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.

Peixe-zebra foram levantadas e produzido de acordo com o procedimento padrão; experimentos foram aprovados pela Universidade de Pittsburgh Animal Care Institucional e Comitê de Uso. 1. Preparação de Embriões / larvas <ol><li> Para realizar cruzamentos programados, configurar adulto do sexo masculino e feminino Tg (fabp10a: CFP-NTR) s931 peixes hemizigóticas ou homozigotos O / N e colocar um divisor entre eles. Remover este divisor na manhã seguinte quando o acasalamento é desejado. Coletar embriões esticando a água usando um filtro fino malha de plástico. </li><li> Ligue o filtro de cabeça para baixo e lavar a superfície da malha com um bom fluxo de água ovo dispensada de uma garrafa squeeze. Transferir 100 embriões em uma placa de 100 mm de petri com 25 ml de água ovo. Mantenha há mais de 100 embriões por placa de petri e criá-los a 28 ° C. </li><li> Para inibir a pigmentação, adicionar feniltioureia (PTU) para as placas de petri antes de 10 horas pós-fertilização (HPF), e levantar embryos / larvas até 80 hpf. A concentração final do PTU é de 0,2 mm. CUIDADO: PTU é tóxico. Use de acordo com as diretrizes de manejo adequadas. </li><li> Anestesiar as larvas por adição de 3-amino benzóico éster etílico do ácido (tricaina), a uma concentração final de 0,016%, em placas de petri. Em seguida, utilizando um microscópio de epifluorescência, larvas tipo PCP-positiva. Use larvas com um tamanho semelhante fígado e descartar aqueles com uma menor ou maior do fígado. NOTA: qualquer larvas PCP-positivo, independentemente de intensidade, pode ser utilizado porque não há nenhuma diferença na extensão da ablação de hepatócitos entre as larvas hemizigótico e homozigoto. CUIDADO: tricaina é tóxico. Use de acordo com as diretrizes de manejo adequadas. </li><li> Retire a água ovo contendo tricaina e adicione água ovo suplementado com 0,2 mM PTU. Manter a embriões / larvas a 28 ° C. </li></ol> 2. Tratamento Mtz <ol><li> Prepare fresco solução a 10 mM Mtz dissolvendo 0,171 g de pó em 1 Mtz00 ml de água de ovo suplementado com 0,2% de DMSO e 0,2 mM de UTP. Para dissolver completamente o Mtz, misturar a solução à temperatura ambiente com agitação rápida durante 20-30 minutos. Para ajudar a solubilizar Mtz Mtz e para melhorar a permeação para as larvas, adicione DMSO ao preparar Mtz. CUIDADO: A exposição prolongada ou aumento da concentração de Mtz é tóxico. Use de acordo com as diretrizes de manejo adequadas. </li><li> Separe as larvas em grupos controle e experimentais, transferindo número desejado de larvas em duas diferente 100 mm prato de petri ou multi-bem placa. Para o grupo experimental, manter as larvas em solução Mtz 10 mM; para o grupo controle, mantê-los em água ovo contendo 0,2% de DMSO. </li><li> Cobrir as placas ou pratos de petri contendo a solução Mtz com folha de alumínio para evitar o foto-inactivação da MTZ, e incuba-se a 28 ° C. A duração do tratamento depende da Mtz fase larval e a linha transgénica. </li><li> Para observar a ablação de hepatócitos grave semelhante no <em&gt; Tg (fabp10a: NTR-PCP) linha, tratar as larvas com a solução de Mtz ou 3,5 dias pós-fertilização (dpf) (para uma duração de 36 h) ou a partir de 5 dpf (para uma duração de 24 horas). </li></ol> 3. Análise da regeneração hepática biliar-driven <ol><li> No final do período de ablação, remover a solução Mtz das placas. Lavar as larvas tratadas com Mtz por adição de 25 ml de água ovo. Agitar a placa de 2-3 vezes para lavar as larvas antes de descartar a água do ovo. Repetir três vezes e, em seguida, mergulhar as larvas mais uma vez em água de ovo. Para evitar o edema do coração e a morte larval em larvas tratadas com Mtz, adicionar uma concentração mais baixa de tricaina, a uma concentração final de 0,008%, para imobilizar as larvas. </li><li> Para a análise do fígado, fígado examinar o tamanho das larvas tratadas com Mtz sob um microscópio de epifluorescência, com o filtro de PCP. Avaliar os níveis de ablação de hepatócitos com base no tamanho relativo do fígado: grande (não-ablação; 0-10% de larvas), forma (parcialmente ablação; 10-20%), e muito pequena (totalmente ablação; 80-90%). </li><li> Retirar as larvas com fígados não ablacionadas ou parcialmente extirpadas. Apenas manter larvas com uma pequena fígado, o que é indicativo de ablação hepatócitos grave. Para a análise de fígado na regeneração 0 ponto de tempo hr (R0h), colher as larvas após Mtz washout e PCP classificação. Caso contrário, mantenha as larvas classificadas em água ovo suplementado com PTU 0,2 mM até que esteja pronto para a colheita. </li><li> Transferir larvas recolhidas para um tubo de microcentrífuga de 1,5 ml e substituir a água de ovo com a solução de fixação de formaldeído a 3% em tampão PEM. Incubar o tubo num rotor O / N a 4 ° C. </li><li> Substituir a solução de fixação com 1 ml de PBSDT e rodar o tubo contendo as larvas no rotor durante 5 min. </li><li> Transferir as larvas fixo numa placa de Petri contendo 100 milímetros PBSDT usando uma pinça e, remover manualmente gema e barbatanas peitorais sob um microscópio de dissecação. Transfira até tp 20 larvas dissecado em um tubo de 1,5 ml. </li> &lt;li&gt; Substitua PBSDT solução com 1 ml de solução de bloqueio e balançar o tubo num agitador rotativo à temperatura ambiente durante 2 h. </li><li> Substituir solução de bloqueio com 100 ul de solução contendo os anticorpos primários de bloqueio. Lentamente balançar o tubo num rotor O / N a 4 ° C. </li><li> Remover a solução de anticorpo primário e lava-se com PBSDT 5 vezes durante 10 min de cada vez. Em seguida, adiciona-se 100 ul de solução de bloqueio contendo anticorpos secundários e rocha que num rotor a TA durante 2 h. </li><li> Lavar cinco vezes com PBSDT durante 10 min de cada vez, à TA por balanço num rotor. </li><li> Orientar as larvas lateralmente sobre uma lâmina de microscópio e adicionar uma gota de meio de montagem. Cobri-los cuidadosamente com uma tampa de vidro e selar a tampa de vidro com um esmalte de unha. Agora, ele está pronto para a microscopia confocal. </li></ol>

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Os autores declaram que não têm interesses financeiros concorrentes.

Grave, mas não leve, ablação de hepatócitos provoca a regeneração do fígado-driven biliar. Por conseguinte, após o tratamento de lavagem e Mtz, é importante para examinar o tamanho do fígado das larvas tratadas com Mtz, que pode ser avaliada pela fluorescência intrínseca do PCP fabp10a: NTR-PCP transgene. Uma vez que uma pequena percentagem das larvas tratadas com Mtz exibirá não excisada (0-5%) ou parcialmente ablação (10-20%) os fígados, é essencial para resolver e apenas analisar as larvas ...

O fígado é um órgão altamente regenerativa. Durante a regeneração do fígado, regenerar hepatócitos, as células do parênquima do fígado, são derivados de hepatócitos pré-existentes (regeneração hepática-driven hepatócitos) ou BEC (regeneração hepática-driven biliar) 1,2. A lesão hepática geralmente provoca a proliferação de hepatócitos pré-existentes; no entanto, quando a proliferação de hepatócitos é comprometida, CEBs pode contribuir para hepatócitos 2-4. Estes dois modos de regeneração do fígado são clinicamente significativa. Após a remoção cirúrgica de uma porção do fígado humano (por exemplo, devido a tumores do fígado ou dadores vivos de fígado), hepatócitos no fígado proliferam restante para recuperar a massa do fígado perdida. Em contraste, em doentes com doenças hepáticas graves, a proliferação dos hepatócitos é grandemente comprometida, de modo a que as CEBs ou células progenitoras do fígado (LPCs) parecem contribuir para a regeneração de hepatócitos 5,6. O modelo de roedor 2/3 hepatectomia parcial, em quehepatócitos proliferam para recuperar a massa hepática perdido, tem contribuído significativamente para a compreensão atual de-driven hepatócito regeneração hepática 7,8. No entanto, não existe um modelo roedor válido no qual os hepatócitos em regeneração são derivados principalmente de CEBs. Embora vários modelos de toxina de roedor fígado levou à identificação de-driven biliar regeneração hepática 2-4, estudos recentes em rastreio de linhagem ratos indicam uma contribuição mínima de CEBs para regenerar hepatócitos nestes modelos 9,10. Alguns dos modelos de lesão de fígado de roedores, incluindo hepatectomia parcial 11-13 e induzida por acetaminofeno danos no fígado 14,15, ter sido aplicada a peixes-zebra e levou à identificação de novos genes ou vias envolvidas na regeneração do fígado. No entanto, hepatócito-driven, mas não BEC-driven, a regeneração do fígado ocorre nesses modelos de lesão do fígado de peixe-zebra. Portanto, um modelo de fígado romance em que BECs contribuir amplamente para se regeneraing hepatócitos é necessária para uma melhor compreensão da regeneração hepática BEC-driven. Os objetivos gerais do modelo de ablação de hepatócitos descritos aqui são (1) para gerar um modelo de fígado em que BECs contribuir amplamente para os hepatócitos em regeneração, e (2) elucidar os mecanismos moleculares e celulares subjacentes regeneração hepática BEC-driven. Nossa hipótese é que a gravidade da lesão determina o modo de regeneração do fígado; Assim, previmos que a regeneração hepática-driven biliar iniciaria após lesão hepática grave. Para testar esta hipótese, desenvolvemos um modelo de fígado de peixe-zebra, gerando uma linhagem transgênica, Tg (fabp10a: CFP-NTR) s931, que é altamente expressa nitrorredutase bacteriana (NTR) fundido com a proteína fluorescente ciano (PCP), sob a fabp10a específicas do hepatócito promotor. Desde NTR converte a pró-droga não tóxica, metronidazol (Mtz), em um fármaco citotóxico, ele destina-se somente a ablates NTR-células que expressam 16-18, neste caso, os hepatócitos. Ao manipular a duração do tratamento Mtz, a extensão da ablação de hepatócitos pode ser controlada. Usando esse modelo, que recentemente informou que após a perda de hepatócitos grave, BECs extensivamente dar origem a regeneração hepatócitos 19, que foi ainda confirmada por outros dois estudos independentes 20,21. Portanto, em comparação com o acima mencionado roedor e modelos de lesão de fígado de peixe-zebra, o nosso modelo de ablação dos hepatócitos é mais vantajoso para estudar a regeneração hepática BEC-dirigido. Este protocolo descreve o procedimento para a realização de experimentos de regeneração hepática utilizando o modelo de ablação de hepatócitos peixe-zebra. Este modelo será adequado para determinar os mecanismos subjacentes a regeneração hepática-driven biliar e para telas químicas para identificar pequenas moléculas que pode reprimir ou aumentam a regeneração do fígado.

<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

O fígado tem uma grande capacidade para se regenerar. Os hepatócitos, as células parenquimatosas do fígado, pode regenerar em uma de duas maneiras: a regeneração hepática hepatocyte- ou accionada por biliar. Na regeneração hepática-driven hepatócitos, os hepatócitos em regeneração são derivados de hepatócitos pré-existentes, ao passo que, na regeneração-driven biliar, hepatócitos em regeneração são derivadas de células epiteliais das vias biliares (CEBs). Para a regeneração hepática-driven hepatócito, existem modelos de roedores excelentes que têm contribuído significativamente para a compreensão atual da regeneração hepática. No entanto, não existe tal modelo de roedor existe para regeneração hepática-driven biliar. Nós recentemente relatado em um modelo de fígado de peixe-zebra em que BECs extensivamente dar origem a hepatócitos após a perda de hepatócitos grave. Neste modelo, os hepatócitos são especificamente ablação por um meio de farmacogenética. Aqui nós apresentamos em detalhes os métodos para retirar hepatócitos e analisar o processo de regeneração hepática BEC-driven.Este modelo de ablação específica do hepatócito pode ser ainda usado para descobrir os mecanismos moleculares e celulares subjacentes da regeneração hepática-driven biliar. Além disso, estes métodos podem ser aplicados para telas químicas para identificar moléculas pequenas que aumentam ou suprimem a regeneração do fígado.

MTZ tratamento durante 36 h (A36h, A representa ablação) 3,5-5 dpf reduzida drasticamente o tamanho do fígado (Figura 1B). Depois Mtz washout, considerado como o início de regeneração hepática (R), forte fabp10a: CFP-NTR e fabp10a: expressão dsRed reapareceu no prazo de 30 horas (Figura 1B). A rede biliar intra-hepático, tal como avaliado por expressão de Alcam que está presente na membrana de CEBs 22, mas inicialmente recolhido foi restabelecida ...

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.

Ablação específicas do hepatócito em Zebrafish para estudar a regeneração do fígado-driven biliar

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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Regeneration and Repair

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8.5K visualizações.  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.

Vídeo: 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.

A regeneração renal em Zebrafish Adulto por lesão induzida pela gentamicina

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

Biologia do Desenvolvimento

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.

Uso do Sistema de Teton para estudar os mecanismos moleculares de Regeneração Zebrafish

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.

Desenvolvimento de um modelo de fígado fibrótica induzida por etanol no peixe-zebra para estudar Progenitor mediada por células de regeneração de hepatócitos

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.

Surgical Ablation Ensaio para Estudar Eye Regeneração em Planárias

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.

Análises histológicas de lesão hepática alcoólica aguda em peixe-zebra

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 o estudo da regeneração em 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.

Quantificando o tamanho do fígado em zebrafish larval usando microscopia de campo brilhante

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.

Ablação e Regeneração a laser baseada em microscópio confocal em células interneuromast baseadas em 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.

Examinando a regeneração muscular em modelos de zebrafish de doença muscular

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

Diferenciação e estimulação mecânica de células endoteliais 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 em destaque: Estudando células endoteliais derivadas de hiPSC cultivadas sob estimulação mecânica 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.