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.

Poisson zèbre ont été soulevées et élevés selon la procédure standard; expériences ont été approuvés par l&#39;Université de Pittsburgh institutionnel de protection des animaux et l&#39;utilisation Comité. 1. Préparation d&#39;embryons / larves <ol><li> Pour mener accouplements chronométrés, mis en place adulte mâle et femelle Tg (fabp10a: CFP-NTR) S931 poissons hémizygotes ou homozygotes O / N et placer une séparation entre eux. Retirer ce diviseur le lendemain matin lorsque l&#39;accouplement est souhaitée. Recueillir des embryons en forçant l&#39;eau en utilisant une maille fine passoire en plastique. </li><li> Tourner la crépine à l&#39;envers et rincer la surface de maille avec un mince filet d&#39;eau d&#39;œuf distribué à partir d&#39;un flacon souple. Transférer 100 embryons dans une boîte de Pétri de 100 mm avec 25 ml d&#39;eau de l&#39;œuf. Gardez pas plus de 100 embryons par boîte de Pétri et les élever à 28 ° C. </li><li> Pour inhiber la pigmentation, ajouter phénylthiourée (PTU) dans les boîtes de Pétri avant 10 h après la fécondation (HPF), et d&#39;élever embryos / larves jusqu&#39;au 80 HPF. La concentration finale de 0,2 mM est PTU. ATTENTION: PTU est toxique. Utiliser conformément aux directives de manipulation appropriées. </li><li> Anesthésier les larves par addition de 3-amino benzoïque ester éthylique de l&#39;acide (tricaïne), à ​​une concentration finale de 0,016%, dans les boîtes de Pétri. Puis, en utilisant un microscope à épifluorescence, larves sorte CFP-positif. Utilisez les larves avec une taille de foie et jeter semblable à celles d&#39;un foie plus petit ou plus grand. REMARQUE: les larves de PCP-positif, indépendamment de l&#39;intensité, peut être utilisé car il n&#39;y a aucune différence dans la mesure des hépatocytes ablation entre les larves hémizygote et homozygotes. ATTENTION: tricaïne est toxique. Utiliser conformément aux directives de manipulation appropriées. </li><li> Retirer l&#39;eau d&#39;œuf contenant Tricaine et ajouter de l&#39;eau d&#39;œuf supplémenté avec 0,2 mM PTU. Garder les embryons / larves à 28 ° C. </li></ol> 2. Traitement Mtz <ol><li> Préparer une solution fraîche mM Mtz 10 en dissolvant 0,171 g de poudre Mtz en 100 ml d&#39;eau d&#39;œuf complété avec 0,2% de DMSO et 0,2 mM PTU. Pour dissoudre complètement le Mtz, mélanger la solution à la température ambiante sous agitation rapide pendant 20-30 minutes. Pour aider à solubiliser Mtz et d&#39;améliorer Mtz perméation dans les larves, ajouter DMSO lors de la préparation Mtz. ATTENTION: L&#39;exposition prolongée ou accrue concentration de Mtz est toxique. Utiliser conformément aux directives de manipulation appropriées. </li><li> Séparer les larves dans des groupes de contrôle et expérimentales en transférant nombre désiré de larves dans deux différents plat 100 mm de Pétri ou plaque multi-puits. Pour le groupe expérimental, garder les larves dans une solution 10 mM Mtz; pour le groupe de contrôle, gardez-les dans de l&#39;eau contenant de l&#39;œuf 0,2% de DMSO. </li><li> Couvrir les plaques ou les boîtes de Pétri contenant la solution Mtz avec du papier d&#39;aluminium pour éviter la photo-inactivation de Mtz, et incuber à 28 ° C. La durée du traitement dépend de la Mtz stade larvaire et la lignée transgénique. </li><li> Pour observer similaire sévère ablation des hépatocytes dans le <em&gt; Tg (fabp10a: CFP-NTR) en ligne, traiter les larves avec la solution Mtz soit de 3,5 jours après la fécondation (DPF) (pour une durée de 36 h) ou de 5 dpf (pour une durée de 24 heures). </li></ol> 3. Analyse des voies biliaires axée sur la régénération du foie <ol><li> A la fin de la période d&#39;ablation, on élimine la solution Mtz des plaques. Laver les larves Mtz traitée par addition de 25 ml d&#39;eau de l&#39;oeuf. Agiter la plaque 2-3 fois pour laver les larves avant de jeter l&#39;eau d&#39;œuf. Répétez trois fois et puis plonger les larves une fois de plus dans l&#39;eau d&#39;œuf. Pour éviter l&#39;œdème cardiaque et la mort larvaire chez les larves traitées Mtz, ajouter une plus faible concentration de tricaïne, à une concentration finale de 0,008%, pour immobiliser les larves. </li><li> Pour l&#39;analyse du foie, examiner la taille du foie des larves Mtz traité sous un microscope à épifluorescence avec le filtre de la PCP. Évaluer les niveaux d&#39;ablation d&#39;hépatocytes fonction de la taille relative du foie: un grand (non-ablation; 0-10% larves), moyen (partiellement ablation; 10-20%) et très faible (entièrement ablation; 80 à 90%). </li><li> Retirer les larves avec des foies non soumises à une ablation ou partiellement ablation. Seulement conserver des larves avec une minuscule foie, qui est indicative d&#39;ablation sévère des hépatocytes. Pour l&#39;analyse du foie à la régénération 0 point de temps de h (R0H), récolter les larves après Mtz lavage et de la PCP tri. Sinon, conservez les larves triés dans l&#39;eau d&#39;œuf supplémenté avec 0,2 mM PTU avant d&#39;être prêt pour la récolte. </li><li> Transfert larves récoltées dans un tube de microcentrifugation de 1,5 ml et remplacer l&#39;eau d&#39;oeuf avec la solution de fixation de 3% de formaldehyde dans du tampon PEM. Incuber le tube sur un rotateur O / N à 4 ° C. </li><li> Remplacer la solution de fixation avec 1 ml de PBSDT et faire tourner le tube contenant les larves sur le rotateur pendant 5 min. </li><li> Transférer les larves fixe dans une boîte de Pétri de 100 mm contenant PBSDT et en utilisant une pince, retirer manuellement jaune et pectorales sous un microscope de dissection. Transférer jusqu&#39;à 20 larves tp disséqué dans un tube de 1,5 ml. </li> &lt;li&gt; Remplacer solution PBSDT avec 1 ml de solution de blocage et de rock du tube sur un rotateur à température ambiante pendant 2 h. </li><li> Remplacer la solution de blocage avec 100 ul de solution de blocage contenant des anticorps primaires. Lentement balancer le tube sur un rotateur O / N à 4 ° C. </li><li> Retirer la solution d&#39;anticorps primaire et laver avec PBSDT 5 fois pendant 10 min à chaque fois. Puis ajouter 100 ul de la solution de blocage contenant des anticorps secondaires et le rock sur un agitateur à température ambiante pendant 2h. </li><li> Laver 5 fois avec PBSDT pour 10 min à chaque fois à la température ambiante en basculant sur un rotateur. </li><li> Orientez les larves latéralement sur une lame de microscope et ajouter une goutte de milieu de montage. Les couvrir soigneusement avec un couvercle en verre et sceller le couvercle en verre avec un polissoir à ongles. Maintenant, il est prêt pour la microscopie confocale. </li></ol>

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Les auteurs déclarent qu&#39;ils ont aucun intérêt financier concurrentes.

Sévère, mais pas doux, hépatocytes ablation suscite conduit biliaire-régénération du foie. Par conséquent, après un traitement Mtz et lavage, il est important d&#39;examiner la taille du foie de la larve Mtz-traité, qui peut être évaluée par fluorescence intrinsèque de la PCP fabp10a: CFP-NTR transgène. Comme un faible pourcentage de larves traitées Mtz affiche non-ablatée (0-5%) ou partiellement ablation (10-20%) des foies, il est essentiel de régler et d&#39;analyser seulement les larves avec...

Le foie est un organe très régénérative. Au cours de la régénération du foie, régénération des hépatocytes, les cellules du parenchyme du foie, sont dérivés des hépatocytes pré-existantes (entraînée hépatocytes régénération du foie (BEC) ou entraîné biliaire de régénération du foie) 1,2. Des lésions hépatiques suscite habituellement la prolifération des hépatocytes pré-existante; cependant, lorsque la prolifération des hépatocytes est compromise, BEC peut contribuer à hépatocytes 2-4. Ces deux modes de régénération du foie sont cliniquement significatives. Après l&#39;ablation chirurgicale d&#39;une partie du foie humain (par exemple, en raison de tumeurs du foie ou des donneurs vivants de foie), les hépatocytes dans le foie restant prolifèrent pour récupérer la masse du foie perdu. En revanche, chez des patients atteints de maladies hépatiques graves, la prolifération des hépatocytes est fortement compromise, de sorte que BEC ou de cellules souches du foie (LPC) semblent contribuer à la régénération des hépatocytes 5,6. Le modèle de hépatectomie partielle rongeurs 2/3, dans lequelhépatocytes prolifèrent pour récupérer la masse du foie perdu, a largement contribué à la compréhension actuelle de entraîné hépatocytes de foie de régénération 7,8. Cependant, il n&#39;y a pas modèle de rongeur valide dans lequel les hépatocytes régénération proviennent principalement de BEC. Bien que plusieurs modèles de rongeurs de la toxine du foie ont conduit à l&#39;identification de conduit biliaire du foie régénération 2-4, des études récentes de la lignée de traçage chez les souris indiquent une contribution minimale de BEC pour la régénération des hépatocytes dans ces modèles 9,10. Certains des modèles de lésions hépatiques de rongeurs, y compris une hépatectomie partielle et induite par l&#39;acétaminophène 11-13-14,15 dommages au foie, ont été appliquées pour le poisson zèbre et conduit à l&#39;identification de nouveaux gènes ou des voies impliquées dans la régénération du foie. Cependant, les hépatocytes entraînée, mais pas BEC-entraînée, la régénération du foie se produit dans ces modèles de lésion du foie de poisson zèbre. Par conséquent, un modèle de lésion du foie roman dans lequel CAE contribuent largement à regenerating hépatocytes est nécessaire pour une meilleure compréhension du BEC-entraîné la régénération du foie. Les objectifs globaux du modèle d&#39;ablation des hépatocytes décrits ici sont (1) pour générer un modèle de lésion du foie dans laquelle CAE contribuent largement aux hépatocytes de régénération, et (2) élucider les mécanismes moléculaires et cellulaires sous-jacents BEC-driven la régénération du foie. Nous émettons l&#39;hypothèse que la gravité des blessures détermine le mode de régénération du foie; ainsi, nous avons prédit que conduit biliaire-régénération du foie lancerait lors d&#39;une lésion des hépatocytes sévère. Pour tester cette hypothèse, nous avons développé un modèle de lésion du foie de poisson zèbre en générant une lignée transgénique, Tg (fabp10a: CFP-NTR) S931, qui exprime très nitroreductase bactérienne (NTR) fusionnée avec une protéine fluorescente cyan (CFP) dans le cadre du fabp10a spécifique hépatocytes promoteur. Depuis NTR convertit le promédicament non toxique, le métronidazole (Mtz), en un médicament cytotoxique, il ne l&#39;ablation destiné NTR-cellules exprimant 16-18, dans ce cas, les hépatocytes. En manipulant la durée du traitement Mtz, l&#39;étendue des hépatocytes ablation peut être contrôlée. En utilisant ce modèle, nous avons récemment signalé que sur une perte sévère des hépatocytes, BEC donne lieu à beaucoup hépatocytes régénération 19, qui a été confirmée par deux autres études indépendantes 20,21. Par conséquent, par rapport à l&#39;rongeur ci-dessus et les modèles de lésions du foie de poisson zèbre, notre modèle d&#39;ablation des hépatocytes est plus avantageux pour l&#39;étude BEC-driven la régénération du foie. Ce protocole décrit la procédure pour effectuer des expériences de régénération du foie en utilisant le modèle hépatocytes d&#39;ablation poisson zèbre. Ce modèle sera appropriée pour déterminer les mécanismes sous-jacents conduit biliaire-régénération du foie et pour les écrans chimiques pour identifier de petites molécules qui peuvent réprimer ou d&#39;augmenter la régénération du foie.

<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

Le foie a une grande capacité à se régénérer. Les hépatocytes, les cellules du parenchyme du foie, peut régénérer en une de deux manières: ou conduit biliaire hepatocyte--régénération du foie. Dans entraîné hépatocytes la régénération du foie, régénération des hépatocytes sont dérivés d&#39;hépatocytes préexistantes, alors que, dans la régénération conduit biliaire, de régénération des hépatocytes sont dérivées de cellules epitheliales biliaires (BEC). Pour entraîné hépatocytes la régénération du foie, il ya d&#39;excellents modèles de rongeurs qui ont contribué de manière significative à la compréhension actuelle de la régénération du foie. Cependant, rien de tel modèle de rongeur existe pour entraînée bilio-régénération du foie. Nous avons récemment rapporté sur un modèle de blessure de foie de poisson zèbre dans lequel BEC donne lieu à beaucoup hépatocytes sur une perte sévère des hépatocytes. Dans ce modèle, les hépatocytes sont spécifiquement ablation par un moyen pharmacogénétiques. Ici, nous présentons en détail les méthodes pour l&#39;ablation des hépatocytes et d&#39;analyser le processus de régénération du foie BEC-driven.Ce modèle d&#39;ablation spécifiques hépatocytes peut encore être utilisé pour découvrir les mécanismes moléculaires et cellulaires sous-jacents de conduit biliaire-régénération du foie. En outre, ces méthodes peuvent être appliquées à des écrans chimiques pour identifier de petites molécules qui inhibent ou augmentent la régénération du foie.

Mtz traitement pendant 36 heures (A36h, A représente ablation) de 3,5 à 5 dpf réduit considérablement la taille du foie (figure 1B). Après Mtz lavage, considéré comme le début de la régénération du foie (R), forte fabp10a: CFP-NTR et fabp10a: expression DsRed réapparu dans les 30 heures (figure 1B). Le réseau des voies biliaires intra-hépatique, tel qu&#39;évalué par l&#39;expression de Alcam qui est présente dans la membrane du BEC 22, d&#3...

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.

Ablation spécifique des hépatocytes chez le poisson zèbre pour étudier conduit biliaire-Régénération hépatique

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 vues.  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.

Vidéo: 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.

Régénération rénale à l&#39;âge adulte poisson zèbre par gentamicine blessures induites

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

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Biologie du développement

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.

Utilisation du système de Teton pour étudier les mécanismes moléculaires de la régénération du poisson zèbre

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.

Développement d&#39;un fibrotique du foie Modèle induite de l&#39;éthanol en Zebrafish pour étudier de cellules souches médiée hépatocytes Régénération

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.

Chirurgical Ablation Essai pour étude de la régénération des yeux dans planaires

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.

Analyses histologiques de lésions hépatiques alcooliques aiguës dans le poisson zébré

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.

Méthodes pour l’étude de la régénération de 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.

Quantifier la taille du foie chez le poisson zèbre larvaire à l'aide de la microscopie Brightfield

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.

Essai confocal d’ablation et de régénération au microscope dans les cellules interuromast de Zebrafish

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.

Examen de la régénération musculaire dans les modèles de poisson-zèbre de maladie musculaire

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

Différenciation et stimulation mécanique de cellules endothéliales dérivées de hiPSC

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.

Pleins feux sur l’auteur : Étude de cellules endothéliales dérivées de hiPSC cultivées sous stimulation mécanique à médiation fluidique 

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.