Diese Arbeit wurde vom NIH R01 HD050901 zu MCM und American Cancer Society Postdoctoral Fellowship # PF-05-041 bis 01-DDC zur TG unterstützt.

<ol><li> Ein männlicher Zebrafisch wird zuerst zerlegt werden, gefolgt von einer weiblichen Fische. Vor Beginn der Dissektion, betäuben ein Fisch in 0,2% Tricaine und dann einschläfern es durch Inkubation in Eiswasser für 15 Minuten. </li><li> Beginnen Sie mit dem leicht klopfen die Fische trocken auf einem Papiertuch und legt es auf dem Seziertisch Matte. Äußerlich haben Zebrafisch einzigen dorsalen, Schwanz-und Afterflossen und gepaart Brust-und Bauchflossen (Abbildung 1). <img src="/files/ftp_upload/1717/1717fig1.jpg" alt="Abbildung 1" /> Abbildung 1. Erwachsene männliche Fische mit Flossen gekennzeichnet. </li><li> Pin die Fische auf die Matte Sezieren durch den fleischigen Teil des Schwanzes und der ventrale Teil der Augenhöhle. </li><li> Snip die Haut an den Bauch des Fisches knapp vor der Afterflosse. Schneiden Sie die Haut und darunter liegende Muskulatur entlang der Bauch von der Afterflosse, die Operculum (die harte Abdeckung über der Kiemen) (Abbildung 2, Stufe 1). <img src="/files/ftp_upload/1717/1717fig2.jpg" alt="Abbildung 2" /> Abbildung 2. Steps bei der Beseitigung von Haut und Körper Wand Muskulatur der inneren Organe freizulegen. </li><li> Dann entfernen Sie das Operculum und die Brustflosse, einschließlich der Schultergürtel (die dicken, knöchernen Region an der Basis des fin) (Abbildung 2, Schritt 2). Schneiden Sie die Haut und darunter liegende Muskulatur zu Beginn von oben der nun freiliegenden Kiemen hinten an der Seite des Fisches und dann auf der Afterflosse (Abb. 2, Schritt 3). </li><li> Entfernen Sie vorsichtig die Haut und darunter liegende Muskulatur von der Seite des Fisches. Viele der inneren Organe sind jetzt sichtbar (Abbildung 3). <img src="/files/ftp_upload/1717/1717fig3.jpg" alt="Abbildung 3" /> Abbildung 3. Erwachsene männliche Fische mit Körperwand und Muskulatur entfernt ermöglicht die Visualisierung der inneren Organe. </li><li> Die Hoden sind lang, weiß, gepaart Organe, die an der dorsalen Körperwand befestigt sind. Entfernen eines Hodens und legen Sie sie in eine Schüssel mit PBS. Untersuchen Sie die Hoden mit reflektiertem Licht zu den Samenkanälchen (Abbildung 4), die Zysten enthalten mit verschiedenen Stadien der Entwicklung von Keimzellen aus spermatagonia zu Spermatiden (Leal et al., 2009) zu visualisieren. <img src="/files/ftp_upload/1717/1717fig4.jpg" alt="Abbildung 4" /> Abbildung 4. Dissection eines Hodens. Die weißen rundlichen Strukturen sind die Samenleiter. </li><li> Dann entfernen Sie den Magen-Darm-System aus der Körperhöhle des Fisches. Die Leber kann durch seine Größe, gelappt Morphologie, bräunlichen Farbe und umfangreiche Vaskularisierung identifiziert werden. Die Gallenblase, eine grüne durchscheinende Flüssigkeit gefüllten Sack, und die Milz, die leuchtend rot erscheint, sind in den Eingeweiden gefunden. </li><li> Separate den Darm aus dem Rest der Organe und auszustrecken. Die vorderen, mittleren und hinteren Regionen des Darms werden durch die Höhe der epithelialen Falten (Wallace et al., 2005) definiert. </li><li> Legen Sie ein Stück des Darms in PBS und beobachten Sie die epithelialen Falten mit Durchlicht. </li><li> Als nächstes untersuchen die Schwimmblase. Die Schwimmblase besteht aus einer hinteren Kammer, die an der Speiseröhre über den pneumatischen Kanal, und eine vordere Kammer, die an das Innenohr durch den Weberschen Apparat (Finney et al., 2006) ist in Verbindung steht. </li><li> Entfernen und Entsorgen der Schwimmblase. Unpin die Fische und re-pin es Bauchseite bis zu den Nieren, die sich entlang der dorsalen Körperwand befindet sezieren. Die Niere ist ein durchsichtiges Rosa-Struktur mit der dorsalen Aorta und pigmentierten Zellen assoziiert. Die Niere ist in Kopf, Körper und Schwanz Regionen (Abbildung 5) unterteilt. Dissect ein Stück der Niere und legen Sie sie in PBS. Tease neben dem Nierengewebe der Nierentubuli zu offenbaren. <img src="/files/ftp_upload/1717/1717fig5.jpg" alt="Abbildung 5" /> Abbildung 5. Lage der Kopf, Körper und Schwanz Nieren entlang der dorsalen Körperwand. </li><li> Sezieren einen weiblichen Fisch. Wie zuvor beschrieben, euthanize die Fische in Eiswasser und pat es trocken ist, bevor Pinning es dem Sezieren Matte. Entfernen Sie die Haut von der Seite des Fisches, wie zuvor gezeigt. Der Eierstock ist ein bilobed Struktur, die in der Leibeshöhle durch einen vaskularisierten Mesovarium ausgesetzt ist. </li><li> Nehmen Sie einen Lappen des Eierstocks, legen Sie sie in PBS, und prüfen Sie mit Durchlicht. Die Eizellen können neben gehänselt werden mit feinen Nadeln und dann inszeniert (Abbildung 6, Selman et al., 1993). Stage I Eizellen sind ca. 10 bis 150 Mikrometer groß und durchscheinend. Stage II-Oozyten sind etwa 150 bis 350 Mikrometer groß und definiert durch das Auftreten von kortikalen Granula. Stadium III Oozyten sind 350 bis 750 Mikrometer groß und undurchsichtig sind aufgrund der Ansammlung von Dotter. Reifen Stadium IV und V Eizellen sind lichtdurchlässig und in der Regel nicht in den Eierstöcken der Frauen, die vor kurzem gepaart gefunden haben. <img src="/files/ftp_upload/1717/1717fig6.jpg" alt="Abbildung 6" /> Abbildung 6. Live Stadium I, II, III und Eizellen unter einem beobachtetenBinokular mit Durchlicht. </li><li> Als nächstes zerlegen das Herz aus dem Fisch. Das Herz befindet sich posterior und ventral der Kiemen. Beginnen Sie mit dem Herausschneiden des Herzens und all das umliegende Gewebe und es in PBS. Sorgfältig sezieren weg das Gewebe rund um das Herz, darauf achten, daß die zarte Atrium Schäden. </li><li> Setzen Sie den sezierten Herzen in Ringer-Lösung, um den Herzschlag zu beobachten. Identifizieren Sie die Atrium, Ventrikel und Bulbus arteriosus (Abb. 7, Hu et al., 2001). <img src="/files/ftp_upload/1717/1717fig7.jpg" alt="Abbildung 7" /> Abbildung 7. Dissected erwachsenen Herzens. </li><li> Füllen Sie das Sezieren, indem das Gehirn aus dem Fisch. Beginnen Sie mit der unpinning die Fische und das Entfernen der Kopf mit einer Rasierklinge. Entfernen Sie so viel Weichgewebe wie möglich von der Bauchseite des Schädels mit einer Pinzette. Entfernen Sie die Augen mit kleinen Feder Schere. Brechen Sie den Schädel und entfernen Sie die Knochen aus dem ventralen Seite des Gehirns. Setzen Sie nun den Kopf in eine Schüssel mit PBS und die Haut abziehen und Schädelknochen von der dorsalen Seite des Gehirns. </li><li> Identifizieren Sie die Riechkolben, Telencephalon, Habenula, Tectum opticum, Cerebellum und Medulla (Abbildung 8, Wullimann et al, 1996;. Schilling, 2002). <img src="/files/ftp_upload/1717/1717fig8.jpg" alt="Abbildung 8" /> Abbildung 8. Dissected erwachsenen Zebrafisch Gehirn. Dorsalansicht, anterior nach oben. </li></ol>

<ol>
	<li>Finney, J. L., Robertson, G. N., McGee, C. A., Smith, F. M., Croll, R. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+Autonomic+Innervation+of+the+Swim+Bladder+in+the+Zebrafish+(Danio+rerio).">Structure and Autonomic Innervation of the Swim Bladder in the Zebrafish (Danio rerio).</a> J Comp Neurol. 495, 587-606 (2006).</li><li>Hu, N., Yost, H. J., Clark, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cardiac+morphology+and+blood+pressure+in+the+adult+zebrafish.">Cardiac morphology and blood pressure in the adult zebrafish.</a> Anat Rec. 264, 1-12 (2001).</li><li>Leal, M. C., Cardoso, E. R., N&#243;brega, R. H., Batlouni, S. R., Bogerd, J., Fran&#231;a, L. R., Schulz, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histological+and+stereological+evaluation+of+zebrafish+(Danio+rerio)+spermatogenesis+with+an+emphasis+on+spermatogonial+generations.">Histological and stereological evaluation of zebrafish (Danio rerio) spermatogenesis with an emphasis on spermatogonial generations.</a> Biol Reprod. 81, 177-187 (2009).</li><li>Schilling, T. F., 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=The+morphology+of+larval+and+adult+zebrafish.">The morphology of larval and adult zebrafish.</a> Zebrafish: A Practical Approach (The Practical Approach Series). , (2002).</li><li>Selman, K., Wallace, R., Sarka, A., Qi, X. <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+oocyte+development+in+the+Zebrafish,+Brachydanio+rerio.">Stages of oocyte development in the Zebrafish, Brachydanio rerio.</a> J. Morphol. 218, 203-224 (1993).</li><li>Wallace, K. N., Akhter, S., Smith, E. M., Lorent, K., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Intestinal+growth+and+differentiation+in+zebrafish.">Intestinal growth and differentiation in zebrafish.</a> Mech Dev. 122, 157-173 (2005).</li><li>Wullimann, M. F., Rupp, B., Reichert, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Neuroanatomy of the zebrafish brain : a topological atlas. , (1996).</li></ol>

Tierversuche wurden in Übereinstimmung mit den Richtlinien und Vorschriften her von der University of Pennsylvania Institutional Animal Care und Verwenden Ausschusses durchgeführt.

Reagenzien: 0,2% Tricaine (Ethyl-3-Aminobenzoesäure) 200 mg Pulver Tricaine 97,9 ml DD-Wasser ~ 1 ml 1 M Tris (pH 9) Der pH-Wert auf 7,0 Phosphat-gepufferte Kochsalzlösung (PBS) 4,0 g NaCl 0,1 g KCl 100 ml 0,1 M PO 4-Puffer, pH 7,3 150 ml dH 2 O Ringer-Lösung 6,7 g NaCl 0,2 g KCl 0,2 g CaCl 2 1,2 g Hepes 1 LH 2 O Der pH-Wert auf 7,2 Ausstattung: Dissecting Gericht mit Matte Electron Microscopy Sciences Katalog # 70540 Vannas Frühjahr Schere Feine Science Tools Katalog Nr. 91500-09

dissection of organs from the adult zebrafish

In den letzten 20 Jahren hat sich der Zebrafisch zu einem leistungsfähigen Modell-Organismus für das Verständnis der Entwicklung von Wirbeltieren und Krankheit. Obwohl experimentelle Analyse des Embryos und Larven ist sehr umfangreich und die Morphologie ist gut dokumentiert, sind die Beschreibungen der erwachsenen Zebrafisch Anatomie und Studien über die Entwicklung der erwachsenen Strukturen und Organe, zusammen mit Techniken für die Arbeit mit Erwachsenen fehlen. Die Organe der Larve eine bedeutende Veränderung in ihrer gesamten Struktur, Morphologie und anatomische Lage während des Larven zu erwachsenen Übergang. Äußerlich entwickelt die transparente Larve ihren charakteristischen Erwachsenen gestreiften Pigment Muster und gepaart Bauchflossen, während intern die Organe zu unterziehen massives Wachstum und Umbau. Darüber hinaus entwickelt die bipotentialen Geschlechtsdrüse Primordium entweder in Hoden oder Eierstock. Dieses Protokoll enthält viele Organe des Erwachsenen und zeigt Methoden für die Präparation des Gehirns, Gonaden, Magen-Darm-System, Herz und Niere des erwachsenen Zebrafisch. Die sezierten Organe können in-situ-Hybridisierung, Immunhistochemie, Histologie, RNA-Extraktion, Protein-Analyse und andere molekulare Techniken eingesetzt werden. Dieses Protokoll wird in die Erweiterung der Studien im Zebrafisch zu helfen, den Umbau des larvalen Organe, die Morphogenese von Organen spezifisch für die Erwachsenen und andere Untersuchungen der erwachsenen Organsysteme umfassen.

Watch this Scientific Journal Video about Dissection of Organs from the Adult Zebrafish at JoVE.com

Dissection of Organs from the Adult Zebrafish

Dieses Protokoll beschreibt ein Verfahren zur Identifizierung und seziert Organe von den erwachsenen Zebrafisch.

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 ...

dissection-of-organs-from-the-adult-zebrafish

Research

JoVE Journal

Biology

103.9K Aufrufe.  University of Pennsylvania-School of Medicine. Dieses Protokoll beschreibt ein Verfahren zur Identifizierung und seziert Organe von den erwachsenen Zebrafisch.

Serie: Dissection of Organs from the Adult Zebrafish

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.

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 ...

Forschung

Biologie

Dissection of Hippocampal Dentate Gyrus from Adult Mouse

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its remarkable neuronal cell plasticity, and its involvement in epilepsy, neurodegenerative diseases, and psychiatric disorders. The hippocampus is composed of distinct regions; the dentate gyrus, which comprises mainly granule neurons, and Ammon's horn, which comprises mainly pyramidal neurons, and the two regions are connected by both anatomic and functional circuits. Many different mRNAs and proteins are selectively expressed in the dentate gyrus, and the dentate gyrus is a site of adult neurogenesis; that is, new neurons are continually generated in the adult dentate gyrus. To investigate mRNA and protein expression specific to the dentate gyrus, laser capture microdissection is often used. This method has some limitations, however, such as the need for special apparatuses and complicated handling procedures. In this video-recorded protocol, we demonstrate a dissection technique for removing the dentate gyrus from adult mouse under a stereomicroscope. Dentate gyrus samples prepared using this technique are suitable for any assay, including transcriptomic, proteomic, and cell biology analyses. We confirmed that the dissected tissue is dentate gyrus by conducting real-time PCR of dentate gyrus-specific genes, tryptophan 2,3-dioxygenase (TDO2) and desmoplakin (Dsp), and Ammon's horn enriched genes, Meis-related gene 1b (Mrg1b) and TYRO3 protein tyrosine kinase 3 (Tyro3). The mRNA expressions of TDO2 and Dsp in the dentate gyrus samples were detected at obviously higher levels, whereas Mrg1b and Tyro3 were lower levels, than those in the Ammon's horn samples. To demonstrate the advantage of this method, we performed DNA microarray analysis using samples of whole hippocampus and dentate gyrus. The mRNA expression of TDO2 and Dsp, which are expressed selectively in the dentate gyrus, in the whole hippocampus of alpha-CaMKII+/- mice, exhibited 0.037 and 0.10-fold changes compared to that of wild-type mice, respectively. In the isolated dentate gyrus, however, these expressions exhibited 0.011 and 0.021-fold changes compared to that of wild-type mice, demonstrating that gene expression changes in dentate gyrus can be detected with greater sensitivity. Taken together, this convenient and accurate dissection technique can be reliably used for studies focused on the dentate gyrus.

A dissection technique for removal of the dentate gyrus from adult mouse under a stereomicroscope was demonstrated in this video-recorded protocol.

Dissection der hippocampalen von erwachsenen Maus

The hippocampus is one of the most widely studied areas in the brain because of its important functional role in memory processing and learning, its ...

Intraperitoneal Injection into Adult Zebrafish

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, this method makes it difficult to know how much reagent is absorbed or taken up per fish. Some experimental questions, particularly those related to metabolic studies, may be better addressed by delivering a defined quantity to each fish, based on weight. Here we present a method for intraperitoneal (IP) injection into adult zebrafish. Injection is into the abdominal cavity, posterior to the pelvic girdle. This procedure is adapted from veterinary methods used for larger fish. It is safe, as we have observed zero mortality. Additionally, we have seen bleeding at the injection site in only 5 out of 127 injections, and in each of those cases the bleeding was brief, lasting several seconds, and the quantity of blood lost was small. Success with this procedure requires gentle handling of the fish through several steps including fasting, weighing, anesthetizing, injection, and recovery. Precautions are required to minimize stress throughout the procedure. Our precautions include using a small injection volume and a 35G needle. We use Cortland salt solution as the vehicle, which is osmotically balanced for freshwater fish. Aeration of the gills is maintained during the injection procedure by first bringing the fish into a surgical plane of anesthesia, which allows slow operculum movements, and second, by holding the fish in a trough within a water-saturated sponge during the injection itself. We demonstrate the utility of IP injection by injecting glucose and monitoring the rise in blood glucose level and its subsequent return to normal. As stress is known to increase blood glucose in teleost fish, we compare blood glucose levels in vehicle-injected and non-injected adults and show that the procedure does not cause a significant rise in blood glucose.

We demonstrate intraperitoneal injection into adult zebrafish. We use a 10 &mu;l NanoFil microsyringe controlled by a Micro4 controller and UltraMicroPump III. This demonstration includes the use of cold water as an anesthetic.

Intraperitoneale Injektion in Adult Zebrafisch

A convenient method for chemically treating zebrafish is to introduce the reagent into the tank water, where it will be taken up by the fish. However, ...

Dissection of Oenocytes from Adult Drosophila melanogaster

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides protection against desiccation and other environmental challenges. Several of these cuticular hydrocarbon (CHC) compounds also function as semiochemical signals, and as such mediate pheromonal communications between members of the same species, or in some instances between different species, and influence behavior. Specialized cells referred to as oenocytes are regarded as the primary site for CHC synthesis. However, relatively little is known regarding the involvement of the oenocytes in the regulation of the biosynthetic, transport, and deposition pathways contributing to CHC output. Given the significant role that CHCs play in several aspects of insect biology, including chemical communication, desiccation resistance, and immunity, it is important to gain a greater understanding of the molecular and genetic regulation of CHC production within these specialized cells. The adult oenocytes of D. melanogaster are located within the abdominal integument, and are metamerically arrayed in ribbon-like clusters radiating along the inner cuticular surface of each abdominal segment. In this video article we demonstrate a dissection technique used for the preparation of oenocytes from adult D. melanogaster. Specifically, we provide a detailed step-by-step demonstration of (1) how to fillet prepare an adult Drosophila abdomen, (2) how to identify the oenocytes and discern them from other tissues, and (3) how to remove intact oenocyte clusters from the abdominal integument. A brief experimental illustration of how this preparation can be used to examine the expression of genes involved in hydrocarbon synthesis is included. The dissected preparation demonstrated herein will allow for the detailed molecular and genetic analysis of oenocyte function in the adult fruit fly.

Dissection of Oenocytes from Adult Drosophila melanogaster

In insects, the oenocytes produce cuticular hydrocarbon compounds. These compounds protect against desiccation and facilitate chemical communication. Here we demonstrate a dissection technique used to isolate the oenocytes from adult Drosophila melanogaster, and illustrate how this preparation can be utilized to study genes involved in hydrocarbon synthesis.

Präparation Oenocytes von Adult Drosophila melanogaster</em

In Drosophila melanogaster, as in other insects, a waxy layer on the outer surface of the cuticle, composed primarily of hydrocarbon compounds, provides ...

Transplantation of Cells Directly into the Kidney of Adult Zebrafish

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic diseases1. However, most organs are not easily accessible, necessitating the need to develop surgical methods to gain access to these structures. In this video article, we describe a method for transplanting cells directly into the kidney of adult zebrafish, a popular model to study regeneration and disease2. Recipient fish are pre-conditioned by irradiation to suppress the immune rejection of the injected cells3. We demonstrate how the head kidney can be exposed by a lateral incision in the flank of the fish, followed by the injection of cells directly in to the organ. Using fluorescently labeled whole kidney marrow cells comprising a mixed population of renal and hematopoietic precursors, we show that nephron progenitors can engraft and differentiate into new renal tissue - the gold standard of any cell-based regenerative therapy. This technique can be adapted to deliver purified stem or progenitor cells and/or small molecules to the kidney as well as other internal organs and further enhances the zebrafish as a versatile model to study regenerative medicine.

Cell transplantation is an essential technique for studying tissue regeneration and for developing cell-based therapies of disease. We demonstrate here a microsurgical technique that permits the transplantation of genetically labeled cells directly into the kidney of adult zebrafish fish.

Die Transplantation von Zellen direkt in die Niere von Adult Zebrafisch

Regenerative medicine based on the transplantation of stem or progenitor cells into damaged tissues has the potential to treat a wide range of chronic ...

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 ...

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.

Gavaging Adult Zebrafisch

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