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 border="0" cellpadding="0" cellspacing="0" width="565">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:307px;">Confocal microscope</td>
			<td style="width:187px;">Carl Zeiss&#160;</td>
			<td style="width:72px;">LSM510&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DMSO</td>
			<td>Sigma</td>
			<td>D8418&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glycerol</td>
			<td>USB</td>
			<td>US16374</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Hyclone Fetal Calf Serum&#160;</td>
			<td>GE Healthcare Life Sciences</td>
			<td>SH30073</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nicotinamide</td>
			<td>Sigma</td>
			<td>N0636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#946;-Nicotinamide adenine dinucleotide hydrate</td>
			<td>Sigma</td>
			<td>N1636</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Nitro blue tetrazolium</td>
			<td>Promega</td>
			<td>S380C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Nusieve GTG</td>
			<td>Lonza</td>
			<td>50081</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td>Sigma</td>
			<td>P6148</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phenylthiourea</td>
			<td>Sigma&#160;</td>
			<td>P7629</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Phosphate buffered saline</td>
			<td>Sigma</td>
			<td>P4417-100Tab</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PYREX Spot Plate</td>
			<td>Corning</td>
			<td>7220-85</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Reef Salt</td>
			<td>AZOO</td>
			<td>AZ28001</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">trans-Dehydroandrosterone</td>
			<td>Sigma</td>
			<td>D4000</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Triton X-100</td>
			<td>Sigma</td>
			<td>T8787</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tween 20</td>
			<td>Sigma</td>
			<td>P9416</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vibratome</td>
			<td>Leica</td>
			<td>VT1000M</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

Universidad de Cartagena UDEC

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

Capturing Tissue Repair in Zebrafish Larvae with Time-lapse Brightfield Stereomicroscopy

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two layers of skin that enclose undifferentiated mesenchyme, and because the larval tail fin regenerates rapidly within 2-3 days. Using this system, we demonstrate a method for capturing the repair dynamics of the amputated tail fin with time-lapse video brightfield stereomicroscopy. We demonstrate that fin amputation triggers a contraction of the amputation wound and extrusion of cells around the wound margin, leading to their subsequent clearance. Fin regeneration proceeds from proximal to distal direction after a short delay. In addition, developmental growth of the larva can be observed during all stages. The presented method provides an opportunity for observing and analyzing whole tissue-scale behaviors such as fin development and growth in a simple microscope setting, which is easily adaptable to any stereomicroscope with time-lapse capabilities.

We present a protocol for capturing the dynamics of zebrafish larval tail fin regeneration on a whole-tissue scale using brightfield-based stereomicroscopy. This technique enables capturing the regeneration dynamics with single cell resolution. This methodology can be adapted to any stereomicroscope equipped with a CCD camera and time-lapse software.

Erfassen von Gewebereparatur in Zebrafisch-Larven mit Zeitrafferhellstereomikroskopie

The zebrafish larval tail fin is ideal for studying tissue regeneration due to the simple architecture of the larval fin-fold, which comprises of two ...

Forschung

Entwicklungsbiologie

Analysis of Zebrafish Larvae Skeletal Muscle Integrity with Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders. In the study of neuromuscular diseases, the integrity of the muscle membrane is a critical disease determinant. To date, numerous neuromuscular conditions display degenerating muscle fibers with abnormal membrane integrity; this is most commonly observed in muscular dystrophies. Evans Blue Dye (EBD) is a vital, cell permeable dye that is rapidly taken into degenerating, damaged, or apoptotic cells; in contrast, it is not taken up by cells with an intact membrane. EBD injection is commonly employed to ascertain muscle integrity in mouse models of neuromuscular diseases. However, such EBD experiments require muscle dissection and/or sectioning prior to analysis. In contrast, EBD uptake in zebrafish is visualized in live, intact preparations. Here, we demonstrate a simple and straightforward methodology for performing EBD injections and analysis in live zebrafish. In addition, we demonstrate a co-injection strategy to increase efficacy of EBD analysis. Overall, this video article provides an outline to perform EBD injection and characterization in zebrafish models of neuromuscular disease.

In this study, we describe a straightforward method to perform Evans Blue Dye (EBD) analysis on zebrafish larvae. This technique is a powerful tool for the characterization of skeletal muscle integrity and delineation of zebrafish models of muscular dystrophy, and is a valuable method for the development of novel therapeutics.

Analyse von Zebrafisch-Larven Skelettmuskel Integrität mit Evans Blue Dye

The zebrafish model is an emerging system for the study of neuromuscular disorders.  In the study of neuromuscular diseases, the integrity of the muscle ...

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 ...

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response to&#160;growth and reproduction. In insects, the larval prothoracic gland (PG) and the adult female ovary play essential roles in biosynthesizing the principal steroid hormones called ecdysteroids. These ecdysteroidogenic organs are innervated from the nervous system, through&#160;which the timing of biosynthesis is affected by environmental cues. Here we describe a protocol for visualizing ecdysteroidogenic organs and their interactive organs in larvae and adults of the fruit fly Drosophila melanogaster, which provides a suitable model system for studying steroid hormone biosynthesis and its regulatory mechanism. Skillful dissection allows us to maintain the positions of ecdysteroidogenic organs and their interactive organs including the brain, the ventral nerve cord, and other tissues. Immunostaining with antibodies against ecdysteroidogenic enzymes, along with transgenic fluorescence proteins driven by tissue-specific promoters, are available to label ecdysteroidogenic cells. Moreover, the innervations of the ecdysteroidogenic organs can also be labeled by specific antibodies or a collection of GAL4 drivers in various types of neurons. Therefore, the ecdysteroidogenic organs and their neuronal connections can be&#160;visualized simultaneously&#160;by immunostaining and transgenic techniques. Finally, we describe how to visualize germline stem cells, whose proliferation and maintenance are controlled by ecdysteroids. This method contributes to comprehensive understanding of steroid hormone biosynthesis and its neuronal regulatory mechanism.

Protocols for Visualizing Steroidogenic Organs and Their Interactive Organs with Immunostaining in the Fruit Fly Drosophila melanogaster

We describe a protocol for dissection, fixation, and immunostaining of steroidogenic organs in Drosophila larvae and adult females to study steroid hormone biosynthesis and its regulatory mechanism. In addition to steroidogenic organs, we visualize the innervation of steroidogenic organs as well as steroidogenic target cells such as germline stem cells.

Protokolle für die Visualisierung Steroidogenic Organe und deren Interactive Organe mit Immunostaining in der Fruchtfliege<em&gt; Drosophila melanogaster</em

In multicellular organisms, a small group of cells is endowed with a specialized function in their biogenic activity, inducing a systemic response ...

A Novel Mammary Fat Pad Transplantation Technique to Visualize the Vessel Generation of Vascular Endothelial Stem Cells

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper vessel development and homeostasis. However, studies on endothelial lineage hierarchy remain elusive due to the lack of tools to gain access as well as to directly evaluate their behavior in vivo. To address this shortcoming, a new tissue model to study angiogenesis using the mammary fat pad has been developed. The mammary gland develops mostly in the postnatal stages, including puberty and pregnancy, during which robust epithelium proliferation is accompanied by extensive vascular remodeling. Mammary fat pads provide space, matrix, and rich angiogenic stimuli from the growing mammary epithelium. Furthermore, mammary fat pads are located outside the peritoneal cavity, making them an easily accessible grafting site for assessing the angiogenic potential of exogenous cells. This work also describes an efficient tracing approach using fluorescent reporter mice to specifically label the targeted population of vascular endothelial stem cells (VESCs) in vivo. This lineage tracing method, coupled with subsequent tissue whole-mount microscopy, enable the direct visualization of targeted cells and their descendants, through which the proliferation capability can be quantified and the differentiation commitment can be fate-mapped. Using these methods, a population of bipotent protein C receptor (Procr) expressing VESCs has recently been identified in multiple vascular systems. Procr+ VESCs, giving rise to both new ECs and pericytes, actively contribute to angiogenesis during development, homeostasis, and injury repair. Overall, this manuscript describes a new mammary fat pad transplantation and in vivo lineage tracing techniques that can be used to evaluate the stem cell properties of VESCs.

This work demonstrates a novel approach to assess the proliferation, differentiation, and vessel-forming potential of vascular endothelial stem cells (VESCs) through mammary fat pad transplantation followed by whole-mount tissue preparation for microscopic observation. A lineage tracing strategy to investigate the behavior of VESCs in vivo is also presented.

Eine neuartige Mamma-Fat-Pad-Transplantationstechnik zur Visualisierung der Gefäßerzeugung von vaskulären endothelialen Stammzellen

Endothelial cells (ECs) are the fundamental building blocks of the vascular architecture and mediate vascular growth and remodeling to ensure proper ...

Live Imaging of Mouse Secondary Palate Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to cleft secondary palate, a common congenital anomaly in humans. Secondary palate fusion has been extensively studied leading to several proposed cellular mechanisms that may mediate this process. However, these studies have been mostly performed on fixed embryonic tissues at progressive timepoints during development or in fixed explant cultures analyzed at static timepoints. Static analysis is limited for the analysis of dynamic morphogenetic processes such a palate fusion and what types of dynamic cellular behaviors mediate palatal fusion is incompletely understood. Here we describe a protocol for live imaging of ex vivo secondary palate fusion in mouse embryos. To examine cellular behaviors of palate fusion, epithelial-specific Keratin14-cre was used to label palate epithelial cells in ROSA26-mTmGflox reporter embryos. To visualize filamentous actin, Lifeact-mRFPruby reporter mice were used. Live imaging of secondary palate fusion was performed by dissecting recently-adhered secondary palatal shelves of embryonic day (E) 14.5 stage embryos and culturing in agarose-containing media on a glass bottom dish to enable imaging with an inverted confocal microscope. Using this method, we have detected a variety of novel cellular behaviors during secondary palate fusion. An appreciation of how distinct cell behaviors are coordinated in space and time greatly contributes to our understanding of this dynamic morphogenetic process. This protocol can be applied to mutant mouse lines, or cultures treated with pharmacological inhibitors to further advance understanding of how secondary palate fusion is controlled.

Here, we present a protocol for live imaging of mouse secondary palate fusion using confocal microscopy. This protocol can be used in combination with a variety of fluorescent reporter mouse lines, and with pathway inhibitors for mechanistic insight. This protocol can be adapted for live imaging in other developmental systems.

Live Imaging der Maus Sekundärer Gaumen Fusion

The fusion of the secondary palatal shelves to form the intact secondary palate is a key process in mammalian development and its disruption can lead to ...

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 ...

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the Notch receptor with ligands from neighboring cells induces activation of the signaling pathway (trans-activation), while interaction with ligands from the same cell inhibits signaling (cis-inhibition). Proper balance between trans-activation and cis-inhibition helps establish optimal levels of Notch signaling in some contexts during animal development. Because of the overlapping expression domains of Notch and its ligands in many cell types and the existence of feedback mechanisms, studying the effects of a given post-translational modification on trans- versus cis-interactions of Notch and its ligands in vivo is difficult. Here, we describe a protocol for using Drosophila S2 cells in cell-aggregation assays to assess the effects of knocking down a Notch pathway modifier on the binding of Notch to each ligand in trans and in cis. S2 cells stably or transiently transfected with a Notch-expressing vector are mixed with cells expressing each Notch ligand (S2-Delta or S2-Serrate). Trans-binding between the receptor and ligands results in the formation of heterotypic cell aggregates and is measured in terms of the number of aggregates per mL composed of &#62;6 cells. To examine the inhibitory effect of cis-ligands, S2 cells co-expressing Notch and each ligand are mixed with S2-Delta or S2-Serrate cells and the number of aggregates is quantified as described above. The relative decrease in the number of aggregates due to the presence of cis-ligands provides a measure of cis-ligand-mediated inhibition of trans-binding. These straightforward assays can provide semi-quantitative data on the effects of genetic or pharmacological manipulations on the binding of Notch to its ligands, and can help deciphering the molecular mechanisms underlying the in vivo effects of such manipulations on Notch signaling.

Cell Aggregation Assays to Evaluate the Binding of the Drosophila Notch with Trans-Ligands and its Inhibition by Cis-Ligands

Complexity of in vivo systems makes it difficult to distinguish between the activation and inhibition of Notch receptor by trans- and cis-ligands, respectively. Here, we present a protocol based on in vitro cell-aggregation assays for qualitative and semi-quantitative evaluation of the binding of Drosophila Notch to trans-ligands vs cis-ligands.

Handy-Aggregation-Assays, die Bindung der Drosophila Kerbe mit Transzu bewerten-Liganden und ihre Hemmung von Cis-Liganden

Notch signaling is an evolutionarily conserved cell-cell communication system used broadly in animal development and adult maintenance. Interaction of the ...

Following Endocardial Tissue Movements via Cell Photoconversion in the Zebrafish Embryo

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the field of developmental biology. The discovery and development of photoconvertible proteins have greatly aided our understanding of these dynamic changes by providing a method to optically highlight cells and tissues. However, while photoconversion, time-lapse microscopy, and subsequent image analysis have proven to be very successful in uncovering cellular dynamics in organs such as the brain or the eye, this approach is generally not used in the developing heart due to challenges posed by the rapid movement of the heart during the cardiac cycle. This protocol consists of two parts. The first part describes a method for photoconverting and subsequently tracking endocardial cells (EdCs) during zebrafish atrioventricular canal (AVC) and atrioventricular heart valve development. The method involves temporally stopping the heart with a drug in order for accurate photoconversion to take place. Hearts are allowed to resume beating upon removal of the drug and embryonic development continues normally until the heart is stopped again for high-resolution imaging of photoconverted EdCs at a later developmental time point. The second part of the protocol describes an image analysis method to quantify the length of a photoconverted or non-photoconverted region in the AVC in young embryos by mapping the fluorescent signal from the three-dimensional structure onto a two-dimensional map. Together, the two parts of the protocol allows one to examine the origin and behavior of cells that make up the zebrafish AVC and atrioventricular heart valve, and can potentially be applied for studying mutants, morphants, or embryos that have been treated with reagents that disrupt AVC and/or valve development.

This protocol describes a method for the photoconversion of Kaede fluorescent protein in endocardial cells of the living zebrafish embryo that enables the tracking of endocardial cells during atrioventricular canal and atrioventricular heart valve development.

Folgenden endokardialen Gewebe Bewegungen über Zelle Ladungszustand des Zebrafish Embryos

During embryogenesis, cells undergo dynamic changes in cell behavior, and deciphering the cellular logic behind these changes is a fundamental goal in the ...

Visualizing the Node and Notochordal Plate In Gastrulating Mouse Embryos Using Scanning Electron Microscopy and Whole Mount Immunofluorescence

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is the formation of the transient organizers, the node and notochordal plate, from cells that have passed through the primitive streak. The proper formation of these signaling centers is essential for the development of the body plan and techniques to visualize them are of high interest to mouse developmental biologists. The node and notochordal plate lie on the ventral surface of gastrulating mouse embryos around embryonic day (E) 7.5 of development. The node is a cup-shaped structure whose cells possess a single slender cilium each. The proper subcellular localization and rotation of the cilia in the node pit determines left-right asymmetry. The notochordal plate cells also possess single cilia albeit shorter than those of the node cells. The notochordal plate forms the notochord which acts as an important signaling organizer for somitogenesis and neural patterning. Because the cells of the node and notochordal plate are transiently present on the surface and possess cilia, they can be visualized using scanning electron microscopy (SEM). Among other techniques used to visualize these structures at the cellular level is whole mount immunofluorescence (WMIF) using the antibodies against the proteins that are highly expressed in the node and notochordal plate. In this report, we describe our optimized protocols to perform SEM and WMIF of the node and notochordal plate in developing mouse embryos to help in the assessment of tissue shape and cellular organization in wild-type and gastrulation mutant embryos.

The node and notochordal plate are transient signaling organizers in developing mouse embryos that can be visualized using several techniques. Here, we describe in detail how to perform two of the techniques to study their structure and morphogenesis: 1) scanning electron microscopy (SEM); and 2) whole mount immunofluorescence (WMIF).

Visualisierung der Knoten und Notochordal Platte In Gastrulating Maus-Embryonen mittels Rasterelektronenmikroskopie und ganze Mount Immunfluoreszenz

The post-implantation mouse embryo undergoes major shape changes after the initiation of gastrulation and morphogenesis. A hallmark of morphogenesis is ...