Diese Arbeit wurde durch die Interne Research Program des National Human Genome Research Institute, National Institutes of Health unterstützt.

Teil 1: Vorbereitung des Gewebes <ol><li> Euthanize der erwachsenen Zebrafisch mit gepufferter MS-222 (Ethyl-3-Aminobenzoesäure Methansulfonat Salz, 0,03%). </li><li> Enthaupten die Fische. Schneiden Sie den Unterkiefer, platzieren Sie den Kopf auf die Zerlegung Pad mit der Bauchseite auf und fixieren das Gewebe mit Insekten Pins. Entfernen Sie die Weichteile ventral auf den Schädel und knacken den ventralen Teil des Schädels Kapsel mit einer feinen Pinzette unter einer Dissektionsmikroskop. </li><li> Fix der Fischkopf in Paraformaldehyd (4%) bei 4 ° C über Nacht. </li><li> Spülen Sie den festen Fischkopf in PBS (Phosphate Buffered Saline, 1x) für 3-mal, jeweils 5 Minuten Zeit. </li></ol> Teil 2: Gross Dissektion des Innenohrs <ol><li> Den Fisch den Kopf auf die Zerlegung Pad mit der Bauchseite nach oben. Immobilisieren sie mit Insekten Pins. Alle folgenden Schritte werden unter der Dissektionsmikroskop durchgeführt. </li><li> Mit einer feinen Pinzette, entfernen Sie die Schädelknochen über das Innenohr und dann vorsichtig herausziehen Innenohrgeweben: Säckchen, lagenae und Schläuchen. Der Sacculus und Lagena sind eng miteinander verbinden und so ziehen zusammen, während der Schlauch (anterior des Sacculus und Lagena), wird sehr wahrscheinlich vom anderen Ende Organe während der Präparation gelöst werden. </li><li> Legen Sie die Innenohrgeweben in PBS (1x). Wenn keine weiteren feinen Sezierung erforderlich ist, spülen Sie das Gewebe 2-3 mal mit PBS (1x) und es kann auch für andere Experimente, z. B. Gefrierschnitte verwendet werden. </li></ol> Teil 3: Fine Dissektion des Innenohrs Sinnesepithelien <ol><li> Die feinen Sezierung kann in einem Tropfen PBS (1x) auf einen sauberen Glasobjektträger durchgeführt werden. </li><li> Entfernen Sie die Utriculardrüsen Otolithen aus dem Utriculus mit zwei Paaren von feinen Pinzette, und, falls gewünscht, schneiden Sie die nicht-sensorische Epithelgewebe und die Innervation der Epithelien mit einer feinen Pinzette und eine Nadel Klinge. </li><li> Entfernen Sie die lagenar Otolithen vor der Trennung saccular und lagenar Epithelien während versuchen, die ganze Sacculus so intakt wie möglich zu halten. Legen Sie das Gewebe in der PBS-drop mit Bauchseite auf. Verwenden Sie die Nadel Klinge sorgfältig zwischen den Sacculus und Lagena geschnitten. Schneiden Sie die übermäßige non-sensorischen Epithel-Gewebe und die innvervation mit feinen Pinzette und Nadel Klinge, falls gewünscht. </li></ol> Teil 4: Fluoreszenzfärbung der Haarzellen in der Sinnesepithelien <ol><li> Spülen Sie den Sinnesepithelien mit PBT (1XPBS und 1% Triton X-100) für 3-mal, jeweils 5 min. </li><li> Inkubieren Sie die Epithelien mit fluophore-markiertem Phalloidin (zB Alexa Flour 488 Phalloidin, 1:1000 verdünnt) für 30 min bei Raumtemperatur. </li><li> Spülen Sie den Sinnesepithelien mit PBT für 3-mal, jeweils 10 Minuten Zeit. </li><li> Montieren Sie die Epithelien auf eine Folie mit Eindeckmedium und überprüfen Sie die gefärbten Gewebeschnitten unter dem Fluoreszenz-Mikroskop mit Filtern der richtigen Wellenlänge. </li><li> Nach der erfolgreichen Präparation und Färbung, können intakte Epithelien mit starken Haarzelle Bündel Färbung unter dem Mikroskop (Abb. 1) zu sehen. </li></ol><img src="/files/ftp_upload/1211/fig1.png" alt="Abbildung 1" /> Abbildung 1. Haarzellen des sackförmigen Sinnesepithel wurden mit Alexa Flour 488 Phalloidin (grün) gefärbt. Die Gewebeprobe wurde seziert und gefärbt wie in der Zeitung erwähnt. Mehrere Bilder wurden an verschiedenen Stellen der Probe entnommen und gefliesten zusammen mit Adobe Photoshop 7.0. Maßstab = 150 um.

In diesem Protokoll beschrieben wir die grobe und feine Zerlegung des Innenohrs Sinnesepithelien der erwachsenen Zebrafisch, die uns Gewebeprobe zur Untersuchung der zellulären und subzellulären Eigenschaften der Sinnesepithelien vorbereiten können. Um die Durchführung einer erfolgreichen Präparation der allererste Schritt, Fixierung, ist sehr wichtig. Stellen Sie sicher, dass Sie immer frisch zubereitet Paraformaldehydlösung weil alte Lösung in unter-Fixierung der Gewebe, die schwer ...

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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>MS222 (Ethyl 3-aminobenzoate methanesulfonate salt)</td>
<td>Reagent</td>
<td>Sigma</td>
<td>A5040</td>
<td>&nbsp;</td>
<tr>
<td>PBS (Phosphate-buffered Saline, 10X)</td>
<td>Reagent</td>
<td>Mediatech</td>
<td>46-013-CM</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde</td>
<td>Reagent</td>
<td>Sigma</td>
<td>P-6148</td>
<td>&nbsp;</td>
<tr>
<td>Triton X-100</td>
<td>Reagent</td>
<td>Sigma</td>
<td>T8787</td>
<td>&nbsp;</td>
<tr>
<td>Alexa Fluor 488 phalloidin</td>
<td>Reagent</td>
<td>Invitrogen</td>
<td>A12379</td>
<td>&nbsp;</td>
<tr>
<td>VECTASHIELD Hard Set Mounting Medium with DAPI</td>
<td>Reagent</td>
<td>Vector Laboratories</td>
<td>H-1500</td>
<td>&nbsp;</td>
<tr>
<td>Dissection microscope</td>
<td>Microscope</td>
<td>Nikon</td>
<td>SMZ1000</td>
<td>&nbsp;</td>
<tr>
<td>Insect pins</td>
<td>Tool</td>
<td>Fine Science Tools</td>
<td>26000-40</td>
<td>&nbsp;</td>
<tr>
<td>Scissors</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5880</td>
<td>&nbsp;</td>
<tr>
<td>Tweezers</td>
<td>Tool</td>
<td>Roboz</td>
<td>RS-5045</td>
<td>&nbsp;</td>
<tr>
<td>Needle blade</td>
<td>Tool</td>
<td>Sharpoint</td>
<td>78-6810</td>
<td>&nbsp;</td>
<tr>
<td>Fluorescent microscope</td>
<td>Microscope</td>
<td>Zeiss</td>
<td>Axiovert 200M</td>
<td>&nbsp;</td>
<tr>
<td>Dissection pad</td>
<td>Tool</td>
<td>eNasco</td>
<td>SB07840M</td>
<td>&nbsp;</td>
<tr>
<td>Zebrafish</td>
<td>Animal</td>
<td>various</td>
<td>n/a</td>
<td>&nbsp;</td>		</tbody>
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gross and fine dissection of inner ear sensory epithelia in adult zebrafish (danio rerio)

Neurosensorische Epithelien im Innenohr sind die entscheidenden Strukturen für die Hör-und Gleichgewichtsstörungen Funktionen. Daher ist es wichtig, die zellulären und molekularen Eigenschaften der Epithelien, die vor allem von zwei Arten von Zellen zusammengesetzt sind zu verstehen: Haarzellen (HC) und Stützzellen (SCS). Hier wählen wir an das Innenohr Sinnesepithelien bei erwachsenen Zebrafisch nicht nur studieren, weil der epithelialen Strukturen stark in allen Wirbeltieren studiert konserviert sind, sondern auch, weil die erwachsenen Zebrafisch ist in der Lage, Kohlenwasserstoffe, eine Fähigkeit, die Säugetiere verlieren kurz nach der Geburt zu regenerieren. Wir verwenden das Innenohr von adulten Zebrafisch als Modellsystem, um die Mechanismen des Innenohrs HC Regeneration bei erwachsenen Wirbeltieren, die hilfreich für die klinische Therapie des Hörens / Balance Defizite in der menschlichen als Folge der HC Verlust könnte zu studieren. Hier zeigen wir, wie auf grobe und feine Sezieren des Innenohres Sinnesepithelien bei erwachsenen Zebrafisch zu tun. Der Brutto-Dissektion entfernt die umgebenden Gewebe des Innenohrs und ist hilfreich für die Vorbereitung Gewebeschnitten, die uns die detaillierte Struktur der Sinnesepithelien untersuchen können. Die feinen Sezierung bereinigt die nicht sensorisch-epithelialen Geweben der einzelnen Epithel und ermöglicht es uns, die Heterogenität der ganzen Epithel leicht prüfen, in ganz-mount epithelialen Proben.

Watch this Scientific Journal Video about Gross and Fine Dissection of Inner Ear Sensory Epithelia in Adult Zebrafish Danio rerio at JoVE.com

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

Das Innenohr Sinnesepithel der erwachsenen Zebrafisch ist ein gutes Modell für das Verständnis der Mechanismen der Haare Zellregeneration bei erwachsenen Wirbeltieren. Dieses Protokoll zeigt die feinen Sezierung der Epithelien, durch die wir Gewebeproben zur Untersuchung der regenerativen Veranstaltungen auf zellulärer und subzellulärer Ebene können.

Gross und Fine Dissection of Inner Ear Sinnesepithelien in Adult Zebrafisch ( Danio rerio)

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

gross-fine-dissection-inner-ear-sensory-epithelia-adult-zebrafish

Research

JoVE Journal

Biology

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

16.3K Aufrufe.  National Human Genome Research Institute. Das Innenohr Sinnesepithel der erwachsenen Zebrafisch ist ein gutes Modell für das Verständnis der Mechanismen der Haare Zellregeneration bei erwachsenen Wirbeltieren. Dieses Protokoll zeigt die feinen Sezierung der Epithelien, durch die wir Gewebeproben zur Untersuchung der regenerativen Veranstaltungen auf zellulärer und subzellulärer Ebene können.

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

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of the stomach is defined by the mouth opening and the posterior end by the bottom of the pylorus. Along the dorsal side of the stomach lies the stomatogastric nervous system (STNS). This nervous system, which contains rhythmic networks that underlie feeding behavior, is an established model system for studying rhythm generating networks and neuromodulation 1,2. While it is possible to study this system in vivo 3, the STNS continues to produce its rhythmic activity when isolated in vitro. In order to study this system in vitro the stomach must be removed from the animal. This video article describes how the stomach can be dissected from the American lobster. In an accompanying video article4 we demonstrate how the STNS can be isolated from the stomach.

Gross Dissection of the Stomach of the Lobster, Homarus Americanus

We describe the gross dissection of the stomach of the American lobster (Homarus americanus).

Gross Dissection der Magen der Lobster, Homarus americanus</em

The stomach of the American lobster (Homarus americanus) is located in the cephalothorax, between the rostrum and the cervical groove. The anterior end of ...

Forschung

Biologie

Retro-orbital Injection in Adult Zebrafish

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is often the most effective and noninvasive form of delivery of an agent of interest. In the zebrafish (Danio rerio), IV injection of drugs has long been a challenge because of the small vessel diameter. This has also proved a significant hurdle for the injection of cells during hematopoeitic stem cell transplantation. Historically, injections into the bloodstream were done directly through the heart. However, this intra-cardiac procedure has a very high mortality rate as the heart is often punctured during injection leaving the fish prone to infection, massive blood loss or fatal organ damage. Drawing on our experience with the mouse, we have developed a new injection procedure in the zebrafish in which the injection site is behind the eye and into the retro-orbital venous sinus. This retro-orbital (RO) injection technique has been successfully employed in both the injection of drugs in the adult fish as well as transplantation of whole kidney marrow cells. RO injection has a much lower mortality rate than traditional intra-cardiac injection. Fish that are injected retro-orbitally tend to bleed less following injection and are at a much lower risk of injury to a major organ like the heart. Further, when performed properly, injected cells and/or drugs quickly enter the bloodstream allowing compounds to exert their effect on the whole fish and kidney cells to easily home to their niche. Thus, this new injection technique minimizes mortality while allowing efficient delivery of material into the bloodstream of adult fish. Here we exemplify this technique by retro-orbital injection of Tg(globin:GFP) cells into adult casper fish as well as injection of a red fluorescent dye (dextran, Texas Red ) into adult casper fish. We then visualize successful injections by whole animal fluorescence microscopy.

Here we show how to do retro-orbital injection in adult zebrafish.

Retro-Orbital-Injection in Adult Zebrafisch

Drug treatment of whole animals is an essential tool in any model system for pharmacological and chemical genetic studies. Intravenous (IV) injection is ...

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 von Organen aus dem Adult Zebrafisch

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

Direct Delivery of MIF Morpholinos Into the Zebrafish Otocyst by Injection and Electroporation Affects Inner Ear Development

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the eye, brain, and somites of zebrafish. The advantage of electroporation over other methods of genetic manipulation is that specific tissues can be targeted, both spatially and temporally, for the introduction of macromolecules by the application of electrical current. Here we describe the use of electroporation for transfecting mif and mif-like morpholinos into the tissues of the developing inner ear of the zebrafish. In past studies, mif morpholino injected into embryos at the 1- to 8-cell stage resulted in widespread morphological changes in the nervous system and eye, as well as the ear. By targeting the tissues of the inner ear at later stages in development, we can determine the primary effects of MIF in the developing inner ear, as opposed to secondary effects that may result from the influence of other tissues. By using phalloidin and acetylated tubulin staining to study the morphology of neurons, neuronal processes, and hair cells associated with the posterior macula, we were able to assess the efficacy of electroporation as a method for targeted transfection in the zebrafish inner ear. The otic vesicles of 24hpf embryos were injected with morpholinos and electroporated and were then compared to embryos that had received no treatment or had been only injected or electroporated. Embryos that were injected and electroporated showed a decrease in hair cell numbers, decreased innervation by the statoacoustic ganglion (SAG) and fewer SAG neurons compared with control groups. Our results showed that direct delivery of morpholinos into otocysts at later stages avoids the non-specific nervous system and neural crest effects of morpholinos delivered at the 1-8 cell stage. It also allows examination of effects that are directed to the inner ear and not secondary effects on the ear from primary effects on the brain, neural crest or periotic mesenchyme.

A method to deliver morpholinos directly into the zebrafish otocyst at 24hpf has been developed. Using microinjection of morpholinos into the lumen of otic vesicle and electroporation to effect penetration, we were able to bypass the effect of morpholinos on the brain and obtain effects specific to the inner ear.

Direkte Lieferung von MIF Morpholinos Into the Zebrafisch Otocyst durch Injektion und Elektroporation Beeinflusst Inner Ear Entwicklung

In recent years, electroporation has become a popular technique for in vivo transfection of DNA, RNA, and morpholinos into various tissues, including the ...

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.

Präparation des Adult Zebrafisch 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 ...

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

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development (see recent reviews1-6), however, work examining post-embryonic through adult cardiac development has been limited7-10. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis11. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5mm standard length (SL) with hearts of 100&mu;m ventricle length (VL), to adults, with SL of 30mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques.
We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)twu34 (12), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

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

A clear, standardized method for dissection and isolation of the zebrafish heart at multiple developmental stages are described. Annotation and quantification techniques are also discussed.

Herz Dissection in Larven, juvenile und adulte Zebrafisch, 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 ...

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.

In-vivo-Elektroporation von Morpholinos in die Adult Zebrafisch Retina

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

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration of adult tissues, such as the heart1, retina2, spinal cord3, optic nerve4, sensory hair cells5, and fins6.
The zebrafish fin is a relatively simple appendage that is easily manipulated to study multiple stages in epimorphic regeneration. Classically, fin regeneration was characterized by three distinct stages: wound healing, blastema formation, and fin outgrowth. After amputating part of the fin, the surrounding epithelium proliferates and migrates over the wound. At 33 &deg;C, this process occurs within six hours post-amputation (hpa, Figure 1B)6,7. Next, underlying cells from different lineages (ex. bone, blood, glia, fibroblast) re-enter the cell cycle to form a proliferative blastema, while the overlying epidermis continues to proliferate (Figure 1D)8. Outgrowth occurs as cells proximal to the blastema re-differentiate into their respective lineages to form new tissue (Figure 1E)8. Depending on the level of the amputation, full regeneration is completed in a week to a month.
The expression of a large number of gene families, including wnt, hox, fgf, msx, retinoic acid, shh, notch, bmp, and activin-betaA genes, is up-regulated during specific stages of fin regeneration9-16. However, the roles of these genes and their encoded proteins during regeneration have been difficult to assess, unless a specific inhibitor for the protein exists13, a temperature-sensitive mutant exists or a transgenic animal (either overexpressing the wild-type protein or a dominant-negative protein) was generated7,12. We developed a reverse genetic technique to quickly and easily test the function of any gene during fin regeneration.
Morpholino oligonucleotides are widely used to study loss of specific proteins during zebrafish, Xenopus, chick, and mouse development17-19. Morpholinos basepair with a complementary RNA sequence to either block pre-mRNA splicing or mRNA translation. We describe a method to efficiently introduce fluorescein-tagged antisense morpholinos into regenerating zebrafish fins to knockdown expression of the target protein. The morpholino is micro-injected into each blastema of the regenerating zebrafish tail fin and electroporated into the surrounding cells. Fluorescein provides the charge to electroporate the morpholino and to visualize the morpholino in the fin tissue.
This protocol permits conditional protein knockdown to examine the role of specific proteins during regenerative fin outgrowth. In the Discussion, we describe how this approach can be adapted to study the role of specific proteins during wound healing or blastema formation, as well as a potential marker of cell migration during blastema formation.

In vivo Electroporation of Morpholinos into the Regenerating Adult Zebrafish Tail Fin

We describe a method to conditionally knockdown the expression of a target protein during adult zebrafish fin regeneration. This technique involves micro-injecting and electroporating antisense oligonucleotide morpholinos into fin tissue, which allows testing the protein&#x2019;s role in various stages of fin regeneration, including wound healing, blastema formation, and regenerative outgrowth.

In-vivo-Elektroporation von Morpholinos in das Regenerations Adult Zebrafisch Leitwerk

Certain species of urodeles and teleost fish can regenerate their tissues. Zebrafish have become a widely used model to study the spontaneous regeneration ...

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.

Regelmäßige Pflege und Wartung eines Zebrafisch (<em&gt; Danio rerio</em&gt;) Labor: Eine Einführung

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 Modellierung des Morbid Human Genome mit Danio rerio

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

Gross und Fine Dissection of Inner Ear Sinnesepithelien in Adult Zebrafisch ( Danio rerio)

Zusammenfassung

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