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.

El pez cebra se plantearon y criado de acuerdo con el procedimiento estándar; experimentos fueron aprobados por la Universidad de Pittsburgh Institucional Cuidado de Animales y el empleo Comisión. 1. Preparación de embriones / larvas <ol><li> Para llevar a cabo apareamientos cronometrados, creado varón adulto y hembra Tg (fabp10a: PPC-NTR) S931 peces hemizygous o homocigotos S / N y colocar un divisor entre ellos. Eliminar este divisor a la mañana siguiente cuando se desea apareamiento. Recoger los embriones por el esfuerzo del agua usando un colador de malla fina de plástico. </li><li> Gire el filtro al revés y enjuagar la superficie de malla con una fina corriente de agua de huevo dispensado desde un frasco. Transferir 100 embriones en un 100 mm placa de Petri con 25 ml de agua huevo. Mantenga no más de 100 embriones por placa de Petri y criarlos a 28 ° C. </li><li> Para inhibir la pigmentación, añada feniltiourea (PTU) en las placas de Petri antes de 10 horas después de la fecundación (HPF), y elevar embryos / larvas hasta 80 HPF. La concentración final de PTU es 0,2 mM. PRECAUCIÓN: La PTU es tóxico. Use de acuerdo con las directrices de manejo apropiadas. </li><li> Anestesiar las larvas mediante la adición de 3-amino éster etílico del ácido benzoico (tricaína), a una concentración final de 0,016%, en las placas de Petri. Luego, utilizando un microscopio de epifluorescencia, larvas de tipo PPC-positivo. Utilice las larvas con un tamaño de hígado similar y descartar aquellos con un hígado más pequeño o más grande. NOTA: cualquier larvas CFP-positiva, independientemente de la intensidad, se puede utilizar porque no hay diferencia en la extensión de la ablación de hepatocitos entre las larvas hemicigotos y homocigotos. PRECAUCIÓN: tricaína es tóxico. Use de acuerdo con las directrices de manejo apropiadas. </li><li> Eliminar el agua de huevo contiene tricaína y añadir agua huevo suplementado con 0,2 mM PTU. Mantenga el embriones / larvas a 28 ° C. </li></ol> 2. Tratamiento Mtz <ol><li> Preparar fresca solución mM Mtz 10 disolviendo 0,171 g de polvo Mtz en 1Agua de huevo 00 ml complementado con 0,2% mM PTU DMSO y 0,2. Para disolver completamente el Mtz, mezclar la solución a TA con agitación rápida durante 20-30 minutos. Para ayudar a solubilizar Mtz y para mejorar Mtz permeabilidad en las larvas, añada DMSO al preparar Mtz. PRECAUCIÓN: La exposición prolongada o aumento de la concentración de Mtz es tóxico. Use de acuerdo con las directrices de manejo apropiadas. </li><li> Separar las larvas en los grupos de control y experimentales mediante la transferencia de número deseado de larvas en dos diferentes 100 mm plato de Petri o placa de múltiples pocillos. Para el grupo experimental, mantener las larvas en 10 mM solución Mtz; para el grupo control, mantenerlos en agua huevo que contiene 0,2% de DMSO. </li><li> Cubrir las placas o platos de petri que contiene la solución Mtz con papel de aluminio para evitar la foto-inactivación de Mtz, y se incuba a 28 ° C. La duración del tratamiento Mtz depende de la etapa larval y la línea transgénica. </li><li> Observar la ablación hepatocitos severa similar en el <em&gt; Tg (fabp10a: PPC-NTR) de la línea, el tratamiento de las larvas con la solución Mtz ya sea 3,5 días después de la fertilización (dpf) (con una duración de 36 horas) o de 5 dpf (con una duración de 24 horas). </li></ol> 3. Análisis de la impulsada por biliar regeneración hepática <ol><li> Al final del periodo de ablación, eliminar la solución Mtz de las placas. Lavar las larvas tratadas-Mtz mediante la adición de 25 ml de agua huevo. Agitar la placa de 2-3 veces para lavar las larvas antes de desechar el agua de huevo. Repetir tres veces y luego sumergir las larvas una vez más en agua de huevo. Para evitar edema corazón y la muerte de las larvas en las larvas tratadas-Mtz, añadir una menor concentración de tricaína, a una concentración final de 0,008%, para inmovilizar las larvas. </li><li> Para el análisis de hígado, examinar el tamaño del hígado de las larvas tratadas con Mtz bajo un microscopio de epifluorescencia con el filtro de la PPC. Evaluar los niveles de ablación de hepatocitos en función del tamaño relativo del hígado: grande (no ablación; 0-10% larvas), medio (parcialmente ablación; 10-20%), y muy pequeña (totalmente ablación; 80-90%). </li><li> Retire las larvas con hígados no ablación parcial o ablación. Sólo mantener las larvas con una pequeña hígado, que es indicativa de la ablación de hepatocitos grave. Para el análisis de hígado en la regeneración 0 punto temporal hr (R0h), la cosecha de las larvas después de Mtz lavado y PPC clasificación. De lo contrario, mantenga las larvas ordenados en agua huevo suplementado con 0,2 mM PTU hasta el momento de la cosecha. </li><li> Traslado larvas recolectadas en un tubo de microcentrífuga de 1,5 ml y reemplazar el agua de huevo con la solución de fijación de 3% de formaldehído en tampón PEM. Incubar el tubo en un rotador O / N a 4 ° C. </li><li> Reemplazar la solución de fijación con 1 ml de PBSDT y girar el tubo que contiene las larvas en el rotador durante 5 min. </li><li> Transfiera las larvas fijo en una placa de Petri de 100 mm que contiene PBSDT y el uso de fórceps, quitar manualmente yema y aletas pectorales bajo un microscopio de disección. Traslado hasta TP 20 larvas diseccionado en un tubo de 1,5 ml. </li> &lt;li&gt; Sustituir solución PBSDT con 1 ml de solución de bloqueo y el rock el tubo en un rotador a temperatura ambiente durante 2 horas. </li><li> Reemplazar solución de bloqueo con 100 l de solución de bloqueo que contiene anticuerpos primarios. Poco a poco el rock el tubo en un rotador O / N a 4 ° C. </li><li> Retirar la solución de anticuerpo primario y lavar con PBSDT 5 veces durante 10 min cada vez. A continuación, añadir 100 ul de solución de bloqueo que contiene anticuerpos secundarios y el rock en un rotador a temperatura ambiente durante 2 horas. </li><li> Lavar 5 veces con PBSDT de 10 min cada vez a RT oscilando en un rotador. </li><li> Orientar las larvas lateralmente sobre un portaobjetos de microscopio y añadir una gota de medio de montaje. Cubrir con cuidado con una cubierta de vidrio y sellar la cubierta de vidrio con un pulidor de uñas. Ahora, está listo para la microscopía confocal. </li></ol>

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Los autores declaran que no tienen intereses financieros en competencia.

Severa, pero no leve, la ablación de hepatocitos provoca la regeneración hepática biliar impulsada. Por lo tanto, después del tratamiento Mtz y de lavado, es importante para examinar el tamaño del hígado de las larvas tratadas-Mtz, que puede ser evaluada por fluorescencia intrínseca de la PPC fabp10a: CFP-NTR transgén. Desde un pequeño porcentaje de las larvas tratadas-Mtz mostrará no separada por ablación (0-5%) o parcialmente ablación (10-20%) hígados, es esencial para ordenar y sólo analizar la...

El hígado es un órgano altamente regenerativa. Durante la regeneración del hígado, la regeneración de los hepatocitos, las células del parénquima del hígado, se derivan de los hepatocitos preexistentes (regeneración hepática hepatocitos impulsada) o BEC (regeneración hepática biliar impulsada) 1,2. La lesión hepática por lo general provoca la proliferación de los hepatocitos preexistentes; sin embargo, cuando se ve comprometida la proliferación de hepatocitos, las BEC puede contribuir a los hepatocitos 2-4. Estos dos modos de regeneración del hígado son clínicamente significativos. Tras la eliminación quirúrgica de una porción del hígado humano (por ejemplo, a causa de tumores en el hígado o donantes de hígado vivos), los hepatocitos en el hígado restante proliferan para recuperar la masa hepática perdido. Por el contrario, en pacientes con enfermedades hepáticas graves, la proliferación de hepatocitos se ve comprometida en gran medida, de modo que las BEC o células progenitoras del hígado (LPC) parecen contribuir a la regeneración de los hepatocitos 5,6. El modelo de roedor 2/3 hepatectomía parcial, en la quehepatocitos proliferan para recuperar la masa hepática perdido, ha contribuido de manera significativa a la comprensión actual de hepatocitos impulsada por la regeneración del hígado 7,8. Sin embargo, no existe un modelo de roedores válida en la que los hepatocitos en regeneración se derivan principalmente de las BEC. Aunque varios modelos de roedores de la toxina del hígado condujeron a la identificación de biliar impulsada por la regeneración del hígado 2-4, los estudios de linaje rastreo recientes en ratones indican una contribución mínima de las BEC a la regeneración de los hepatocitos en estos modelos 9,10. Algunos de los modelos de lesión del hígado de roedores, incluyendo la hepatectomía parcial 11-13 y daño hepático inducido por acetaminofeno-14,15, se han aplicado a pez cebra y llevado a la identificación de nuevos genes o vías implicadas en la regeneración del hígado. Sin embargo, impulsado hepatocitos, pero no conducido-BEC, la regeneración del hígado se produce en estos modelos de lesión de hígado de pez cebra. Por lo tanto, un modelo de lesión hepática novela en la que BEC contribuyen ampliamente a regenerating hepatocitos es necesario para una mejor comprensión de la regeneración del hígado impulsado por la BEC. Los objetivos generales del modelo de ablación hepatocitos describen aquí son (1) para generar un modelo de lesión hepática en la que BEC contribuyen ampliamente a los hepatocitos en regeneración, y (2) a dilucidar los mecanismos moleculares y celulares que subyacen a la regeneración del hígado impulsado por la BEC. La hipótesis de que la gravedad de la lesión determina el modo de regeneración del hígado; por lo tanto, predijo que la regeneración hepática biliar impulsada iniciaría tras la lesión del hepatocito severo. Para probar esta hipótesis, hemos desarrollado un modelo de lesión hepática pez cebra mediante la generación de una línea transgénica, Tg (fabp10a: PPC-NTR) S931, que expresa altamente nitrorreductasa bacteriana (NTR) fusionada con la proteína fluorescente cian (PPC) en el marco del fabp10a específico de hepatocitos promotor. Desde NTR convierte el profármaco no tóxico, metronidazol (Mtz), en un fármaco citotóxico, se extirpa sólo el destinado NTR-las células que expresan 16-18, en este caso, los hepatocitos. Mediante la manipulación de la duración del tratamiento Mtz, la extensión de la ablación de los hepatocitos puede ser controlada. El uso de este modelo, se informó recientemente que en caso de pérdida de hepatocitos grave, BEC ampliamente dan lugar a hepatocitos en regeneración 19, lo que fue confirmado por otros dos estudios independientes 20,21. Por lo tanto, en comparación con el roedor antes mencionado y modelos de lesión de hígado de pez cebra, nuestro modelo de ablación de hepatocitos es más ventajoso para el estudio de la regeneración del hígado impulsado por la BEC. Este protocolo se describe el procedimiento para llevar a cabo experimentos de regeneración hepática mediante el modelo de ablación hepatocitos pez cebra. Este modelo será apropiado para determinar los mecanismos subyacentes a la regeneración hepática biliar impulsada y para pantallas químicos para identificar pequeñas moléculas que pueden reprimir o aumentar la regeneración del hígado.

<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

El hígado tiene una gran capacidad de regeneración. Los hepatocitos, las células parenquimatosas del hígado, se puede regenerar en una de dos maneras: la regeneración del hígado hepatocyte- o biliar impulsada. En la regeneración de los hepatocitos del hígado impulsada, los hepatocitos en regeneración se derivan de hepatocitos preexistentes, mientras que, en la regeneración biliar impulsada, los hepatocitos en regeneración se derivan de las células epiteliales biliares (BEC). Para la regeneración del hígado hepatocitos impulsada, hay excelentes modelos de roedores que han contribuido de manera significativa a la comprensión actual de la regeneración del hígado. Sin embargo, no existe tal modelo de roedores para la regeneración hepática biliar impulsada. Recientemente hemos informado en un modelo de lesión hepática pez cebra en el que las BEC ampliamente dan lugar a hepatocitos en caso de pérdida de hepatocitos grave. En este modelo, los hepatocitos se ablación específicamente por un medio de farmacogenéticos. Aquí presentamos en detalle los métodos para la ablación de los hepatocitos y analizar el proceso de regeneración del hígado impulsado por la BEC.Este modelo de ablación específico de hepatocitos se puede utilizar más para descubrir los mecanismos moleculares y celulares subyacentes de la regeneración hepática biliar impulsada. Además, estos métodos pueden ser aplicados a las pantallas químicos para identificar pequeñas moléculas que aumentan o inhiben la regeneración del hígado.

Tratamiento Mtz para 36 hr (A36h, A significa ablación) 3,5 a 5 dpf reducido drásticamente el tamaño del hígado (Figura 1B). Después Mtz lavado, considerado como el comienzo de la regeneración del hígado (R), fuerte fabp10a: PPC-NTR y fabp10a: Expresión dsRed reapareció dentro de 30 horas (Figura 1B). La red biliar intrahepático, como se evaluó por la expresión de Alcam que está presente en la membrana de las BEC 22, colapsado inicialmente pero ...

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.

Ablación específico de hepatocitos en pez cebra para estudiar biliar impulsada regeneración hepática

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

Research

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JoVE Journal

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Cell Biology

Developmental Biology

Unit 1

Regeneration and Repair

SCIENTISTS IN ACTION

8.5K vistas.  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.

Video: 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.

La regeneración renal en adultos de pez cebra por gentamicina lesión inducida

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

Investigación

Biología del Desarrollo

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.

El uso del Sistema de Teton para estudiar los mecanismos moleculares de la regeneración de pez cebra

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.

Desarrollo de un Modelo de hígado fibrótico inducida por etanol en el pez cebra para el Estudio de células progenitoras mediada por la regeneración de hepatocitos

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.

La ablación quirúrgica de ensayo para estudio de la regeneración de los ojos en planarias

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.

Análisis histológicos de lesiones hepáticas alcohólicas agudas en peces cebra

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.

Métodos para el estudio de la regeneración en 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.

Cuantificación del tamaño del hígado en el pez cebra larvario mediante la microscopía 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.

Ensayo de regeneración y ablación láser basado en microscopio confocal en células interneuromas de peces cebra

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.

Examinando la regeneración muscular en modelos de pez cebra de enfermedad muscular

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

Diferenciación y estimulación mecánica de células endoteliales derivadas 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.

Autor destacado: Estudio de células endoteliales derivadas de hiPSC cultivadas bajo estimulación mecánica mediada por fluidos 

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

Ablación específico de hepatocitos en pez cebra para estudiar biliar impulsada regeneración hepática