Ce travail est soutenu par un fonds de démarrage à partir du Département des sciences biologiques à l&#39;Université Purdue.

Partie 1: Préparatifs avant microdissection <ol><li> Solutions <ol class="lalpha"><li> E3 moyenne 4 (5 mM de NaCl, 0,17 mM de KCl, 0,33 mM de CaCl2 et 0,33 mM MgSO 4). </li><li> Une solution de Ringer, 5 (116 mM NaCl, 2,9 mM de KCl, 1,8 mM CaCl 2, 5 mM d&#39;HEPES, pH 7,2), stérilisée par filtration. </li><li> NaOH 5N. </li></ol></li><li> Gravure chimique des aiguilles de tungstène <ol class="lalpha"><li> Fixez un petit bécher contenant NaOH 5N sur une boîte de Petri par de l&#39;argile. </li><li> Attacher un trombone sur le côté du vase, afin qu&#39;il entre en contact avec la solution de NaOH. </li><li> Connectez l&#39;électrode négative d&#39;une alimentation DC pour le trombone et l&#39;électrode positive à une extrémité d&#39;une pièce courte (~ 2cm) du fil de tungstène. </li><li> Enveloppez l&#39;autre extrémité du fil de tungstène avec une petite boule d&#39;argile et trempette cette fin dans la solution de NaOH. </li><li> Augmenter la tension à environ 3V et tremper le fil de haut en bas dans la solution de NaOH jusqu&#39;à une vitesse de gravure de bons est atteint. Une aiguille dans une bonne qualité peuvent généralement être gravés en 10 minutes. </li><li> Le fil de tungstène exposée à la solution deviendra progressivement plus minces. Quand la boule d&#39;argile tombe, de l&#39;autre côté du fil qui est encore attaché à l&#39;électrode positive aura une forme aiguille pointue. </li><li> Rincer l&#39;aiguille forte avec une solution de Ringer ou E3 pour enlever les dépôts de sel. </li><li> Fixez l&#39;aiguille à un applicateur en bois par un ruban ou un porte-aiguille pour une manipulation facile. </li></ol></li><li> Les plaques de culture <ol class="lalpha"><li> Le Falcon 60 x 15 mm plaques en polystyrène de culture prêts à l&#39;emploi pour la microdissection de la rétine. </li><li> Pour disséquer RPE-joint la rétine, écraser deux embryons complètement dans une plaque de culture contenant la solution de Ringer pour se débarrasser de l&#39;adhésivité de la surface 30 minutes avant la dissection. Peu avant la dissection, laver abondamment la plaque avec une solution de Ringer fraîches. </li></ol></li><li> Collecte des embryons et de la tenue <ol class="lalpha"><li> Zebrafish adultes sont maintenues comme décrit 4, 5. </li><li> Séparez les poissons d&#39;élevage en cuves parent statiques paires par un diviseur de la nuit avant la reproduction. </li><li> Enlevez le séparateur après la lumière ambiante est activée dans le lendemain matin. Laisser les parents à traverser à intervalles de 10 minutes toutes les demi-heures. Séparez les poissons après chaque passage. </li><li> Maintenir les embryons prélevés sur chaque passage dans le milieu de l&#39;E3 séparément à 28 ° C. </li><li> Embryons au stade de 10 à 12 heures après fécondation (HPF) et aime revenir immédiatement avant la dissection au niveau des points de temps spécifique, selon les critères établis 1, 6. </li></ol></li></ol> Partie 2: Dissection - retrait du cerveau et une exposition oculaire <ul><li> Les procédures décrites dans cette section sont communes pour disséquer la rétine (partie 3A) et RPE-joint la rétine (partie 3B). </li><li> Tous les dissections fines sont effectuées par un bout de la pince Dumont et assistée par une autre paire de pince Dumont pour le positionnement des tissus. Une aiguille de tungstène chimiquement gravé peut être utilisé pour des manipulations fines, si nécessaire. </li><li> Tous les dissections fines sont effectuées à un grossissement de 8x ~ 5 sous un microscope Olympus SZX16 équipé d&#39;un objectif 1X ou équivalent. </li><li> Maintenir les embryons dans le milieu de l&#39;E3 dans une paillasse étuve à 28 ° C à côté de la loupe pour accéder facilement embryons lors de la dissection. </li><li> La platine du microscope est chauffé à 28 ° C par une plaque thermo pour minimiser l&#39;influence de la fluctuation de la température sur l&#39;expression génique. </li></ul><ol><li> Transfert d&#39;un embryon à un stade précis de développement d&#39;une solution de Ringer dans un Falcon 60 X 15 mm plaque de culture préparé comme décrit dans la Partie 1 # 3. </li><li> Couper la tête avec une partie du tronc antérieur rapidement du corps. </li><li> Pin l&#39;extrémité postérieure de ce tissu sur une plaque de culture avec la pince aider. </li><li> Ouvrez la tête sur le côté dorsal de départ du front antérieur. </li><li> Enlever le cerveau de sorte que le côté médial de l&#39;œil est exposé et est orientée vers le haut pour des manipulations supplémentaires. </li></ol> Partie 3A: dissection rétiniennes 1 <ol><li> Préparer l&#39;embryon comme décrit dans la partie 2. </li><li> Brosser soigneusement l&#39;EPR exposé sur la face médiale de la balle des yeux qui est orientée vers le haut par le bout de la pince jusqu&#39;à ce qu&#39;une petite ouverture à la rétine est perçue. </li><li> Poursuivre le brossage et l&#39;épluchage d&#39;action jusqu&#39;à la face médiale de la rétine est presque tous exposés. Évitez de gratter et de poinçonnage de la rétine exposée. </li><li> Pour supprimer l&#39;EPR latérale sur la rétine qui est maintenant tournée vers le bas, approche qui et brosse à un angle de 45 ° environ de la surface de plaque de culture. </li><li> Retirez l&#39;EPR qui est attaché relativement fermement à l&#39;ora serrata en épinglant la partie détachée de l&#39;EPR à la cPlaque ulture pour la dissection, puis soulevez délicatement la rétine. </li><li> Rouler la rétine au fond de la boîte de culture pour nettoyer l&#39;EPR résiduels. <ol class="lalpha"><li> Nous avons remarqué que cette plaque notamment Falcon culture en polystyrène a une adhésion préférentielle des RPE pendant environ 20 minutes une fois l&#39;embryon est écrasé. Cette propriété est utilisée pour l&#39;enlèvement des restes EPR. Cependant, les cellules rétiniennes ne collent à la surface dans une moindre mesure, qui peuvent être inspectés de près à fort grossissement. On doit trouver un équilibre entre l&#39;exhaustivité de l&#39;enlèvement RPE et l&#39;intégrité de la rétine. </li><li> L&#39;objectif adhère souvent à la plaque de culture et se détache de la rétine au cours du laminage. Parfois, il est nécessaire de détacher le cristallin par une aiguille de tungstène gravé avec une action tourbillonnante sur la surface de la lentille. </li></ol></li><li> Ces procédures peuvent réussir à éliminer l&#39;EPR de la rétine sans pour autant compromettre l&#39;intégrité du tissu. Ceci est indiqué par une bonne morphologie générale (figure 1B et B &#39;) et l&#39;histologie (figure 1B &quot;), une flèche, en particulier, la préservation de la matrice extracellulaire entre la couche des photorécepteurs et l&#39;EPR est exceptionnellement bonne (figure 1B.&quot;; comparer avec l&#39;histologie de l&#39;embryon entier (figure 1A &quot;)). </li></ol> Partie 3B: RPE-attachée de dissection rétiniennes 3 <ol><li> Préparer l&#39;embryon comme décrit dans la partie 2. </li><li> Pin la tête à la plaque de culture par la pince assistant. Soulevez doucement l&#39;œil du côté latéral postérieur et rouler sur le côté antérieur. <ol class="lalpha"><li> La choroïde présomptifs et le tissu scléral à l&#39;extérieur de la couche RPE sont relativement étroitement attachée à la peau et peut être surtout décollée en roulant les yeux attentivement du côté antérieur. </li></ol></li><li> Retirez la lentille avec une aiguille de tungstène après le côté latéral de l&#39;oeil est exposé et quand l&#39;œil est encore attaché à la peau. </li><li> Ces procédures peuvent réussir à préserver la couche d&#39;EPR entier avec la rétine. Choroïde présomptif et le tissu scléral peut être éliminé en grande partie (figure 1C, C &#39;et C &quot;). </li></ol> Partie 4: collecte d&#39;échantillons de tissus pour des travaux d&#39;ARN <ol><li> Les échantillons peuvent être disséqués recueillies dans TRIzol dans un tube de micro sans RNase pour la caractérisation aval ARN comme décrit précédemment 1. </li></ol> Les résultats représentatifs <img src="/files/ftp_upload/2028/2028fig1.jpg" alt="Figure 1" /> La figure 1 (A). Latérale et (A &#39;) vue dorsale d&#39;une tête de poisson zèbre larvaire à 52 HPF avant dissection. (A &quot;) La section correspondante histologique de la tête des larves dans (A) et (A &#39;) (B). Latérale et (B&#39;) vue dorsale d&#39;une rétine disséqués à 54 HPF. La surface de la rétine est intacte des deux latéraux et des vues dorsale. (B &quot;) La section correspondante histologique de la rétine disséqué en (B) et (B &#39;). La structure de la rétine et le laminage rétine est intacte. En particulier, la matrice extracellulaire, entre la couche des photorécepteurs et l&#39;EPR (A &quot;, flèches) a été bien conservé dans la rétine disséqué (B&quot;, flèches). (C) et latérale (C &#39;) vue médiale d&#39;un disséqué RPE-attachée rétine à 52 HPF. EPR couche était intact et continu, qui a également été indiqué par la coupe histologique des tissus disséqués (C &quot;). La zone blanche dans C &#39;est le nerf optique (flèche). Pour l&#39;histologie, les échantillons de tissus ont été prélevés et fixés dans 4% paraformaldéhyde. Plastic enrobage et de sectionnement de ces échantillons ont été réalisées comme décrit 3. Barre d&#39;échelle = 50 microns.

<ol>
	<li>Leung, Y. F., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retina.">Gene expression profiling of zebrafish embryonic retina.</a> Zebrafish. 2, 269-283 (2005).</li><li>Leung, Y. F., Ma, P., Link, B. A., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Factorial+microarray+analysis+of+zebrafish+retinal+development.">Factorial microarray analysis of zebrafish retinal development.</a> Proc Natl Acad Sci U S A. 105, 12909-12914 (2008).</li><li>Leung, Y. F., Ma, P., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+zebrafish+embryonic+retinal+pigment+epithelium+in+vivo.">Gene expression profiling of zebrafish embryonic retinal pigment epithelium in vivo.</a> Invest Ophthalmol Vis Sci. 48, 881-890 (2007).</li><li>Nusslein-Volhard, C., Dahm, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Zebrafish : a practical approach. , (2002).</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> , (2000).</li><li>Kimmel, C. B., Ballard, W. W., Kimmel, S. R., Ullmann, B., Schilling, T. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stages+of+embryonic+development+of+the+zebrafish.">Stages of embryonic development of the zebrafish.</a> Dev Dyn. 203, 253-310 (1995).</li><li>Fadool, J. M., Dowling, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Zebrafish:+a+model+system+for+the+study+of+eye+genetics.">Zebrafish: a model system for the study of eye genetics.</a> Prog Retin Eye Res. 27, 89-110 (2008).</li></ol>

Expériences sur les animaux ont été effectuées en conformité avec les directives et règlements établis par les soins des animaux et du Comité de Purdue utilisation.

Microdissection de tissus oculaires poisson zèbre peut effectivement obtenir des rétines intactes et RPE-attachée rétines. Cette aide substantielle des études d&#39;expression se rapportant à un tissu oculaire spécifique (ie la rétine ou RPE). En fait, nous avons utilisé avec succès ces procédures pour obtenir des profils d&#39;expression ARN de la rétine entière 1 et 3 EPR. L&#39;utilité de ces profils est fortement soutenue par notre récente identification des voies et des familles...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Cordless pestle motor</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-370</td>
<td>&nbsp;</td>
<tr>
<td>DC power supply</td>
<td>&nbsp;</td>
<td>Lascar</td>
<td>PSU130</td>
<td>Any DC supply would work. The specific voltage of a different machine will need further optimization.</td>
</tr>
<tr>
<td>Disposable pestle &amp; microtube, 1.5 mL (DNase, RNase and pyrogen-free)</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>47747-366</td>
<td>These are used for tissue collection in TRIzol for expression analysis.</td>
</tr>
<tr>
<td>Dumont #5 forceps, Tips: 0.05 x 0.01mm, Inox</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>500341</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Dumont #5SF forceps, Tips: 0.025 x 0.005mm, Inox</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>11252-00</td>
<td>Fine tip dimension is desirable but is not inflexible, as one may need to sharpen the tip from time to time.</td>
</tr>
<tr>
<td>Falcon polystyrene culture plates, 60 X 15 mm</td>
<td>&nbsp;</td>
<td>BD Biosciences</td>
<td>351007</td>
<td>These plates are used as dissection plates.</td>
</tr>
<tr>
<td>Olympus SZX16 Stereomicroscope</td>
<td>&nbsp;</td>
<td>Olympus</td>
<td>SZX16</td>
<td>Any stereomicroscope would work. We used Leica stereomicroscope in previous studies1-3 without any issues. We also use the 1X objective exclusively for the dissection even though we have a 2X objective installed. </td>
</tr>
<tr>
<td>Sharpening stone</td>
<td>&nbsp;</td>
<td>Fine Science Tools</td>
<td>29008-01</td>
<td>Use this to sharpen the tip of the forceps if necessary</td>
</tr>
<tr>
<td>Thermo plate</td>
<td>&nbsp;</td>
<td>Tokai Hit</td>
<td>MATS-U55SZX2B</td>
<td>This is used to maintain the temperature of the tissue throughout dissection and minimize the influence of temperature fluctuation on gene expression. We also put the whole microscope in an environmentally controlled room at 28&deg;C during dissection in previous studies1-3 with good success.</td>
</tr>
<tr>
<td>Trizol, 100 mL</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15596-026</td>
<td>&nbsp;</td>
<tr>
<td>tungsten wire, 0.015 inch diameter</td>
<td>&nbsp;</td>
<td>World Precision Instruments</td>
<td>TGW1510</td>
<td>&nbsp;</td>
<tr>
<td>Wooden Applicator</td>
<td>&nbsp;</td>
<td>Puritan</td>
<td>807</td>
<td>This is used for holding the chemically-etched tungsten needle.</td>
</tr>		</tbody>
		</table>
		</div>

microdissection of zebrafish embryonic eye tissues

Le poisson zèbre est un modèle animal populaires pour la recherche sur le développement des yeux en raison de sa rapidité<em&gt; Ex utero</em&gt; Le développement et la fécondité bon. En trois jours après la fécondation (DPF), les larves se montrent de la première réponse visuelle. De nombreux gènes ont été identifiés pour contrôler un développement de l&#39;œil bon, mais nous sommes loin d&#39;une compréhension complète de l&#39;architecture génétique sous-jacente. Whole profilage de l&#39;expression des gènes du génome est un outil utile pour élucider génétique du réseau de réglementation pour le développement des yeux. Cependant, la petite taille de l&#39;œil embryonnaire chez le poisson zèbre, il est difficile d&#39;obtenir des tissus de l&#39;œil intact et pur pour l&#39;analyse de l&#39;expression. Par exemple, la longueur antéro-postérieure de l&#39;oeil entre le jour 2 et 3 est seulement environ 200-300 um, tandis que le diamètre de la lentille est um moins 100. En outre, l&#39;épithélium pigmentaire rétinien (EPR) qui sous-tendent la rétine est juste un épithélium simple couche. Alors que les profils d&#39;expression génique peuvent être obtenus à partir de l&#39;embryon entier, ils ne représentent pas exactement l&#39;expression de ces tissus. Par conséquent le tissu pur doit être obtenu pour un profil d&#39;expression génique réussie de développement de l&#39;œil. Pour répondre à cette question, nous avons développé une approche de la rétine intacte et microdissect rétine avec RPE jointe de dpf 1-3, qui couvrent les grandes étapes de la morphogenèse des yeux. Toutes les procédures qui peut être fait avec une pince fine et fournitures de laboratoire générale en vertu de stéréomicroscopes standard. Pour la dissection de la rétine, l&#39;EPR à une seule couche est enlevée et décollée par brossage action et l&#39;adhésion préférentielle des restes EPR à la surface de la plaque de culture pour la dissection. Pour RPE-joint la dissection de la rétine, l&#39;adhésion de l&#39;EPR à la plaque de dissection est retiré avant la dissection de telle sorte que l&#39;EPR peut être complètement conservé avec la rétine. Une action de levage attentive de ce tissu peut séparer efficacement la choroïde et la sclère présomptive. La lentille peut être retirée dans les deux cas par une aiguille de tungstène gravé chimiquement. En bref, notre approche peut obtenir des tissus oculaires intact et a été utilisé avec succès pour étudier les profils expression tissu-spécifique de la rétine de poisson zèbre<sup&gt; 1, 2</sup&gt; Et l&#39;épithélium pigmentaire rétinien<sup&gt; 3</sup&gt;.

Watch this Scientific Journal Video about Microdissection of Zebrafish Embryonic Eye Tissues at JoVE.com

Microdissection of Zebrafish Embryonic Eye Tissues

Cet article décrit une approche pour le poisson-zèbre microdissect rétines avec et sans l&#39;épithélium pigmentaire rétinien attachés, de un à trois embryons jours post-fécondation.

Microdissection de tissus embryonnaires Zebrafish Eye

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post ...

microdissection-of-zebrafish-embryonic-eye-tissues

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16.0K vues.  Purdue University. Cet article décrit une approche pour le poisson-zèbre microdissect rétines avec et sans l'épithélium pigmentaire rétinien attachés, de un à trois embryons jours post-fécondation.

Vidéo: Microdissection of Zebrafish Embryonic Eye Tissues

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Microdissection laser des tissus de mammifères

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

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Biologie

Live Imaging of the Zebrafish Embryonic Brain by Confocal Microscopy

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the developing zebrafish brain (Gutzman et al, 2008). Such analysis affords a new understanding of the underlying cell biology required for development of the 3D structure of the vertebrate brain, and significantly increases our ability to study neural tube morphogenesis. The embryonic zebrafish brain is shaped beginning at 18 hours post fertilization (hpf) as the ventricles within the neuroepithelium inflate. By 24 hpf, the initial steps of neural tube morphogenesis are complete. Using the method described here, embryos at the one cell stage are injected with mRNA encoding membrane-targeted green fluorescent protein (memGFP). After injection and incubation, the embryo, now between 18 and 24 hpf, is mounted, inverted, in agarose and imaged by confocal microscopy. Notably, the zebrafish embryo is transparent making it an ideal system for fluorescent imaging. While our analyses have focused on the midbrain-hindbrain boundary and the hindbrain, this method could be extended for analysis of any region in the zebrafish to a depth of 
80-100 &mu;m.

In this video, we demonstrate a method by which to analyze the developing vertebrate brain in live zebrafish embryos at single cell resolution by confocal microscopy. This includes the method by which we inject the single-cell zebrafish embryo and subsequently mount and image the developing brain.

En direct l&#39;imagerie du cerveau de poisson zèbre embryonnaires par microscopie confocale

In this video, we demonstrate the method our lab has developed to analyze the cell shape changes and rearrangements required to bend and fold the ...

Lens Transplantation in Zebrafish and its Application in the Analysis of Eye Mutants

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development can be addressed. The zebrafish is useful for genetic studies due to several advantageous characteristics, including small size, high fecundity, short lifecycle, and ease of care. Lens development occurs rapidly in zebrafish. By 72 hpf, the zebrafish lens is functionally mature [3]. Abundant genetic and molecular resources are available to support research in zebrafish. In addition, the similarity of the zebrafish eye to those of other vertebrates provides basis for its use as an excellent animal model of human defects[4-7]. Several zebrafish mutants exhibit lens abnormalities, including high levels of cell death, which in some cases leads to a complete degeneration of lens tissues [8]. To determine whether lens abnormalities are due to intrinsic causes or to defective interactions with the surrounding tissues, transplantation of a mutant lens into a wild-type eye is performed. Using fire-polished metal needles, mutant or wild-type lenses are carefully dissected from the donor animal, and transferred into the host. To distinguish wild-type and mutant tissues, a transgenic line is used as the donor. This line expresses membrane-bound GFP in all tissues, including the lens. This transplantation technique is an essential tool in the studies of zebrafish lens mutants.

Lens development involves interactions with other tissues. Several zebrafish eye mutants are characterized by an abnormally small lens size. Here we demonstrate a lens transplantation experiment to determine whether this phenotype is due to intrinsic causes or defective interactions with tissues that surround the lens.

Objectif transplantation chez le poisson zèbre et son application dans l&#39;analyse de mutants Eye

The lens plays an important role in the development of the optic cup[1,2]. Using the zebrafish as a model organism, questions regarding lens development ...

Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups of cells in a larger context are altered.  Watching how one cell interacts with and behaves in a unique environment is essential to characterizing cell functions.  Determining how the localized misexpression of a specific protein influences surrounding cells provides insightful information on the roles that protein plays in a variety of developmental processes.  Our lab uses the zebrafish model system to uniquely combine genetic approaches with classical transplantation techniques to generate genotypic or phenotypic chimeras.  We study neuron-glial cell interactions during the formation of forebrain commissures in zebrafish. This video describes a method that allows our lab to investigate the role of astroglial populations in the diencephalon and the roles of specific guidance cues that influence projecting axons as they cross the midline. Due to their transparency zebrafish embryos are ideal models for this type of ectopic cell placement or localized gene misexpression. Tracking transplanted cells can be accomplished using a vital dye or a transgenic fish line expressing a fluorescent protein. We demonstrate here how to prepare donor embryos with a vital dye tracer for transplantation, as well as how to extract and transplant cells from one gastrula staged embryo to another.  We present data showing ectopic GFP+ transgenic cells within the forebrain of zebrafish embryos and characterize the location of these cells with respect to forebrain commissures.  In addition, we show laser scanning confocal timelapse microscopy of Alexa 594 labeled cells transplanted into a GFP+ transgenic host embryo.  These data provide evidence that gastrula staged transplantation enables the targeted positioning of ectopic cells to address a variety of questions in Developmental Biology.

Zebrafish cell transplantation enables the combination of genetics and embryology to generate tissue specific chimeras. This video demonstrates gastrula staged cell transplantations that have allowed our lab to investigate the roles of astroglial populations and specific guidance cues during commissure formation in the forebrain.

Tissu ciblé chimères embryonnaires: Zebrafish cellulaire Gastrula transplantation

Certain fundamental questions in the field of developmental biology can only be answered when cells are placed in novel environments or when small groups ...

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolement d&#39;ARN à partir de synthèse d&#39;ADNc embryonnaires Zebrafish et pour analyser l&#39;expression génique

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Microdissection of Black Widow Spider Silk-producing Glands

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of developing offspring 1,2. Spiders accomplish these tasks by spinning several distinct fiber types that have diverse mechanical properties. Such specialization of fiber types has occurred through the evolution of different silk-producing glands, which function as small biofactories. These biofactories manufacture and store large quantities of silk proteins for fiber production. Through a complex series of biochemical events, these silk proteins are converted from a liquid into a solid material upon extrusion.
Mechanical studies have demonstrated that spider silks are stronger than high-tensile steel 3. Analyses to understand the relationship between the structure and function of spider silk threads have revealed that spider silk consists largely of proteins, or fibroins, that have block repeats within their protein sequences 4. Common molecular signatures that contribute to the incredible tensile strength and extensibility of spider silks are being unraveled through the analyses of translated silk cDNAs. Given the extraordinary material properties of spider silks, research labs across the globe are racing to understand and mimic the spinning process to produce synthetic silk fibers for commercial, military and industrial applications. One of the main challenges to spinning artificial spider silk in the research lab involves a complete understanding of the biochemical processes that occur during extrusion of the fibers from the silk-producing glands.
Here we present a method for the isolation of the seven different silk-producing glands from the cobweaving black widow spider, which includes the major and minor ampullate glands [manufactures dragline and scaffolding silk] 5,6, tubuliform [synthesizes egg case silk] 7,8, flagelliform [unknown function in cob-weavers], aggregate [makes glue silk], aciniform [synthesizes prey wrapping and egg case threads] 9 and pyriform [produces attachment disc silk] 10. This approach is based upon anesthetizing the spider with carbon dioxide gas, subsequent separation of the cephalothorax from the abdomen, and microdissection of the abdomen to obtain the silk-producing glands. Following the separation of the different silk-producing glands, these tissues can be used to retrieve different macromolecules for distinct biochemical analyses, including quantitative real-time PCR, northern- and western blotting, mass spectrometry (MS or MS/MS) analyses to identify new silk protein sequences, search for proteins that participate in the silk assembly pathway, or use the intact tissue for cell culture or histological experiments.

Here we describe an efficient strategy to remove the silk-producing glands from the abdomen of female black widow spiders. This procedure allows the rapid isolation of the seven distinct silk-producing glands in a highly purified fashion, an important process for investigators studying spider silk production and fiber assembly.

Microdissection de Black Widow Soie d&#39;araignée des glandes productrices

Modern spiders spin high-performance silk fibers with a broad range of biological functions, including locomotion, prey capture and protection of ...

Immunostaining of Dissected Zebrafish Embryonic Heart

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics 1,2. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects 3. The expression of any gene can be manipulated via morpholino technology or RNA injection 4. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis 5. 
Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue 6. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. 
 Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. 
Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals.

A rapid way to conduct immunostaining of zebrafish embryonic heart is described. Compared to the whole mount immunostaining approach, this method dramatically increases the penetration of the antibodies, which allows obtaining high resolution images that reveal cellular/subcellular structures in the heart within a much reduced processing time.

Immunocoloration des Disséqué coeur embryonnaire Zebrafish

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology ...

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 ...

A System for ex vivo Culturing of Embryonic Pancreas

The pancreas controls vital functions of our body, including the production of digestive enzymes and regulation of blood sugar levels1. Although in the past decade many studies have contributed to a solid foundation for understanding pancreatic organogenesis, important gaps persist in our knowledge of early pancreas formation2. A complete understanding of these early events will provide insight into the development of this organ, but also into incurable diseases that target the pancreas, such as diabetes or pancreatic cancer. Finally, this information will generate a blueprint for developing cell-replacement therapies in the context of diabetes.
 During embryogenesis, the pancreas originates from distinct embryonic outgrowths of the dorsal and ventral foregut endoderm at embryonic day (E) 9.5 in the mouse embryo3,4. Both outgrowths evaginate into the surrounding mesenchyme as solid epithelial buds, which undergo proliferation, branching and differentiation to generate a fully mature organ2,5,6. Recent evidences have suggested that growth and differentiation of pancreatic cell lineages, including the insulin-producing &beta;-cells, depends on proper tissue-architecture, epithelial remodeling and cell positioning within the branching pancreatic epithelium7,8. However, how branching morphogenesis occurs and is coordinated with proliferation and differentiation in the pancreas is largely unknown. This is in part due to the fact that current knowledge about these developmental processes has relied almost exclusively on analysis of fixed specimens, while morphogenetic events are highly dynamic.
 Here, we report a method for dissecting and culturing mouse embryonic pancreatic buds ex vivo on glass bottom dishes, which allow direct visualization of the developing pancreas (Figure 1). This culture system is ideally devised for confocal laser scanning microscopy and, in particular, live-cell imaging. Pancreatic explants can be prepared not only from wild-type mouse embryos, but also from genetically engineered mouse strains (e.g. transgenic or knockout), allowing real-time studies of mutant phenotypes. Moreover, this ex vivo culture system is valuable to study the effects of chemical compounds on pancreatic development, enabling to obtain quantitative data about proliferation and growth, elongation, branching, tubulogenesis and differentiation. In conclusion, the development of an ex vivo pancreatic explant culture method combined with high-resolution imaging provides a strong platform for observing morphogenetic and differentiation events as they occur within the developing mouse embryo.

A System for ex vivo Culturing of Embryonic Pancreas

Here, we describe a method for isolation, culture and manipulation of mouse embryonic pancreas. This represents an excellent ex vivo system for studying various aspects of pancreatic development, including morphogenesis, differentiation and growth. Pancreatic bud explants can be cultured for several days and used in a range of different applications, including whole-mount immunofluorescence and live imaging.

Un système d&#39;<em&gt; Ex vivo</emLa culture&gt; de pancréas embryonnaire

The pancreas controls vital functions of our body,  including the production of digestive enzymes and regulation of blood sugar  levels1. Although in the ...

Laser-inflicted Injury of Zebrafish Embryonic Skeletal Muscle

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections (bupivacaine, cardiotoxin or notexin), muscle transplantations (denervation-devascularization induced regeneration), intensive exercise, but also murine muscular dystrophy models such as the mdx mouse (for a review of these approaches see 1). In zebrafish, genetic approaches include mutants that exhibit muscular dystrophy phenotypes (such as runzel2 or sapje3) and antisense oligonucleotide morpholinos that block the expression of dystrophy-associated genes4. Besides, chemical approaches are also possible, e.g. with Galanthamine, a chemical compound inhibiting acetylcholinesterase, thereby resulting in hypercontraction, which eventually leads to muscular dystrophy5. However, genetic and pharmacological approaches generally affect all muscles within an individual, whereas the extent of physically inflicted injuries are more easily controlled spatially and temporally1. Localized physical injury allows the assessment of contralateral muscle as an internal control. Indeed, we recently used laser-mediated cell ablation to study skeletal muscle regeneration in the zebrafish embryo6, while another group recently reported the use of a two-photon laser (822 nm) to damage very locally the plasma membrane of individual embryonic zebrafish muscle cells7.
Here, we report a method for using the micropoint laser (Andor Technology) for skeletal muscle cell injury in the zebrafish embryo. The micropoint laser is a high energy laser which is suitable for targeted cell ablation at a wavelength of 435 nm. The laser is connected to a microscope (in our setup, an optical microscope from Zeiss) in such a way that the microscope can be used at the same time for focusing the laser light onto the sample and for visualizing the effects of the wounding (brightfield or fluorescence). The parameters for controlling laser pulses include wavelength, intensity, and number of pulses.
Due to its transparency and external embryonic development, the zebrafish embryo is highly amenable for both laser-induced injury and for studying the subsequent recovery. Between 1 and 2 days post-fertilization, somitic skeletal muscle cells progressively undergo maturation from anterior to posterior due to the progression of somitogenesis from the trunk to the tail8, 9. At these stages, embryos spontaneously twitch and initiate swimming. The zebrafish has recently been recognized as an important vertebrate model organism for the study of tissue regeneration, as many types of tissues (cardiac, neuronal, vascular etc.) can be regenerated after injury in the adult zebrafish10, 11.

The method presented here comprises the precise injury of live zebrafish embryos with high-energy laser pulses and the subsequent analysis of these injuries and their recovery with time. We also show how genetically labeled single or groups of skeletal muscle cells can be tracked during and after laser light induced damage.

Laser-infligé des blessures de poisson zèbre muscle squelettique embryonnaire

Various experimental approaches have been used in mouse to induce muscle injury with the aim to study muscle regeneration, including myotoxin injections ...

Methods Article

Microdissection de tissus embryonnaires Zebrafish Eye