Name: a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys
Uploaded: 2014-12-02T11:00:00.000+00:00
Duration: 10 min 51 s
Description: Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

Diese Arbeit wurde durch die NIH (DK093625 an LH und DK069909 und DK047757 zu JHL) und die VA (Merit Award I01BX000820 zu JHL). Wir möchten Dr. Michael Pack und Dr. Jie er für die Bereitstellung wesentlicher Unterstützung danken an der Universität von Pennsylvania Zebrafisch-Core.

HINWEIS: Ethik Hinweise: Alle Zebrafisch-Experimente wurden von der Institutional Animal Care und Verwenden Committee an der Eastern Virginia Medical School zugelassen. 1. Morpholino Vorbereitung <ol><li> Design und Synthese Übersetzung blockier (Aug-) und Spleiß-Hemmung (Spleiß-) Antisense-Morpholino (MO) Oligonukleotide für das Gen von Interesse nach Herstellerangaben (1A). Bitte lesen Sie Informationen des Herstellers, die in Tabelle 1. HINWEIS: MOs werden als lyophilisierte Aktien in Glasflaschen geliefert. </li><li> Hinzufügen hochwertigen sterilem Wasser auf die Glasflasche zu resuspendieren MOs zu einer Endkonzentration von 25 ug / ul. Stellen Sie sicher, dass das Oligonukleotid vollständig gelöst ist. Wenn einige solide bleibt, heizen das Fläschchen mit dem Lager Oligonukleotid-Lösung bei 65 ° C für 5 bis 10 Minuten und kurz vortexen. </li><li> Bewahren Sie die MO-Stammlösung bei RT. Lagern Sie diese nicht bei 4 ° C oder -20 °; C, weil geringere Temperaturen können dazu führen, die MO-Oligonukleotids an der Behälterwand zu binden. Messen Sie die Konzentration der Stammlösung mit einem Spektralphotometer jedes Mal eine neue MO-Stammlösung hergestellt wird (siehe Tabelle 1 beziehen). </li><li> Bereiten Sie die MO-Arbeitslösung am Tag der Injektion durch Verdünnung der Stammlösung mit hochgradigen sterilem Wasser auf die gewünschte Dosis. Mit 0,5% Phenol-Rot zu einer Konzentration von 0,05% zu erreichen. Wenn Sie beispielsweise 5 ul Arbeitslösung mit einer Konzentration von 15 ng / nL, fügen 3 ul MO Stammlösung und 0,5 ul 0,5% Phenol-rot bis 1,5 ul Wasser. HINWEIS: Mit dieser Arbeitskonzentration, jeder Tropfen (500 pl) der Injektion enthält 7,5 ng MO und zwei Tropfen enthalten 15 ng von MO. </li></ol> 2. Herstellung der Einspritzvorrichtung <ol><li> Kauf Glas vorgezogen Nadeln (siehe Tabelle 1 für weitere Informationen). Alternativ ziehen Sie das Glas Injektionsnadel with eine Nadel Magnet. </li><li> Schalten Sie den Kompressor und stellen Sie die Druckeinstellung auf 50 psi. Schalten Sie den Binokular-Lichtquelle und Pico Mikroeinspritzpumpe und passen Sie die Einstellungen wie folgt: Halten Druck 20 psi, werfen Druck 10 psi, Zeit-Wert von 2,5 und 100 &amp; mgr; s Palette von Gating. </li><li> Legen Sie das Glas vor, zog Nadel mit 5 ul der Injektionslösung und setzen Sie die Nadel in vertikaler Position mit der Spitze nach unten. Warten Sie, bis die gesamte Lösung der Nadelspitze ohne sichtbare Luftblasen erreicht. Setzen Sie den Nadelhalter in einer passenden Position neben dem Mikroskop und legen Sie die Glasnadel in die Halterung. Ist der Einspritzwinkel um 45 Grad. </li><li> Bringen Sie die Nadelspitze in Sicht unter dem Mikroskop, hoch über der Bühne, und konzentrieren sich auf die dünnsten Bereich der Spitze. Brechen Sie die Nadelspitze mit feinen Spitze Pinzette. </li><li> Legen Sie ein Lineal und ein Rohr oder Schlauch mit Innendurchmesser von 0,15 mm nebeneinander unter dem Mikroskop;Injizieren der Lösung in ein Ende des Kapillarröhrchens einen Flüssigkeitssäule bilden. Stellen Sie die Auswurfzeit alle 17 Tropfen je 1 mm Länge Flüssigkeitssäule zu erreichen, wird das Volumen jeder Tropfen ist 1 nl (Tropfenvolumen = π x Radius 2 x Länge (der Flüssigkeitssäule) / count (Tropfen). HINWEIS: Ein Tropfen mit einem Durchmesser von 0,1 mm ergibt einen Injektionsvolumen von etwa 500 pl (Tropfenvolumen = 4/3 x π x Radius 3). </li></ol> 3. Herstellung der befruchteten Zebrafischembryonen und Injektion mit Morpholinos <ol><li> Transgenen Zebrafisch Zuchtpaare nach den veröffentlichten Protokollen für Standard Zebrafischhaltung und Wartung: bis Tg (GFP wt1b) ein. Sammeln Embryonen nach Naturbrut, und halten sie in einer 10 cm Petrischale mit E3 Wasser gefüllt (5 mM NaCl, 0,17 mm KCl, 0,33 mM CaCl 2, 0,33 mM MgSO 4). Starten MO Injektion sofort 13. Überwachen Embryo Morphologie unter dem Mikroskop und stellen Sie sicher, MO Injektion ist im Ein-Zell-Stadium, oder spätestens am Vierzellstadium. <ol><li> Transfer der Embryonen in einen 10-cm-Petrischale mit Agar Tröge keilförmig. Saugen Sie das zusätzliche E3 Wasser und drücken Sie vorsichtig die Embryonen in die Tröge. </li><li> Bearbeiten Sie die Embryonen mit der Mikropipette 1-Zell-Stadium Embryos unter dem Binokular visualisieren. Durchdringen die Chorion und dann das Eigelb, ein oder zwei Tropfen MO in den Dotter (1 Tropfen = 500 pl) zu injizieren. Übertragen Sie die injizierten Embryonen zu einer 10 cm Petrischale mit E3 Wasser und Inkubation bei 28,5 ° C. </li></ol></li><li> Nach den Injektionen, entfernen Sie die toten Embryonen und notieren Sie die Anzahl der injizierten Embryonen. Ersetzen Sie die E3 Wasser in der Schale alle 24 bis 48 Stunden. </li></ol> 4. Phänotyp Rettungsversuche <ol><li> Auszuwählen, die ein Ortholog von einer anderen Spezies, die eine andere primäre Basispaarstruktur (üblicherweise 3-7 Basenpaar Mismatches) aufweist und daher beständig gegen das MO, zur Rettung. Beispielsweise wird in diesem Experiment haben wir unsMaus weil die Maus Wnt5a mRNA-Sequenz unterscheidet sich von der Zebrafisch-Sequenz. </li><li> Entwerfen eines Primers mit der Vorwärtsprimer beginnend beim Translationsstelle und der Reverse-Primer in der Nähe des Endes der mRNA codierende Region eingestellt. Hinzufügen eines T7-Promotorsequenz am 5&#39;-Ende des Vorwärtsprimer-Sequenz. In diesem Experiment verwendeten wir die folgenden Primersequenzen; vorwärts: 5&#39;-taa tac gac tca cta Tag GGA cta tga tgc tgc tga agc tga a- 3 &#39;und umgekehrt: 5&#39;-TCA ctt gca gac gta ctg gtc- 3&#39;. </li><li> Durchführen einer 50 ul Polymerase-Kettenreaktion (PCR) mit den Primersatz (0,5 &amp; mgr; M von jedem Primer) vorstehend ausgebildet und 0,1 &amp; mgr; g Matrizen-DNA, die das Maus Wnt5a cDNA-Sequenz mit der folgenden PCR-Programm: 1 Zyklus bei 95 ° C für 5 min; 35 Zyklen von 95 ° C (30 sec), 58,5 ° C (30 sec), 72 ° C (1 min); und abschließende Extension bei 72 ° C für 7 min. </li><li> Nach Abschluss ter Zyklus ausführen 2 ul des PCR-Produkts auf 1% Agarosegel, um sicherzustellen, dass die Band die richtige Größe. Man reinige das PCR-Produkt mit einem PCR-Purification-Kit nach den Anweisungen des Herstellers. Überprüfen DNA-Konzentration mit einem Spektralphotometer. Speicherung des gereinigten PCR-Produkts bei -20 ° C oder direkt in verkappter RNA Transkription Verwendung im nächsten Schritt. </li><li> Synthetisieren in vitro mRNA mit Cap mit einem verkappten RNA-Synthese-Kits nach den Anweisungen des Herstellers. Verdünnen Sie die mRNA in 20 ul RNase freies Wasser und Bestimmung der Konzentration. Aliquot der RNA und bei -80 ° C. </li><li> Am Tag der Injektion, bereiten eine Arbeitslösung der mRNA mit RNase frei sterilem Wasser. Man beachte, daß 40 pg ist im allgemeinen die höchste RNA Dosis, bei der unspezifische oder toxische Wirkung des RNA treten nicht auf. Halten Sie die Arbeitslösung auf Eis. Spritzen Sie die Embryos mit MO und dann die Rettung mRNA, oder Co-spritzen beide zusammen. Wenn beispielsweise die Konzentration der mRNA prepared in Schritt 4.5 ist 0,6 ug / ul, fügen 0,67 ul mRNA (0,4 ug) bis 4,33 &amp; mgr; l RNase-freies Wasser bis zu einer Endkonzentration von 0,08 ug / ul zu ergeben, und ein Tropfen (500 pl) jeder Injektion enthält 40 pg mRNA. Bereiten MO, wie in Schritt 3 beschrieben. </li></ol> 5. Vorbereitung der injizierten Embryonen für Fluorescent Imaging <ol><li> Nach der Inkubation bei 28,5 ° CO / N, fügen Sie 0,003% N-Phenylthioharnstoff (PTU) mit dem E3 Wasser Melanisierung, die Beobachtung der Vorniere Struktur mittels Fluoreszenzmikroskopie wirkt verhindern. Allerdings PTU nicht vor Gastrulation abgeben, weil es die frühe embryonale Entwicklung betrifft. </li><li> Bei 48 HPF, manuell dechorionate die Embryonen mit feinen Pinzette. Nehmen Sie Bilder von 48 und 72 HPF Embryonen unter einem Licht Dissektionsmikroskop. Hinweis: Diese Bilder sind ohne Narkose entnommen werden. </li><li> Etwa 10 Minuten vor der unter dem Fluoreszenzmikroskop Bildgebung, betäuben die Embryonen, indem Sie sie in10 cm Petrischale mit 160 ug / ml gepufferte Tricaine. Warten Sie 5-10 Minuten, und dann locker am Zebrafisch zu bestätigen, dass sie sich nicht bewegen und damit die Narkose wirkt. </li><li> Nachdem die Embryos betäubt werden, montieren sie in 3% Methylcellulose und orientieren sie in Bauchlage unter einem Binokular (siehe Tabelle 1). </li><li> Beachten Sie die Vorniere Struktur unter einem Fluoreszenzmikroskop (siehe auch Tabelle 1) und nehmen Sie Bilder mit unterschiedlicher Vergrößerung (10X und 20X). </li></ol>

<ol>
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The authors have nothing to disclose.

Polyzystische Nierenerkrankung (PKD) ist eine der führenden Ursachen für end-stage renal Krankheit beim Menschen und wird durch fortschreitende Zysten, Nieren Erweiterung und abnorme Entwicklung Röhrchen 14 aus. Autosomal dominante PKD (ADPKD) ist eine Erbkrankheit, bei der die Mutation von entweder PKD1 kodiert Polycystin-1 (PC1) oder PKD2, Polycystin-2 (PC2) kodiert, führt zu Zystennieren. Viele andere Gene, vor allem Proteine, die in der primären Zilien gefunden codiert, wird angenommen, dass bei der ...

Zebrafisch (Danio rerio) Embryonen wurden allgemein als Modell für die Untersuchung der Nierenentwicklung und polyzystische Nierenerkrankung verwendet. Es gibt viele Vorteile der Verwendung von Zebrafisch als Tiermodell: Die Durchführbarkeit Studium genetischen Wechselwirkungen, die Fähigkeit, Antisense-Morpholino (MO) für die Proteinzuschlags verwenden, die Möglichkeit, schnell zu testen große Anzahl von Embryos und die Betrachtung Organ Phänotypen lebenden Larven 1. Die Vorniere ist die erste Niere bei Wirbeltieren entwickeln und ist funktional in Zebrafisch-Larven 2. Die Struktur der Zebrafisch pronephros ist relativ einfach im Vergleich zu den Säuger Metanephros, um die dritte und letzte Niere in Säugetieren zu entwickeln. Nephronanalog ist die Arbeitseinheit der Niere, wobei jede humane Nieren enthaltend zwischen 500.000-1.000.000 Nephronen 3,4 und jeder Maus Niere mit ungefähr 13.000 Nephronen 5, was es schwierig macht, einzelne Nephron Struktur beobachten in Mensch oder Maus Nieren. Der Zebrafisch besitzt nur zwei Nephronen und jedes Zebrabärbling Nephron enthält alle Hauptkomponenten im Glomerulus und Tubuli von Mäusen und Menschen und 6 ähnlich speziellen Nierenzelltypen gefunden. Im Vergleich zu anderen Wirbeltiermodellen wie Xenopus, Zebrafisch Nephron mehr ähnelt die Säuger Nephron weil es ein geschlossenes System 7 hat. In den letzten Jahren hat sich die Zebrafisch wurde sequenziert, was die breite Einführung von genetischen Werkzeugen umfangreichen Mutanten Ressourcen und Sammlungen von transgenen Reporterlinien in Zebrafisch-Modelle. Die Zebrafisch Vorniere bildet sich zwischen 12 bis 72 Stunden nach der Befruchtung (HPF) und kann leicht in den durchsichtigen Embryos visualisiert werden. Die Wilms-Tumor Protein WT1 ist ein wesentlicher Faktor für die Nierenentwicklung. Transgenic Zebrafischlinien grün fluoreszierende Protein (GFP) unter der Kontrolle des Promotors wt1b Tg Ausdruck (wt1b: GFP) zeigen GFP expression speziell in pronephric Regionen in Zebrafischembryonen, ab 17 HPF 8. Nephronophthisis (NPHP), eine autosomal-rezessive zystischen Nierenerkrankungen, wird durch Mutationen der Gene NPHP 9 verursacht. NPHP4 durch Morpholino Knockdown verursacht Zystenbildung in der Tg (wt1b: GFP). Fisch 10 Daher ist dieser transgene Fische ein geeignetes Modell zur Beobachtung Nieren Strukturen und Zystenbildung während der Nierenentwicklung. Wichtig ist, dass der Einfluss von Modulatoren der Nierenentwicklung Verwendung dieses Stammes in einer Zeit und Arbeit effizient untersucht werden. Unser Papier beschreibt die Verwendung von Tg (wt1b: GFP) Fisch als Vorbild zu Nierenzystenbildung nach Genmodulation visualisieren. Wir benutzten Start- und Spleißstellen Antisense-MOs um knock down der Wnt5a Gen in Zebrafisch. Wnt5a ist eine nicht-kanonische sekretierte Glykoprotein des Wnt-Familie, die in der Entwicklung von verschiedenen Organen und postnatalen zellulären eine wichtige Rolle spielt,Funktion 11. Wnt5a wirkt durch nicht-kanonischen Wnt-Signalwege, einschließlich der planaren Zellpolarität (PCP) Weg, die gefunden wurde, um eine Rolle in orientierte Zellteilung während der Nierentubuli Dehnung zu spielen. Wnt5a regelt die Wnt / PCP-Weg, indem ein Komplex mit dem Rezeptor ähnliche Tyrosinkinase (Ryk), die weiter transduziert Wnt5a Signalisierung durch Bildung eines Komplexes mit dem VANGL planaren Zellpolarität protein 2 (Vangl2), wodurch Vangl2 Stabilität 12 fördern. Defekte in der PCP-Weg kann in zufälliger Zellteilung verursachen und eine Nierenzystenbildung. Wir nutzten die Tg (wt1b: GFP) Zebrafisch Linie zu Nierenzystenbildung folgenden Wnt5a Zuschlags beobachten. Die Tg (wt1b: GFP) Zebrafisch-Modell ermöglicht die Live-Darstellung und zeitnahe Beobachtung der Nierenstruktur. Nach Wnt5a Zuschlags wurde Nierenzystenbildung gefunden, beginnend bei 24 HPF; bei 72 HPF, könnte Zysten in den Glomeruli und den proximalen Tubuli gefunden werden. Diese Methode könnte auch zu s verwendet werdencreen andere Gene, die Nierenzystenbildung verursachen könnten.

<table><tbody><tr><td>0.5% Phenol-Red</td><td>Sigma</td><td>P0290</td><td>Color indicator for injection</td></tr><tr><td>Morpholino</td><td>Genn Tools, LLC</td><td>(customized)</td><td>Customized designed to the gene of interest</td></tr><tr><td>Pre-pulled needle</td><td>Tritech Research</td><td>MINJ-PP</td><td>If large amount of needle is required, you can also purchase a needle puller and prepare the needle in the lab</td></tr><tr><td>T7 mMessage Kit</td><td>Ambion</td><td>1344</td><td>For in vitro transcription to make capped mRNA for rescue experiment</td></tr><tr><td>N-Phenylthiourea</td><td>Sigma</td><td>P7629</td><td>For prevention of melanization</td></tr><tr><td>Tricaine</td><td>Sigma</td><td>A5040</td><td>Also called ethyl 3-aminobenzoate, for zebrafish anesthesia</td></tr><tr><td>Methyl Cellulose</td><td>Sigma</td><td>M-0387</td><td>For position of zebrafish&amp;nbsp;</td></tr><tr><td>QIAquick PCR Purification Kit&amp;nbsp;</td><td>Qiagen</td><td>28104</td><td>For purification of PCR product for cap RNA synthesis.</td></tr><tr><td>dNTP mix</td><td>Promega</td><td>U1511</td><td>For PCR</td></tr><tr><td>Tag DNA Polymerase</td><td>Invitrogen</td><td>10342-053</td><td>For PCR</td></tr><tr><td>NanoDrop spectrophotometer</td><td>Thermo Scientific</td><td>ND-1000</td><td>For measure morpholino and RNA concentration</td></tr><tr><td>Air Compressor</td><td>Werther International, Inc.</td><td>Panther Compact 106</td><td>Air source for injection</td></tr><tr><td>Pico micro-injection pump</td><td>World Precision Instruments Inc</td><td>PV830 Pnematic PicoPump</td><td>Other types of microinjection system can be used.</td></tr><tr><td>Micro-manuplator</td><td>World Precision Instruments Inc</td><td>MMJR</td><td>Right-handed (MMJL for left handed)</td></tr><tr><td>Needle holder</td><td>World Precision Instruments Inc</td><td>5430-ALL</td><td>To hold needle for micromanipulation</td></tr><tr><td>Dumont Tweezers</td><td>Fine Surgical Tools</td><td>11253-20</td><td>For breaking off the needle tip</td></tr><tr><td>Dissecting microscope</td><td>Leica M205C&amp;nbsp;</td><td></td><td>For observing and imaging zebrafish embryos</td></tr><tr><td>Fluorscence microscope</td><td>Zeiss Axio Obserer D1m &amp;nbsp;</td><td></td><td>For imaging zebrafish pronephros</td></tr><tr><td>Capillary tube (I.D. 0.15 mm)</td><td>VitroCom</td><td>CV1525Q-100</td><td>For measure the volume of each injected drop</td></tr></tbody></table>

a possible zebrafish model of polycystic kidney disease: knockdown of wnt5a causes cysts in zebrafish kidneys

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The molecular mechanisms leading to cyst formation in PKD remain somewhat unclear, but many genes are thought to be involved. Wnt5a is a non-canonical glycoprotein that regulates a wide range of developmental processes. Wnt5a works through the planar cell polarity (PCP) pathway that regulates oriented cell division during renal tubular cell elongation. Defects of the PCP pathway have been found to cause kidney cyst formation. Our paper describes a method for developing a zebrafish cystic kidney disease model by knockdown of the wnt5a gene with wnt5a antisense morpholino (MO) oligonucleotides. Tg(wt1b:GFP) transgenic zebrafish were used to visualize kidney structure and kidney cysts following wnt5a knockdown. Two distinct antisense MOs (AUG - and splice-site) were used and both resulted in curly tail down phenotype and cyst formation after wnt5a knockdown. Injection of mouse Wnt5a mRNA, resistant to the MOs due to a difference in primary base pair structure, rescued the abnormal phenotype, demonstrating that the phenotype was not due to &#8220;off-target&#8221; effects of the morpholino. This work supports the validity of using a zebrafish model to study wnt5a function in the kidney.

Wnt5a niederschlagen wurde durch die Einführung Übersetzung blockieren MO erreicht (August-MO) oder Exon / Intron-Grenze Splice-MO (Splice-MO) auf Zebrafischembryonen an dem einen Zellstadium. Die August-MO zielt auf das Start-Codon und daher hemmt sowohl von Müttern und zygotischen Wnt5a Nachricht. Der Spleiß-MO zielt die dritte Spleißdonorstelle und hemmt nur die zygotischen Transkript Wnt5a (1A). Die Aug- und Spleiß- morphants phenocopied seitig mit mehreren Mängeln, darunter...

Watch this Scientific Journal Video about A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys at JoVE.com

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish.

Eine mögliche Zebrafisch-Modell der Kidney Disease: Knockdown von<em&gt; Wnt5a</em&gt; Ursachen Zysten in Zebrafisch-Nieren

Polycystic kidney disease (PKD) is one of the most common causes of end-stage kidney disease, a devastating disease for which there is no cure. The ...

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Research

JoVE Journal

Medicine

A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

11.1K Aufrufe.  Eastern Virginia Medical School. We describe a method of generating a possible zebrafish model of polycystic kidney disease. We used Tg(wt1b:GFP) fish to visualize kidney structure. Knockdown of wnt5a was by morpholino injection. Pronephric cyst formation after wnt5a knockdown was observed in this GFP transgenic zebrafish. 

Serie: A Possible Zebrafish Model of Polycystic Kidney Disease: Knockdown of wnt5a Causes Cysts in Zebrafish Kidneys

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

Precise Cellular Ablation Approach for Modeling Acute Kidney Injury in Developing Zebrafish

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore adequate kidney function after supportive treatment. However, a better understanding of how nephron cell death and repair occur on the cellular level is required to minimize cell death and to enhance the regenerative process. The zebrafish pronephros is a good model system to accomplish this goal because it contains anatomical segments that are similar to the mammalian nephron. Previously, the most common model used to study kidney injury in fish was the pharmacological gentamicin model. However, this model does not allow for precise spatiotemporal control of injury, and hence it is difficult to study cellular and molecular processes involved in kidney repair. To overcome this limitation, this work presents a method through which, in contrast to the gentamicin approach, a specific Green Fuorescent Protein (GFP)-expressing nephron segment can be photoablated using a violet laser light (405 nm). This novel model of AKI provides many advantages that other methods of epithelial injury lack. Its main advantages are the ability to &#34;dial&#34; the level of injury and the precise spatiotemporal control in the robust in vivo animal model. This new method has the potential to significantly advance the level of understanding of kidney injury and repair mechanisms.

This work presents a new in vivo model of segmental kidney injury using kidney GFP transgenic zebrafish. The model allows for the induction of the targeted ablation of kidney epithelial cells to show the cellular mechanisms of nephron injury and repair.

Präziser zellulärer Ablationsansatz zur Modellierung akuter Nierenverletzungen bei der Entwicklung von Zebrafisch

Acute Kidney Injury (AKI) is a common medical condition with a high mortality rate. With the repair abilities of the kidney, it is possible to restore ...

Shifting Zebrafish Lethal Skeletal Mutant Penetrance by Progeny Testing

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be difficult to study, and can lead to confounding results. The protocol described here is a straightforward breeding paradigm to increase and decrease penetrance in lethal zebrafish skeletal mutants. Because lethal mutants cannot be selectively bred directly, the classic selective breeding strategy of progeny testing is employed. This method also includes protocols for Kompetitive Allele Specific PCR (KASP) genotyping zebrafish and staining larval zebrafish cartilage and bone. Applying the husbandry strategy described here can increase the penetrance of an interesting skeletal phenotype enabling more reproducible results in downstream applications. In addition, decreasing the mutant penetrance through this selective breeding strategy can reveal the developmental processes that most crucially require the function of the mutated gene. While the skeleton is specifically considered here, we propose that this methodology will be useful for all zebrafish mutant lines.

The goal of this protocol is to alter the penetrance of lethal skeletal mutant phenotypes in zebrafish by selective breeding. Lethal mutants cannot be grown to adulthood and bred themselves, therefore this protocol describes a method for tracking and selecting penetrance through multiple generations by progeny testing.

Verschiebung der Zebrafisch letale Skelett mutierte Penetranz um Nachkommen testen

Zebrafish mutant phenotypes are often incompletely penetrant, only manifesting in some mutants. Interesting phenotypes that inconsistently appear can be ...

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 ...

Behavioral And Physiological Analysis In A Zebrafish Model Of Epilepsy

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research have uncovered a large spectrum of genes implicated in various forms of epilepsy, highlighting the heterogeneous nature of this disorder. Appropriate animal models are essential for investigating the pathological mechanisms triggered by genetic mutations implicated in epilepsy and for developing specialized, targeted therapies. In recent years, zebrafish has emerged as a valuable vertebrate organism for modeling epilepsies, with the use of both genetic manipulation and exposure to known epileptogenic drugs, such as pentylenetetrazole (PTZ), to identify novel anti-epileptic therapeutics. Deleterious mutations in the mTOR regulator DEPDC5 have been associated with various forms of focal epilepsies and knock-down of the zebrafish orthologue causes hyperactivity associated with spontaneous seizure-like episodes, as well as enhanced electrographic activity and characteristic turn wheel swimming. Here, we described the method involved in generating the DEPDC5 loss-of-function model and illustrate the protocol for assessing motor activity at 28 and 48 h post fertilization (hpf), as well as a method for recording field activity in the zebrafish optic tectum. An illustration of the effect of the epileptogenic drug PTZ on neuronal activity over time is also provided.

Here, we present a protocol for the development and the characterization of a zebrafish model of epilepsy resulting from the transient inhibition of the DEPDC5 gene.

Verhaltens- und physiologische Analyse in einem Zebrafischmodell der Epilepsie

Epilepsy represents one of the most common neurological disorders, affecting an estimated 50 million people worldwide. Recent advances in genetic research ...

Neurowissenschaften

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present methods for the development of assays to query potentially clinically significant nonsynonymous changes using in vivo complementation in zebrafish. Zebrafish (Danio rerio) are a useful animal system due to their experimental tractability; embryos are transparent to enable facile viewing, undergo rapid development ex vivo, and can be genetically manipulated.1 These aspects have allowed for significant advances in the analysis of embryogenesis, molecular processes, and morphogenetic signaling. Taken together, the advantages of this vertebrate model make zebrafish highly amenable to modeling the developmental defects in pediatric disease, and in some cases, adult-onset disorders. Because the zebrafish genome is highly conserved with that of humans (~70% orthologous), it is possible to recapitulate human disease states in zebrafish. This is accomplished either through the injection of mutant human mRNA to induce dominant negative or gain of function alleles, or utilization of morpholino (MO) antisense oligonucleotides to suppress genes to mimic loss of function variants. Through complementation of MO-induced phenotypes with capped human mRNA, our approach enables the interpretation of the deleterious effect of mutations on human protein sequence based on the ability of mutant mRNA to rescue a measurable, physiologically relevant phenotype. Modeling of the human disease alleles occurs through microinjection of zebrafish embryos with MO and/or human mRNA at the 1-4 cell stage, and phenotyping up to seven days post fertilization (dpf). This general strategy can be extended to a wide range of disease phenotypes, as demonstrated in the following protocol. We present our established models for morphogenetic signaling, craniofacial, cardiac, vascular integrity, renal function, and skeletal muscle disorder phenotypes, as well as others.

In Vivo Modeling of the Morbid Human Genome using Danio rerio

Here, we present a systematic approach for developing physiologically relevant, sensitive and specific in vivo assays for interpreting variation in human pathology. Transient genetic manipulation via microinjection of WT and mutant human mRNA and morpholino (MO) antisense oligonucleotides harness the tractability of the developing zebrafish embryo to rapidly assay pathogenic mutations, especially, but not exclusively, in the context of human developmental disorders.

In Vivo Modellierung des Morbid Human Genome mit Danio rerio

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

Biologie

Visualization of Craniofacial Development in the sox10: kaede Transgenic Zebrafish Line Using Time-lapse Confocal Microscopy

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, convergence and extension of facial prominences, and maturation of the craniofacial skeleton. To study the contribution of the cranial neural crest to specific regions of the zebrafish palate a sox10: kaede transgenic zebrafish line was generated. Sox10 provides lineage restriction of the kaede reporter protein to the neural crest, thereby making the cell labeling a more precise process than traditional dye or reporter mRNA injection. Kaede is a photo-convertible protein that turns from green to red after photo activation and makes it possible to follow cells precisely. The sox10: kaede transgenic line was used to perform lineage analysis to delineate CNC cell populations that give rise to maxillary versus mandibular elements and illustrate homology of facial prominences to amniotes. This protocol describes the steps to generate a live time-lapse video of a sox10: kaede zebrafish embryo. Development of the ethmoid plate will serve as a practical example. This protocol can be applied to making a time-lapse confocal recording of any kaede or similar photoconvertible reporter protein in transgenic zebrafish. Furthermore, it can be used to capture not only normal, but also abnormal development of craniofacial structures in the zebrafish mutants.

Visualization of experimental data has become a key element in presenting results to the scientific community. Generation of live time-lapse recording of growing embryos contributes to better presentation and understanding of complex developmental processes. This protocol is a step-by-step guide to cell labeling via photoconversion of kaede protein in zebrafish.

Visualisierung von Craniofacial Entwicklung in den sox10: kaede transgener Zebrafische Zeile mit Zeitraffer-konfokale Mikroskopie

Vertebrate palatogenesis is a highly choreographed and complex developmental process, which involves migration of cranial neural crest (CNC) cells, ...

Analysis of Nephron Composition and Function in the Adult Zebrafish Kidney

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys are composed of functional units known as nephrons, which are highly conserved with other vertebrates, including mammals. Research in zebrafish has recently demonstrated that two distinctive phenomena transpire after adult nephrons incur damage: first, there is robust regeneration within existing nephrons that replaces the destroyed tubule epithelial cells; second, entirely new nephrons are produced from renal progenitors in a process known as neonephrogenesis. In contrast, humans and other mammals seem to have only a limited ability for nephron epithelial regeneration. To date, the mechanisms responsible for these kidney regeneration phenomena remain poorly understood. Since adult zebrafish kidneys undergo both nephron epithelial regeneration and neonephrogenesis, they provide an outstanding experimental paradigm to study these events. Further, there is a wide range of genetic and pharmacological tools available in the zebrafish model that can be used to delineate the cellular and molecular mechanisms that regulate renal regeneration. One essential aspect of such research is the evaluation of nephron structure and function. This protocol describes a set of labeling techniques that can be used to gauge renal composition and test nephron functionality in the adult zebrafish kidney. Thus, these methods are widely applicable to the future phenotypic characterization of adult zebrafish kidney injury paradigms, which include but are not limited to, nephrotoxicant exposure regimes or genetic methods of targeted cell death such as the nitroreductase mediated cell ablation technique. Further, these methods could be used to study genetic perturbations in adult kidney formation and could also be applied to assess renal status during chronic disease modeling.

The zebrafish adult kidney is an excellent system for renal regeneration and disease studies. An essential aspect of such research is the assessment of nephron structure and function. This protocol describes several methodologies that can be implemented to assess nephron tubule composition and to evaluate renal reabsorption.

Analyse der Nephron Zusammensetzung und Funktion im erwachsenen Zebrafisch Nieren

The zebrafish model has emerged as a relevant system to study kidney development, regeneration and disease. Both the embryonic and adult zebrafish kidneys ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Akute Nierenverletzung Modell induziert durch Cisplatin bei erwachsenen Zebrafischen

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Medizin

Evaluation of Zebrafish Kidney Function Using a Fluorescent Clearance Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish kidney develops and functions in almost the same way as humans. A major difference in the construction of the human kidney is the presence of millions of nephrons compared to the zebrafish that has only two. However, simplifying such a complex system into basic functional units has aided our understanding of how the kidney develops and operates. In zebrafish, the midline located glomerulus is responsible for the initial blood filtration into two pronephric tubules that diverge to run bilaterally down the embryonic axis before fusing to each other at the cloaca. The pronephric tubules are heavily populated by motile cilia that facilitate the movement of filtrate along the segmented tubule, allowing the exchange of various solutes before finally exiting via the cloaca2-4. Many genes responsible for CKD, including those related to ciliogenesis, have been studied in zebrafish5. However, a major draw back has been the difficulty in evaluating zebrafish kidney function after genetic manipulation. Traditional assays to measure kidney dysfunction in humans have proved non translational to zebrafish, mainly due to their aquatic environment and small size. For example, it is not physically possible to extract blood from embryonic staged fish for analysis of urea and creatinine content, as they are too small. In addition, zebrafish do not produce enough urine for testing on a simple proteinuria &#8216;dipstick&#8217;, which is often performed during initial patient examinations. We describe a fluorescent assay that utilizes the optical transparency of the zebrafish to quantitatively monitor the clearance of a fluorescent dye, over time, from the vasculature and out through the kidney, to give a read out of renal function1,6-9.

The zebrafish is a popular tool to model chronic kidney disease (CKD). However, their small size makes it impossible to evaluate renal function using traditional methods. We describe a fluorescent dye kidney clearance assay1 that allows quantitative analysis of zebrafish kidney function in CKD.

Bewertung der Zebrafisch Nierenfunktion unter Verwendung einer fluoreszierenden Räumungs Assay

The zebrafish embryo offers a tractable model to study organogenesis and model human genetic disease. Despite its relative simplicity, the zebrafish ...

Eine mögliche Zebrafisch-Modell der Kidney Disease: Knockdown von

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