Name: visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish
Uploaded: 2016-12-21T13:00:00.000+00:00
Duration: 7 min 19 s
Description: Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Wir danken Prof. Christoph Englert und Prof. Didier Stainier zum Verschenken der Tg (wt1b: GFP) LI1 und Tg (kdrl: EGFP) S843 Stämme sind, und die Taiwan Zebrabärbling Core Facility für die Bereitstellung von Tg (kdrl: mCherry) CI5. Diese Studie wurde unterstützt durch Zuschüsse aus Taiwan Ministerium für Wissenschaft und Technologie (96-2628-B-029-002-MY3, unterstützt 101-2313-B-029-001, 102-2628-B-029-002-MY3, 102- 2321-B-400-018).

Ethikerklärung: Alle experimentellen Verfahren auf Zebrabärbling wurden von der Institutional Animal Care und Use Committee von Tunghai University (IRB Zulassung NO 101-12.) Genehmigt und in Übereinstimmung mit den genehmigten Richtlinien durchgeführt. 1. Stammlösungen für 3β -HSD enzymatischen Aktivität Färbung <ol><li> Vorbereitung trans-Dehydroandrosteron [10 mg / ml in Dimethylsulfoxid (DMSO)]. </li><li> Bereiten β-Nicotinamidadenindinucleotid-Hydrat (1,2 mg / ml in 0,1 M Phosphatpuffer, pH 7,2). </li><li> Bereiten Nicotinamid (Vitamin B3, 50 mg / ml in H 2 O). </li><li> Herstellung von 4-Nitroblau-Tetrazolium (50 mg / ml in 70% Dimethylformamid). </li><li> Aliquotieren alle diese Komponenten in Mikrozentrifugenröhrchen und bei -20 ° C. HINWEIS: Nach unserer Erfahrung wiederholtem Einfrieren und Auftauen der Aliquots aller vorgenannten Lösungen haben keinen Einfluss auf die Intensität der 3β-HSD-Färbung. </li></ol> 2. WLoch-Mount-3β-HSD-Färbung HINWEIS: Die 3β-Hsd enzymatische Aktivität ist nachweisbar in Zebrabärbling Embryonen bereits 28 Stunden nach der Befruchtung (hpf) bei Kultivierung bei 28,5 ° C, die mit dem Beginn der mRNA - Expression von hsd3b1 2,7 konsistent ist. Die ganze Montage 3β-HSD Färbeprotokolls in dieser Studie basiert auf einem zuvor beschriebenen Verfahren 8 und wurde in der Entwicklung von Zebrafisch bis zu 7 Tage nach der Befruchtung 9 erfolgreich zu sein bestimmt. Für die vaskuläre Mikroumgebung des Interrenal- steroidogenen Gewebe zu analysieren, Vollmontage 3β-HSD - Färbung muss transgenen Linien exprimieren Endothel-spezifische Fluoreszenz, wie Tg angewendet werden (kdrl: EGFP) S843 14 und Tg (kdrl: mCherry) CI5 15 . Zur Analyse der Interrenal- steroidogenen Gewebe unter Berücksichtigung Zebrabärbling Nierenentwicklung kann 3β-HSD-Färbung sein Anwened auf die Tg (wt1b: GFP) LI1 Fisch 16, wobei die Bildung der Glomeruli und Tubuli pronephric abgegrenzt ist. <ol><li> Behandeln Sie die Entwicklung von Zebrafisch, indem sie in 0,03% Phenylthioharnstoff in Ei Wasser (0,06 mg / L Riff Salz in demineralisiertem Wasser) Inkubation von einer Stufe zwischen 12 und 24 hpf beginnen, Pigmentbildung zu hemmen. </li><li> Fix dechorionated Embryonen oder Larven in (A) 4% Paraformaldehyd (PFA) in phosphatgepufferter Kochsalzlösung (PBS), supplementiert mit 0,1% Tween 20 (PFAT) für 1 Stunde bei Raumtemperatur (RT) oder (B) 2% PFAT über Nacht bei 4 ° C oder für 2 Stunden bei 4 ° C, um 4 h lang bei RT verfolgt. Achtung: PFA ist giftig beim Einatmen und bei Hautkontakt. Es ist schädlich für die Haut, der Schleimhäute, der Augen und der oberen Atemwege. </li><li> Waschen Sie die Embryonen 4x 10 min mit PBS mit 0,1% Tween 20 (PBST) bei RT ergänzt. HINWEIS: Es ist wichtig, nicht die Proben trocknen, während die Waschlösungen zu verändern. </li><li> Frisch herstellen, die staining Lösung vor den Reaktionen (Tabelle 1). Vorbereiten, Wirbel und Zentrifugenteile A und B in getrennten Röhren. HINWEIS: alle Komponenten Hinzufügen gerade in einem einzigen Rohr würde heftige Niederschläge verursachen. </li><li> Fügen Sie die gesamte Lösung von Teil A auf die gesamte Lösung von Teil B, gut mischen die Färbung Lösung herzustellen, und sofort zu verteilen 1 ml in jedes Mikroröhrchen, das fixiert und gewaschen Embryonen. </li><li> Wrap-Mikrozentrifugenröhrchen mit Aluminiumfolie zum Schutz vor Licht und Ort entweder auf einem Rotator oder einem Wippschüttler für sanftes Schütteln bei RT. </li><li> Empirisch bestimmen durch Überwachung chromogenen Signale durch Mikroskopie, die Reaktionszeit. Leichte Fällungen wird im Laufe der Zeit entwickeln; aber sie werden nicht mit dem Reaktionsprozess stören. HINWEIS: Bei Embryonen bei 25 ° C für 4 Stunden bei 28 HPF, Färbung fixiert erzeugt ein klares Signal an die Interrenal- Gewebe. </li><li> Die Reaktion durch 4x Waschen für 10 min in PBST. ichf keine weiteren immunhistochemischen (IHC) Analyse erforderlich ist, post-fix die gefärbten Embryonen mit 4% PFAT für 60 min bei RT. Andernfalls speichert die gewaschenen Embryonen bei 4 ° C bis zur weiteren Verwendung. </li><li> Für Ganz Mount Mikroskopie, deaktivieren Sie die gefärbten, post-fixiert und gewaschen Embryonen mit 50% Glycerin in PBS, orientieren sie mit der Rückenseite auf einer Folie nach oben und beobachten ein aufrechtes Mikroskop unter Hellfeldbeleuchtung verwendet wird. HINWEIS: Um ein hochauflösendes Bild des Interrenal- Gewebemorphologie, flach-Mount-Analyse der 3β-HSD-gefärbt und deyolked Embryo wird empfohlen. Da die Interrenal- steroidogenen Gewebe direkt über dem Dottersack positioniert ist, ermöglicht eine ventrale flach-Mount - Analyse des Interrenal- Gewebe eine direkte und klare Beobachtung der Interrenal- Morphologie 17. </li></ol> 3. IHC Analyse von 3β-HSD-gefärbten Embryonen Hinweis: Für die vaskuläre Mikroumgebung des steroi Analysedogenic Gewebe, die 3β-HSD-gefärbten Embryonen mit einer transgenen Endothel-spezifischen Fluoreszenzreporter werden 12,18 Vibratom Schnitte und anschließender IHC Analyse der Quer unterworfen. <ol><li> Post-Fixierung <ol><li> Post-fix die 3β-HSD-gefärbt und gewaschen Embryonen mit 2% PFA, ergänzt mit 1% Triton X-100 für 1 Stunde bei 4 ° C und waschen 4x 10 min in PBS mit 1% Triton X-100 (PBSTx) bei RT. </li></ol></li><li> Verankerung <ol><li> Bereiten Sie 4% niedrig schmelzenden Agarose durch die Agarose in PBS Auflösung bei 95 ° C, aliquoten in Mikrozentrifugenröhrchen, und lagern bei 4-8 ° C. </li><li> Schmelzen Sie eine aliquote Menge von 4% niedrig schmelzenden Agarose bei 70 ° C für 10 min, und halten Sie dann die aliquoten bei 47 ° C. Einbetten von jedem Embryo in geschmolzenem 4% niedrigschmelzender Agarose in einem frei stehenden Kappe aus einem 1,5 ml Mikrozentrifugenröhrchen, die als Form dient, und lassen, bis sie fest zu kühlen. </li></ol></li><li> Vibratom Sectioning <ol><li> Kleben Sie ein Stück Papier, Klebeband auf die trockene platform des Vibratom. </li><li> Entfernen Sie den gehärteten Agaroseblock aus der Form durch eine Rasierklinge. Bewerben superglue auf das Papierband auf der Plattform, und befestigen Sie die Agarose-Block auf das Papierband, mit der vorderen Seite der Probe nach oben zeigt. Trimmen der Agarose-Block in eine trapezPrismenForm mit ca. 4 mm und 8 mm auf jeder Seite der oberen und unteren Flächen, respectively. </li><li> Spülen Sie die Probe enthaltenden Agarose-Block mit kaltem PBS, ergänzt mit 0,5% Triton X-100 (0,5% PBSTx) durch eine Pipette verwenden. </li><li> Schneiden Sie die Agarose-Block in 100 &amp; mgr; m Abschnitte entsprechend der Bedienungsanleitung des Vibratom. Spülen Sie ständig die Agarose-Block Austrocknen zu verhindern. Da jede Scheibe geschnitten wird, entfernen Sie sie aus dem Vibratom eine 26 G Spritzennadel verwenden und übertragen auf einer Tüpfelplatte kalt 0,5% PBSTx (500 &amp; mgr; l / Well) enthält. </li><li> Sammeln Sie die 3β-HSD-positive Gewebeschnitte von unter einem Präpariermikroskop prüft, und übertragen Sie die Abschnitte durch eine piPette Spitze mit seinem Ende cut-off (ca. 1 mm Innendurchmesser an der Öffnung) in einer 96-Well-Platte mit kaltem 0,5% PBSTx (150 &amp; mgr; l / Well). Die Gewebeproben in der Regel aus dem Agarose-Block nach diesem Schritt distanzieren. </li></ol></li><li> Permeabilisierungs und Waschen <ol><li> Waschen Sie die Scheiben für 15 min in 0,5% PBSTx bei RT, 10 min in PBS 6x, 4x und für 10 min in PBS mit 1% Rinderserumalbumin / 1% DMSO ergänzt / 0,1% Triton X-100 (PBDTx). </li></ol></li><li> Blockierung <ol><li> Inkubieren Scheiben für 1 h in 2% fötalem Kälberserum (FCS) in PBDTx bei RT. </li></ol></li><li> Primäre Antikörper-Reaktion <ol><li> Inkubieren Scheiben für 1,5 Tage in PBDTx einen primären Antikörper enthält (zB polyklonale Kaninchen - Anti-Human - Fibronektin und Maus - anti-human focal adhesion kinase) bei 4 ° C. </li></ol></li><li> Waschen <ol><li> Waschen Sie die Scheiben für 10 min in PBDTx bei RT 6x. </li></ol></li><li> Blockierung <ol><li> Inkubieren Sie die Scheiben für 1 h in 2% FCS in PBDTx bei RT. </li></ol></li><li> Sekundär-Antikörper-Reaktion <ol><li> Inkubieren Scheiben für 1,5 Tage in PBDTx einen sekundären Antikörper (Fluorophor-konjugierten Ziege-anti-Kaninchen- oder anti-Maus-IgG) bei 4 ° C enthält. </li></ol></li><li> Waschen <ol><li> Waschen Sie die Scheiben für 10 min in PBDTx bei RT und 4x für 10 min in PBST bei RT 6x. HINWEIS: Für die konfokale mikroskopische Analyse, deaktivieren Sie die Proben mit 50% Glycerin in PBS. </li></ol></li></ol> 4. Die konfokale Analyse <ol><li> Beachten Sie die ganze Berg Embryo und den Abschnitt mit einem konfokalen Mikroskop mit 10X / 0.5 und 20x / 0,75 Objektiv. </li><li> Zur gleichzeitigen Detektion der Fluoreszenz und dem 3β-Hsd Färbung, stellen Sie den mehrspurigen Modus wie folgt: 488 nm Argon-Laser und 505-530 nm-Bandpassfilter für die Detektion von GFP; 543 nm Helium-Neon-Laser und 560-615 nm-Bandpassfilter für den Nachweis von mCherry; und Lichtkanal für den Nachweis von 3β-Hsd Färbung übertragen.Die Pinholegröße 1 Airey Einheit und die Auflösung beträgt 1.024 x 1.024 Pixel. </li><li> Montieren Sie die z-Stacks (mit 6 &amp; mgr; m-Intervall) als 3D-Projektion und verschmelzen die Projektion und das Hellfeld, mit Hilfe der eingebauten in der konfokalen Software. </li></ol>

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The authors have no competing financial interests to declare.

Die Signalstärke des 3β -HSD-Aktivität erhöht sich im Laufe der Reaktion. Klare Signale der 3β-HSD wurden nach 4 Stunden Reaktions für die Stufen von 28 HPF weiter erkannt. Jedoch erfordert die Reaktionsdauer empirische Bestimmung, abhängig von dem Zweck des Assays. In Fällen, in denen die Färbung über Nacht Verarbeitung erfordert, neigt eine leicht bläuliche Hintergrund auf den Proben zu entwickeln. Dieses Problem kann durch Erhöhen der Fixationsdauer (beispielsweise 4 Stunden in 4% PFAT bei RT) vo...

Die Nebenniere, eine entscheidende Komponente des Hypothalamus-Hypophysen-Nebennieren-Achse, sondert Steroide und koordiniert Steroid-Homöostase und die körperliche Reaktion auf Stress. Die Nebenniere besteht aus der äußeren Rinde, die Steroide in einer Zone spezifisch und innere Medulla, die Katecholamine synthetisiert absondert. Die Interrenal- Drüse in teleosts ist das Gegenstück der Nebenniere bei Säugetieren und ist steroidogenen Interrenal- und chromaffinen Zellen zusammengesetzt aus, die funktionale Äquivalente der Nebennierenrinde und Medulla sind jeweils 1-3. Studien mit der Zebrabärbling - Modell durchgeführt , haben berichtet , dass beide steroidogenen und chromaffinen Zelllinien von molekularen und zellulären Mechanismen gebildet sind ähnlich denen hoch in Säugetieren 1,2. Daher ist der Zebrabärbling ein potenziell leistungsfähiges Modell für genetische Erkrankungen zu studieren, neuroendokrinen Kontrolle und Systembiologie des Hypothalamus-Hypophysen-Nebennieren (Interrenal-) Achse. <p class = &quot;jove_content&quot;&gt; in der Nebenniere, 3β-Hsd katalysiert die Umwandlung von Progesteron aus Pregnenolon, 17α-Hydroxyprogesteron aus 17α-hydroxypregnelolone und Androstendion aus Dehydroepiandrosteron 4,5. 3β-HSD wichtig, alle Klassen der hormonellen Steroide für die Biosynthese, nämlich Progesteron, Glucocorticoide, Mineralocorticoiden, Androgene und Östrogene. Die beiden menschlichen 3β HSD Isoenzyme HSD3B1 und HSD3B2 sind differentiell exprimiert 6. HSD3B1 wird in der Plazenta und peripheren Geweben exprimiert, während HSD3B2 in der Nebennierenrinde und Gonaden exprimiert wird. Menschliche HSD3B1 und HSD3B2 co-Orthologe von Zebrabärbling hsd3b1, die am Interrenal- Gewebe und adulte Gonaden exprimiert wird; Zebrabärbling hsd3b2 ist ein maternal exprimierte Gen , dessen Transkripte verschwinden vor organogenesis 7. Das Protokoll des gesamten Montage 3β-Hsd enzymatischen Aktivitätstest für Zebrabärbling durch Modifizieren Levy-Verfahren entwickelt wurde, eins von Milano et al. , Auf Gefrierschnitten von acht Teleostier Arten 8. Aufgrund der Gewebedurchlässigkeit und optische Transparenz der Entwicklungs Zebrabärbling, Vollmontage 3β-Hsd Histochemie kann erfolgreich für die feste Zebrafischembryo und Larven und speziell die differenzierten Interrenal- Gewebe abzugrenzen verwendet werden. Dieses empfindliche und schnelle Assay wurde auf verschiedenen Mutanten und morphants angewendet wurden verschiedene Arten von Interrenal- dysmorphogenesis demonstriert. Die Interrenal- 3β -HSD - Aktivität im Embryo abwesend wo Spezifikation des Interrenal- Gewebe durch eine bestimmte Zuschlags des FF1b Transkriptionsfaktor gestört ist und verringert so die Interrenal- Differenzierung durch eine Zuschlags des FF1b coregulator Prox1 9,10 betroffen ist. Bemerkenswert ist , kann der 3β-HSD in Mutanten mit schweren frühzeitig Defekte wie einäugige Stecknadelkopf und schielen, wo der 3β-H detektiert werdensd histochemistry umreißt , wie die Interrenal- Zellmigration 11 betroffen. Die Differenzierung des Interrenal- Gewebe wird nicht einmal in der vollständigen Abwesenheit von Blut und Vaskulatur kompromittiert. Deshalb, wie Endothel-abgeleiteten Signale die Entwicklung Interrenal- Organ formen kann 12,13 bestimmt werden. Insgesamt ist dieser histochemischen Assay zur Untersuchung der Spezifikation, Differenzierung und Migration von steroidogenen Zellen im Zebrafisch Modell erfolgreich verwendet wurde. Daher sollte ein effizientes und zuverlässiges Werkzeug für jede genetische oder chemische Bildschirme Targeting Nebennieren- und Interrenal- Organstörungen sein.

<table><tbody><tr><td>Confocal microscope</td><td>Carl Zeiss&amp;nbsp;</td><td>LSM510&amp;nbsp;</td><td></td></tr><tr><td>DMSO</td><td>Sigma</td><td>D8418&amp;nbsp;</td><td></td></tr><tr><td>Glycerol</td><td>USB</td><td>US16374</td><td></td></tr><tr><td>Hyclone Fetal Calf Serum&amp;nbsp;</td><td>GE Healthcare Life Sciences</td><td>SH30073</td><td></td></tr><tr><td>Nicotinamide</td><td>Sigma</td><td>N0636</td><td></td></tr><tr><td>&amp;beta;-Nicotinamide adenine dinucleotide hydrate</td><td>Sigma</td><td>N1636</td><td></td></tr><tr><td>4-Nitro blue tetrazolium</td><td>Promega</td><td>S380C</td><td></td></tr><tr><td>Nusieve GTG</td><td>Lonza</td><td>50081</td><td></td></tr><tr><td>Paraformaldehyde</td><td>Sigma</td><td>P6148</td><td></td></tr><tr><td>Phenylthiourea</td><td>Sigma&amp;nbsp;</td><td>P7629</td><td></td></tr><tr><td>Phosphate buffered saline</td><td>Sigma</td><td>P4417-100Tab</td><td></td></tr><tr><td>PYREX Spot Plate</td><td>Corning</td><td>7220-85</td><td></td></tr><tr><td>Reef Salt</td><td>AZOO</td><td>AZ28001</td><td></td></tr><tr><td>trans-Dehydroandrosterone</td><td>Sigma</td><td>D4000</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma</td><td>T8787</td><td></td></tr><tr><td>Tween 20</td><td>Sigma</td><td>P9416</td><td></td></tr><tr><td>Vibratome</td><td>Leica</td><td>VT1000M</td><td></td></tr></tbody></table>

visualizing the interrenal steroidogenic tissue and its vascular microenvironment in zebrafish

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying differentiated steroidogenic tissues through chromogenic histochemical staining of 3-&#946;-Hydroxysteroid dehydrogenase /&#916;5-4 isomerase (3&#946;-Hsd) activity-positive cells is critical for monitoring the morphology and differentiation of adrenocortical and interrenal tissues in mammals and teleosts, respectively. In the zebrafish model, the optical transparency and tissue permeability of the developing embryos and larvae allow for whole-mount staining of 3&#946;-Hsd activity. This staining protocol, as performed on transgenic fluorescent reporter lines marking the developing pronephric and endothelial cells, enables the detection of the steroidogenic interrenal tissue in addition to the kidney and neighboring vasculature. In combination with vibratome sectioning, immunohistochemistry, and confocal microscopy, we can visualize and assay the vascular microenvironment of interrenal steroidogenic tissues. The 3&#946;-Hsd activity assay is essential for studying the cell biology of the zebrafish interrenal gland because to date, no suitable antibody is available for labeling zebrafish steroidogenic cells. Furthermore, this assay is rapid and simple, thus providing a powerful tool for mutant screens targeting adrenal (interrenal) genetic disorders as well as for determining disruption effects of chemicals on steroidogenesis in pharmaceutical or toxicological studies.

Um zu bestimmen , wie die steroidogenen Interrenal- Gewebe codevelops mit der pronephric Niere Glomeruli und seiner im Entstehen begriffenen Gefäße, die 3β -HSD enzymatische Aktivität Assay auf der doppelt transgenen Tg (wt1b: GFP) durchgeführt wurde , LI1; Tg (kdrl: mCherry) CI5 Embryo bei 34 HPF (Abbildung 1). In diesem Stadium wird die 3β-HSD-positive steroidogenen Gewebe rechts der Mittellinie liegt und unmittelbar kaudal...

Watch this Scientific Journal Video about Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish at JoVE.com

Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Die Interrenal- Drüse in Zebrabärbling ist die Teleosteer Pendant der Säugetiernebenniere. Dieses Protokoll führt , wie das 3-β-Hydroxysteroid - Dehydrogenase (Δ 5-4 Isomerase; 3β-Hsd) zur Durchführung enzymatischer Aktivitätstest, der differenzierte steroidogenen Zellen in der Entwicklungs Zebrabärbling erkennt.

Visualisierung der Interrenal- Steroidogenic Gewebe und seine Vascular Mikromilieu in Zebrabärbling

This protocol introduces how to detect differentiated interrenal steroidogenic cells through a simple whole-mount enzymatic activity assay. Identifying ...

visualizing-interrenal-steroidogenic-tissue-its-vascular

Research

JoVE Journal

Developmental Biology

6.9K Aufrufe.  Tunghai University.  Die Interrenal- Drüse in Zebrabärbling ist die Teleosteer Pendant der Säugetiernebenniere. Dieses Protokoll führt , wie das 3-β-Hydroxysteroid - Dehydrogenase (Δ 5-4 Isomerase; 3β-Hsd) zur Durchführung enzymatischer Aktivitätstest, der differenzierte steroidogenen Zellen in der Entwicklungs Zebrabärbling erkennt. 

Serie: Visualizing the Interrenal Steroidogenic Tissue and Its Vascular Microenvironment in Zebrafish

Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development.
The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed.
In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning&#160;or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance.
The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

The method described here allows time-lapse analysis of organ development in zebrafish embryos by using a fluorescence dissecting microscope capable of performing optical sectioning and simple strategies of readjustment to correct focal and planar drift.

Analyse von Zebrabärbling Nierenentwicklung mit Zeitraffer-Imaging einem Binokular Mit Ausgerüstet für Optische Schnitte

In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method ...

Forschung

Entwicklungsbiologie

Immunostaining Phospho-epitopes in Ciliated Organs of Whole Mount Zebrafish Embryos

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development of all vertebrates. The kinetics and location of signals - even within single cells - in the developing embryo complements the identification of important developmental genes. Immunostaining techniques are described that have been shown to define the kinetics of intracellular and whole animal signals in structures as small as primary cilia. The techniques for fixing, imaging and processing images using a laser-scanning confocal compound microscope can be completed in as few as 36 hr.
Zebrafish (Danio rerio) is a desirable organism for investigators who seek to conduct studies in a vertebrate species that is affordable and relevant to human disease. Genetic knockouts or knockdowns must be confirmed by the loss of the actual protein product. Such confirmation of protein loss can be achieved using the techniques described here. Clues into signaling pathways can also be deciphered by using antibodies that are reactive with proteins that have been post-translationally modified by phosphorylation. Preserving and optimizing the phosphorylated state of an epitope is therefore critical to this determination and is accomplished by this protocol.
This study describes techniques to fix embryos during the first 72 hr of development and co-localize a variety of relevant epitopes with cilia in the Kupffer&#39;s Vesicle (KV), the kidney and the inner ear. These techniques are straightforward, do not require dissection and can be completed in a relatively short period of time. Projecting confocal image stacks into a single image is a useful means of presenting these data.

Techniques are described to immunostain phospho-epitopes in whole zebrafish embryos and then conduct two-color fluorescent confocal localization in cellular structures as small as primary cilia. The techniques for fixing and imaging can define the location and kinetics of the appearance or activation of specific proteins.

Immunfärbung Phospho-Epitope in Ciliated Organe Ganze Berge Zebrafischembryonen

The rapid proliferation of cells, the tissue-specific expression of genes and the emergence of signaling networks characterize early embryonic development ...

Chemie

Dissection of Larval Zebrafish Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex determination system, where several genes distributed throughout the genome collectively determine the sex identities of individual fish. Currently, the genes involved in regulating gonad development and how they work remain elusive. Normally, isolating gonadal tissue is the first step to examine the sex developmental processes. Here, we present a procedure to isolate gonadal tissue from 17 dpf (days post fertilization) and 25 dpf zebrafish larvae. The isolated gonadal tissue may be subsequently examined by morphology and gene expression profiling.

Here, we present a protocol for isolating gonadal tissue of larval zebrafish, which will facilitate investigations of zebrafish sex differentiation and maintenance.

Dissection von Larval Zebrabärbling Gonadal Tissue

Although wild zebrafish possess a ZZ/ZW sex-determination system, domesticated zebrafish have lost the sex chromosome. They utilize a polygenic sex ...

In Vivo Imaging and Quantitation of the Host Angiogenic Response in Zebrafish Tumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A zebrafish xenograft is created by implanting mammalian tumor cells into the perivitelline space of two days-post-fertilization zebrafish embryos, followed by measuring the extent of the angiogenic response observed at an experimental endpoint up to two days post-implantation. The key advantage to this method is the ability to accurately quantitate the zebrafish host angiogenic response to the graft. This enables detailed examination of the molecular mechanisms as well as the host vs tumor contribution to the angiogenic response. The xenografted embryos can be subjected to a variety of treatments, such as incubation with potential anti-angiogenesis drugs, in order to investigate strategies to inhibit tumor angiogenesis. The angiogenic response can also be live-imaged in order to examine more dynamic cellular processes. The relatively undemanding experimental technique, cheap maintenance costs of zebrafish and short experimental timeline make this model especially useful for the development of strategies to manipulate tumor angiogenesis.

The aim of this method is to generate an in vivo model of tumor angiogenesis by xenografting mammalian tumor cells into a zebrafish embryo that has fluorescently-labelled blood vessels. By imaging the xenograft and associated vessels, a quantitative measurement of the angiogenic response can be obtained.

In Vivo Bildgebung und Quantifizierung der Angiogenreaktion des Wirts bei Zebrafischtumor Xenografts

Tumor angiogenesis is a key target of anti-cancer therapy and this method has been developed to provide a new model to study this process in vivo. A ...

Krebsforschung

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and optical transparency. In particular, the zebrafish embryo has become an important organism to study vertebrate kidney organogenesis as well as multiciliated cell (MCC) development. To visualize MCCs in the embryonic zebrafish kidney, we have developed a combined protocol of whole-mount fluorescent in situ hybridization (FISH) and whole mount immunofluorescence (IF) that enables high resolution imaging. This manuscript describes our technique for co-localizing RNA transcripts and protein as a tool to better understand the regulation of developmental programs through the expression of various lineage factors.

Visualizing Multiciliated Cells in the Zebrafish Through a Combined Protocol of Whole Mount Fluorescent In Situ Hybridization and Immunofluorescence

Cilia development is vital to proper organogenesis. This protocol describes an optimized method to label and visualize ciliated cells of the zebrafish.

Visualisierung von Multiciliated Zellen in der Zebrabärbling durch ein kombiniertes Protokoll der ganze Berg Leuchtstofflampen In Situ Hybridisierung und Immunfluoreszenz

In recent years, the zebrafish embryo has emerged as a popular model to study developmental biology due to traits such as ex utero embryo development and ...

Adult Zebrafish Injury Models to Study the Effects of Prednisolone in Regenerating Bone Tissue

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows within roughly two weeks after injury. Furthermore, zebrafish are able to heal bone rapidly after trepanation of the skull, and repair fractures that can be easily introduced into zebrafish bony fin rays. These injury assays represent feasible experimental paradigms to test the effect of administered drugs on rapidly forming bone. Here, we describe the use of these 3 injury models and their combined use with systemic glucocorticoid treatment, which exerts bone inhibitory and immunosuppressive effects. We provide a workflow on how to prepare for immunosuppressive treatment in adult zebrafish, illustrate how to perform fin amputation, trepanation of calvarial bones, and fin fractures, and describe how the use of glucocorticoids affects both bone forming osteoblasts and cells of the monocyte/macrophage lineage as part of innate immunity in bone tissue.

Here, we describe 3 adult zebrafish injury models and their combined use with immunosuppressive drug treatment. We provide guidance on imaging of regenerating tissues and on detecting bone mineralization therein.

Erwachsenen Zebrafisch Verletzungen Modelle zur Untersuchung der Auswirkungen von Prednisolon bei der Regeneration von Knochengewebe

Zebrafish are able to regenerate various organs, including appendages (fins) after amputation. This involves the regeneration of bone, which regrows ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

Quantifizierung der Lebergröße in Larval Zebrafischen mit Brightfield-Mikroskopie

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

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 ...

Biologie

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetes durch die Augen eines Fisches zu studieren: Mikrodissektion, Visualisierung und Analyse von den Erwachsenen tg(fli:EGFP) Zebrafisch Retinal Gefäßsystem

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Medizin

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

The zebrafish embryo is an excellent model for developmental biology research. During embryogenesis, zebrafish develop with a yolk mass, which presents three-dimensional challenges for sample observation and analysis. This protocol describes how to create two-dimensional flat mount preparations of whole mount in situ (WISH) stained zebrafish embryo specimens.

Wohnung Berge Vorbereitung für Beobachtung und Analyse der Zebrafisch-Embryo Proben von Bunt Ganze Berge<em&gt; In situ</em&gt; Hybridisierung

The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model ...