 O distillée </td></tr><tr><td> Boîtes de Pétri </td><td> BD Falcon </td><td> 351029 </td><td></td></tr><tr><td> PCR de 8 tubes bandes </td><td> Etats-Unis scientifique </td><td> 1402-2500 </td><td></td></tr><tr><td> Numérique étrier </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

Le poisson zèbre sont devenus un organisme modèle utile et pratique pour l&#39;étude du développement embryonnaire, le cœur (voir commentaires récents 1-6), cependant, le travail examinant post-embryonnaire par le développement cardiaques adultes a été limité 7-10. Examiner la morphologie changeante du coeur de maturation et le vieillissement sont limitées par le manque de techniques disponibles pour la stadification et isoler les cœurs juvéniles et adultes. Afin d&#39;analyser le développement cardiaque sur la durée de vie du poisson, nous disséquons coeurs poisson zèbre à de nombreux stades et les photographier pour une analyse ultérieure 11. Les caractéristiques morphologiques du cœur peuvent être facilement quantifiés et des coeurs individuels peuvent être analysés ultérieurement par une foule de méthodes standard. Zebrafish croître à des taux variables et de la maturation meilleure corrélation avec la taille des poissons que l&#39;âge, donc, après la fixation, la longueur du poisson, nous photographier et mesurer comme un indicateur de la maturation des poissons. Ce protocole explique deux distincts, la taille de techniques de dissection à charge pour le poisson-zèbre, allant de 3,5 mm de longueur standard des larves (SL) avec des coeurs de la longueur du ventricule 100 microns (VL), aux adultes, avec SL de 30mm et VL 1mm ou plus. Poissons larvaires et adultes ont le corps bien distincts et la morphologie d&#39;organes. Les larves ne sont pas seulement beaucoup plus petit, ils ont moins de pigments et de chaque organe est visuellement très difficiles à identifier. Pour cette raison, nous utilisons des techniques de dissection distinctes. Nous avons utilisé des procédures pré-dissection de fixation, comme nous avons découvert que les cœurs disséqués directement après l&#39;euthanasie ont une morphologie plus variable, avec ballon très lâche et comme oreillettes rapport avec des coeurs enlevés après fixation. Le poisson fixés avant la dissection, de conserver la morphologie in vivo et la position de chambre (données non présentées). En outre, à des fins de démonstration, nous profitons du coeur (infarctus) spécifiques GFP transgéniques Tg (myl7: GFP) twu34 (12), ce qui nous permet de visualiser le cœur entier et est particulièrement utile aux premiers stades de développement lorsque les cardiaques morphologie est moins distinctes à partir des tissus environnants. Dissection du cœur fait une analyse plus approfondie de la biologie cellulaire et moléculaire sous-jacente de développement du cœur et de la maturation en utilisant l&#39;hybridation in situ, immunohistochimie, l&#39;extraction d&#39;ARN ou d&#39;autres méthodes analytiques plus facile en post-embryonnaire du poisson zèbre. Ce protocole fournira une technique précieuse pour l&#39;étude de la maturation et le vieillissement de développement cardiaque.

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

Une méthode claire et standardisée pour la dissection et l&#39;isolement du cœur poisson zèbre à de multiples stades de développement sont décrits. Techniques d&#39;annotation et de quantification sont également discutés.

Dissection du cœur chez les larves, le poisson zèbre juvéniles et adultes, 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

21.3K vues.  Queens College, City University of New York. Une méthode claire et standardisée pour la dissection et l'isolement du cœur poisson zèbre à de multiples stades de développement sont décrits. Techniques d'annotation et de quantification sont également discutés.

Vidéo: 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.

Dissection de SNC larvaire chez 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 ...

Recherche

Biologie

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.

Drosophile larvaire JNM Dissection

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.

Dissection fine et globale de l&#39;épithélium sensoriel de l&#39;oreille interne adultes poisson zèbre ( 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.

Dissection d&#39;organes sur le poisson zèbre pour adultes

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.

Dissection et la coloration des Drosophile Ovaires larvaires

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.

Dissection du rein Zebrafish adultes

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 électroporation in vivo de morpholinos dans la rétine de poisson zèbre adulte

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.

Soins et entretien réguliers d&#39;un poisson zèbre (<em&gt; Danio rerio</em&gt;) Laboratoire: An Introduction

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.

In Vivo modélisation du génome humain morbide utilisant 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 poisson zèbre pour adultes

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