The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

Zebrafisch wurden angehoben und nach Standardverfahren gezüchtet; Experimente wurden von der Universität Pittsburgh Institutional Animal Care und Verwenden Committee genehmigt. 1. Herstellung von Embryos / Larven <ol><li> Um zeitgesteuerte Paarungen, einzurichten erwachsenen männlichen und weiblichen Tg (fabp10a: CFP-NTR) durchführen S931 hemizygot oder homozygot Fische O / N und legen Sie eine Trennlinie zwischen ihnen. Entfernen Teiler am nächsten Morgen, als Gegen gewünscht wird. Sammeln Embryonen durch belasten das Wasser mit einem feinen Kunststoffnetz Sieb. </li><li> Drehen Sie den Filter auf den Kopf und spülen Sie die Netzoberfläche mit einem feinen Strom von Ei Wasser aus einem Squeeze-Flasche abgefüllt. Übertragen Sie 100 Embryonen in eine 100 mm Petrischale mit 25 ml Wasser Ei. Halten Sie nicht mehr als 100 Embryonen pro Petrischale und heben Sie sie auf 28 ° C. </li><li> Pigmentierung hemmen, fügen Phenylthioharnstoff (PTU) in die Petrischalen vor 10 Stunden nach der Befruchtung (HPF), und heben Sie embryos / Larven bis 80 HPF. Die Endkonzentration der PTU beträgt 0,2 mm. ACHTUNG: PTU ist giftig. Verwenden Sie in Übereinstimmung mit den entsprechenden Richtlinien zum Umgang. </li><li> Betäuben die Larven durch Zugabe von 3-Aminobenzoesäure-ethylester (Tricaine) bei einer Endkonzentration von 0,016%, in die Petrischalen. Dann, mit einem Epifluoreszenz-Mikroskop, sortieren GFP-positive Larven. Verwenden Larven mit einer ähnlichen Lebergröße und entsorgen Sie die mit einer kleineren oder größeren Leber. Anmerkung: Jeder GFP-positive Larven, egal Intensität kann verwendet werden, da es keinen Unterschied in dem Ausmaß der Ablation Hepatozyten zwischen der hemizygot homozygoten Larven. ACHTUNG: Tricaine ist giftig. Verwenden Sie in Übereinstimmung mit den entsprechenden Richtlinien zum Umgang. </li><li> Entfernen Sie das Wasser, das Ei Tricaine und fügen Sie Ei Wasser mit 0,2 mM PTU ergänzt. Halten Sie die Embryonen / Larven bei 28 ° C. </li></ol> 2. Mtz Treatment <ol><li> Bereiten Sie frischen 10 mM Mtz durch Auflösen von 0,171 g Mtz Pulver in 100 ml Ei Wasser ergänzt mit 0,2% DMSO und 0,2 mM PTU. Um die Mtz vollständig zu lösen, mischen Sie die Lösung bei RT unter schnellem Rühren für 20-30 Minuten. Um Ihnen zu helfen zu solubilisieren Mtz und Mtz Permeation in die Larven zu verbessern, fügen DMSO bei der Vorbereitung Mtz. ACHTUNG: längerer Exposition oder erhöhte Konzentration von Mtz ist giftig. Verwenden Sie in Übereinstimmung mit den entsprechenden Richtlinien zum Umgang. </li><li> Trenne die Larven in Kontroll- und Versuchsgruppen durch Übertragen gewünschte Anzahl der Larven in zwei 100 mm-Petrischale oder Multi-Well-Platte. Für die Versuchsgruppe, halten Sie die Larven in 10 mM Mtz Lösung; in der Kontrollgruppe, halten sie in Ei-Wasser, das 0,2% DMSO. </li><li> Umfassen die Platten oder Petrischalen MTZ Lösung mit einer Aluminiumfolie, um den Photo Inaktivierung Mtz verhindern, und Inkubieren bei 28 ° C. Die Dauer der Behandlung hängt von Mtz Larvenstadium und der transgenen Linie. </li><li> Ähnliche schwere Hepatozyten-Ablation in das beobachten <em&gt; Tg (fabp10a: CFP-NTR) Linie, behandeln die Larven mit der Mtz Lösung von entweder 3,5 Tage nach der Befruchtung (DPF) (für die Dauer von 36 h) oder von 5 dpf (für die Dauer von 24 h). </li></ol> 3. Analyse der Gallengetriebenen Leberregeneration <ol><li> Am Ende der Ablation Zeitraum, entfernen Sie die Mtz Lösung von den Platten. Durch Zugabe von 25 ml Wasser Ei waschen Sie die MTZ-behandelten Larven. Schwenken Sie die Platte 2-3 mal, um die Larven vor der Entsorgung das Ei Wasser zu waschen. Wiederholen Sie drei Mal und dann tauchen die Larven wieder in Ei Wasser. Zu Herzen Ödeme und zum Tod der Larve in der MTZ-behandelten Larven zu vermeiden, fügen Sie eine niedrigere Konzentration von Tricaine, in einer endgültigen Konzentration von 0,008%, um die Larven zu immobilisieren. </li><li> Für Leberanalyse, Prüfung der Lebergröße der MTZ-behandelten Larven unter einem Epifluoreszenz-Mikroskop mit der GFP-Filter. Beurteilen Hepatozyten Ablation Ebenen auf der Grundlage des relativen Lebergröße: groß (nicht abgetragenen; 0-10% Larven), mittel (teilweise abgetragen, 10-20%) und sehr klein (vollständig abgetragen; 80-90%). </li><li> Entfernen Sie die Larven mit nicht abgetragene oder teilweise abgetragen Lebern. Nur halten Larven mit einer winzigen Leber, die indikativ für schwere Hepatozyten Ablation. Für Leber-Analyse bei der Regeneration 0 hr Zeitpunkt (R0h), ernten die Larven nach Mtz Auswaschen und CFP Sortierung. Ansonsten halten die sortiert Larven im Ei Wasser mit 0,2 mM PTU ergänzt, bis bereit für die Ernte. </li><li> Transfer geerntet Larven in ein 1,5 ml Mikrozentrifugenröhrchen und Ersetzen ei Wasser mit der Fixierungslösung von 3% Formaldehyd in PEM-Puffer. Das Röhrchen auf einem Rotator O / N bei 4 ° C. </li><li> Ersetzen Sie die Fixierungslösung mit 1 ml PBSDT und drehen Sie die Röhre mit den Larven auf dem Rotator für 5 min. </li><li> Übertragen Sie die Fest Larven in eine 100 mm Petrischale mit PBSDT und mit einer Pinzette, Eigelb und Brustflossen unter einem Binokular manuell entfernen. Übertragung von bis tp 20 seziert Larven in ein 1,5-ml-Tube. </li> &lt;li&gt; Ersetzen PBSDT Lösung mit 1 ml Blockierungslösung und Rock das Rohr auf einem Rotator bei RT für 2 h. </li><li> Ersetzen Blockierungslösung mit 100 ul Blockierungslösung mit primären Antikörpern. Langsam schaukeln Sie das Rohr auf einem Rotator O / N bei 4 ° C. </li><li> Entfernen Sie den primären Antikörperlösung und wäscht mit PBSDT 5 mal für 10 Minuten jedes Mal. Dann fügen Sie 100 ul Blockierungslösung, die Sekundärantikörper und schütteln Sie sie auf einem Rotator bei RT 2 h. </li><li> 5 mal mit PBSDT für je 10 min waschen bei RT durch Schaukeln auf einem Rotator. </li><li> Richten Sie die Larven seitlich auf einen Objektträger und einen Tropfen Eindeckmittel. Sorgfältig decken Sie sie mit einem Deckglas und Dichtung das Deckglas mit einem Nagellack. Jetzt ist es bereit für die konfokale Mikroskopie. </li></ol>

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Die Autoren erklären, dass sie keine konkurrierenden finanziellen Interessen.

Ernst, aber nicht mild, entlockt Hepatozyten-Ablation Gallengetriebenen Leberregeneration. Daher wird nach Mtz Behandlung und Auswaschen, ist es wichtig, um die Leber Größe des MTZ-behandelten Larven, die durch intrinsische Fluoreszenz des GFP fabp10a bewertet werden können untersuchen: GFP-Transgen NTR. Da ein kleiner Prozentsatz der MTZ-behandelten Larven wird nicht abgetragenen (0-5%) oder (10-20%) Lebern teilweise abgetragen anzuzeigen, ist es wichtig zu klären, und nur analysieren die Larven m...

Die Leber ist ein hoch regenerative Organ. Während der Leberregeneration, Regeneration von Leberzellen, die parenchymale Zellen der Leber, sind aus vorbestehenden Leberzellen (Hepatozyten getriebenen Leberregeneration) oder BECs (Gallengetriebenen Leberregeneration) 1,2 abgeleitet. Leberschäden hervorruft, in der Regel die Verbreitung von bereits bestehenden Leberzellen; Wenn jedoch Hepatozytenproliferation kompromittiert ist, kann dazu beitragen, BECs Hepatozyten 2-4. Diese beiden Modi der Leberregeneration klinisch signifikant. Nach der operativen Entfernung eines Teils der menschlichen Leber (zB aufgrund von Lebertumoren oder lebenden Leberspender), Hepatozyten im Leberrest vermehren, um die verlorene Lebermasse wiederherzustellen. Dagegen bei Patienten mit schweren Lebererkrankungen, Hepatozytenproliferation stark beeinträchtigt wird, so daß BECs oder Leberstammzellen (LPCs) scheinen zur Regenerierung Hepatozyten 5,6 beizutragen. Das Nagetier 2/3 Teilhepatektomie Modell, in demHepatozyten vermehren, um die Lebermasse verloren zu erholen, hat wesentlich zum derzeitigen Verständnis der Hepatozyten-driven Leberregeneration 7,8 beigetragen. Jedoch gibt es keine gültige Nagetiermodell, in dem Regenerieren Hepatozyten werden hauptsächlich aus BECs abgeleitet. Obwohl mehrere Nagetier Lebertoxin Modellen führte zur Identifizierung von Gallengetriebenen Leberregeneration 2-4, letzten Linienverfolgungsstudien an Mäusen zeigen einen minimalen Beitrag von BECs regenerierendes Leberzellen in diesen Modellen 9,10. Einige der Nagetiermodellen Leberverletzung, einschließlich partieller Hepatektomie 11-13 und Acetaminophen induzierten Leberschaden 14,15 haben Zebrafisch angewandt und führten zur Identifizierung neuer Gene oder Wege bei der Leberregeneration in Verbindung gebracht. Allerdings Hepatozyten-getrieben, aber nicht BEC-getrieben, tritt die Leberregeneration in diesen Zebrafischleberschäden Modelle. Daher ist eine neuartige Leberschäden Modell, in dem BECs weitgehend an Regenerat beitragening Hepatozyten ist für ein besseres Verständnis der BEC getriebenen Leberregeneration benötigt. Die übergeordneten Ziele des Hepatozyten-Ablation Modell hier beschrieben sind (1), um eine Schädigung der Leber Modell, in dem BECs weitgehend zu regenerierenden Leberzellen beitragen, und (2) Aufklärung der molekularen und zellulären Mechanismen BEC-driven Leberregeneration zugrunde zu generieren. Wir vermuten, dass die Schwere der Verletzung bestimmt den Modus der Leberregeneration; So sagten wir voraus, dass Gallengetriebenen Leberregeneration würde von schweren Verletzungen Hepatozyten zu initiieren. Um diese Hypothese zu testen, ein Zebrafischleberschäden Modell entwickelten wir durch die Erzeugung eines transgenen Linie, Tg (fabp10a: CFP-NTR) S931, dass hoch drückt bakterielle Nitroreduktase (NTR) mit cyan fluoreszierende Protein (GFP) fusioniert unter der Hepatozyten-spezifischen fabp10a Promotor. Da NTR wandelt das nicht-toxische Prodrug, Metronidazol (MTZ), in ein zytotoxisches Arzneimittel, abträgt es nur die beabsichtigte NTR-exprimierenden Zellen 16-18, in diesem Fall werden die Hepatozyten. Durch Manipulation der Dauer der MTZ Behandlung kann das Ausmaß der Ablation Hepatozyten gesteuert werden. Mit diesem Modell haben wir vor kurzem berichtet, dass bei schweren Hepatozyten Verlust, BECs weitgehend Anlass zu regenerierenden Leberzellen 19, die durch zwei weitere unabhängige Studien 20,21 bestätigt wurde. Daher kann im Vergleich zu dem vorgenannten Nagetier und Zebrafisch Leberverletzung Modellen ist unsere Hepatozyten Ablation Modell zum Studium BEC getriebenen Leberregeneration vorteilhafter. Dieses Protokoll beschreibt das Verfahren für die Durchführung der Leberregeneration Experimente unter Verwendung der Zebrafisch-Hepatozyten-Ablation-Modell. Dieses Modell wird für die Ermittlung der Mechanismen Gallengetriebenen Leberregeneration zugrunde liegenden und für die chemische Bildschirme, um kleine Moleküle, die zu unterdrücken oder zu erweitern Leberregeneration identifizieren können geeignet sein.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine) powder</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium sulfate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3771</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media (Vectashield)</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 15 mm petri dish&#160;</td>
			<td>Denville Scientific Inc.</td>
			<td>M5300</td>
			<td></td>
		</tr>
		<tr>
			<td>clear nail polish</td>
			<td>Fisher Scientific</td>
			<td>50949071</td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Dumont #5&#160;</td>
		</tr>
		<tr>
			<td>glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-544-1</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-540-A and 12-542-A</td>
			<td>18 x 18 mm</td>
		</tr>
		<tr>
			<td>zn-5 (mouse anti-Alcam)</td>
			<td>ZIRC</td>
			<td>zn-5</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>goat anti-Hnf4a</td>
			<td>Santa Cruz</td>
			<td>sc-6556 (C-19)</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>rat anti-RFP</td>
			<td>Allele Biotechnology</td>
			<td>ACT-CM-MRRFP10</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td>
			<td>Jackson laboratories</td>
			<td>705-605-147</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21240</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Cy3 donkey anti-rat IgG</td>
			<td>Jackson laboratories</td>
			<td>712-165-150</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Leica Microsystems</td>
			<td>S6F</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td>M205FA</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr>
			<td>egg water</td>
			<td></td>
			<td></td>
			<td>0.3 g/L of sea salts &#8216;Instant Ocean&#8217; and 0.5 mM CaSO4 in distilled water.</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>25.6 g Na2HPO4&#183;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.&#160; Autoclave for 20 min at 121&#176;C.</td>
		</tr>
		<tr>
			<td>1X PBSDT</td>
			<td></td>
			<td></td>
			<td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td>
		</tr>
		<tr>
			<td>PEM buffer</td>
			<td></td>
			<td></td>
			<td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td>
		</tr>
	</tbody>
</table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

Die Leber hat eine große Kapazität zu regenerieren. Hepatocyte- oder Gallengetriebenen Leberregeneration: Hepatozyten, die parenchymale Zellen der Leber, in eine von zwei Arten zu regenerieren. In Hepatocyten getriebenen Leberregeneration, regenerieren Hepatozyten aus vorbestehenden Hepatozyten abgeleitet ist, während, in der Gallengetriebenen Regeneration werden Regenerieren von Hepatozyten und Gallen Epithelzellen (BECs) abgeleitet. Für Hepatozyten getriebenen Leberregeneration, gibt es sehr gute Nagermodellen, die wesentlich zum aktuellen Verständnis der Leberregeneration beigetragen haben. Es besteht jedoch keine solche Nagetiermodell für Gallengetriebenen Leberregeneration. Wir haben vor kurzem auf einem Zebrafischleberschäden Modell, in dem BECs weitgehend Anlass zu Hepatozyten bei schweren Hepatozyten-Verlust geben wiesen. In diesem Modell werden die Leberzellen spezifisch durch eine pharmakogenetische Mittel abgetragen. Hier im Detail präsentieren wir die Methoden, um Hepatozyten abzutragen und den BEC-driven Leberregenerationsprozess zu analysieren.Diese Hepatozyten-spezifische Ablation Modell kann weiter verwendet werden, um die zugrunde liegenden molekularen und zellulären Mechanismen der Gallengetriebenen Leberregeneration entdecken werden. Darüber hinaus können diese Verfahren, um chemische Bildschirmen angewendet werden, um kleine Moleküle, erweitern oder die Regeneration der Leber zu unterdrücken identifizieren.

MTZ Behandlung für 36 h (A36h, A steht für Ablation) von 3,5 bis 5 dpf drastisch reduziert Lebergröße (1B). CFP-NTR und fabp10a:: Nach dem Auswaschen Mtz, als Beginn der Leberregeneration (R), starke fabp10a als dsRed Ausdruck erschien innerhalb von 30 h (1B). Die intrahepatische Gallen Netzwerk als durch Expression ALCAM die in der Membran von BECs 22 vorliegt beurteilt zunächst zusammenfällt, aber bei 54 Stunden nach der Auswaschung (R54h) (1...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Hepatozyten-spezifische Ablation in Zebrafische, um Gallengetriebenen Leberregeneration Studieren

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or ...

hepatocyte-specific-ablation-zebrafish-to-study-biliary-driven-liver

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Unit 1

Regeneration and Repair

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8.5K Aufrufe.  University of Pittsburgh. To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

Serie: Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

Nieren-Regeneration im erwachsenen Zebrafisch durch Gentamicin induzierte Schädigung

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is ...

Forschung

Entwicklungsbiologie

Use of the TetON System to Study Molecular Mechanisms of Zebrafish Regeneration

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough understanding of the molecular and cellular mechanisms involved requires experimental tools that allow for inducible, tissue-specific manipulation of gene expression or signaling pathways. Therefore, we and others have recently adapted the TetON system for use in zebrafish. The TetON system facilitates temporally and spatially-controlled gene expression and we have recently used this tool to probe for tissue-specific functions of Wnt/beta&#8211;catenin signaling during zebrafish tail fin regeneration. Here we describe the workflow for using the TetON system to achieve inducible, tissue-specific gene expression in the adult regenerating zebrafish tail fin. This includes the generation of stable transgenic TetActivator and TetResponder lines, transgene induction and techniques for verification of tissue-specific gene expression in the fin regenerate. Thus, this protocol serves as blueprint for setting up a functional TetON system in zebrafish and its subsequent use, in particular for studying fin regeneration.

Here we outline the workflow for using the TetON system to achieve tissue-specific gene expression in the adult regenerating zebrafish tail fin.

Die Nutzung der Teton-System, um molekulare Mechanismen bei Zebrafisch Regeneration Studieren

The zebrafish has become a very important model organism for studying vertebrate development, physiology, disease, and tissue regeneration. A thorough ...

Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

Entwicklung einer fibrotischen Leber Modell in Zebrabärbling Ethanol-induzierte zur Untersuchung Progenitor-vermittelte Zell Hepatozyten-Regeneration

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading ...

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

Chirurgische Ablation Assay zur Untersuchung von Augen Regeneration in Planarien

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Histologische Analysen der akuten alkoholischen Leberverletzung in Zebrafisch

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Methoden für die Untersuchung der Regeneration im Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

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 ...

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Konfokale Mikroskop-basierte Laserablation und Regenerationstest in Zebrafisch Interneuromastzellen

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

Untersuchung der Muskelregeneration in Zebrafischmodellen von Muskelerkrankungen

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, ...

Differenzierung und mechanische Stimulation von hiPSC-abgeleiteten Endothelzellen

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Autor im Rampenlicht: Untersuchung von hiPSC-abgeleiteten Endothelzellen, die unter fluidisch vermittelter mechanischer Stimulation kultiviert wurden 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

Methods Article

Hepatozyten-spezifische Ablation in Zebrafische, um Gallengetriebenen Leberregeneration Studieren