Name: development of an ethanol-induced fibrotic liver model in zebrafish to study progenitor cell-mediated hepatocyte regeneration
Uploaded: 2016-05-13T14:00:00
Description: Watch this Scientific Journal Video about Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration at JoVE.com

This work was supported in part by grants from the GTEC (2731336 and 1411318), the NIH (K01DK081351), and the NSF (1354837) to C. H. S. We thank Alem Giorgis for critical reading of the manuscript.

Zebrafish were raised and bred using a standard protocol that meets the criteria of the National Institutes of Health and approved by the Georgia Institute of Technology Institutional Animal Care and Use Committee.&#160;
1. Preparation of Solutions
<ol>
	<li>Prepare 20 L egg water (interchangeably used with &#8216;embryo medium&#8217;)&#160;&#160;to maintain embryonic/larval zebrafish. Dissolve 1.5 g CaSO4 and 6 g instant ocean sea salt in 250 ml distilled water. Pour into a carboy filled with 20 L di...

<ol>
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P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+liver+fibrosis+in+rodents.">Modeling liver fibrosis in rodents.</a> Methods Mol Med. 117, 237-250 (2005).</li><li>Gabele, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+model+of+interactive+effects+of+alcohol+and+high-fat+diet+on+hepatic+fibrosis.">A new model of interactive effects of alcohol and high-fat diet on hepatic fibrosis.</a> Alcohol Clin Exp Res. 35 (7), 1361-1367 (2011).</li><li>Lieber, C. 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R., Saxena, M. T., Mumm, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+zebrafish+chemical+screening+technologies.">Advances in zebrafish chemical screening technologies.</a> Future Med Chem. 4 (14), 1811-1822 (2012).</li><li>Chen, C. H., Durand, E., Wang, J., Zon, L. I., Poss, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=zebraflash+transgenic+lines+for+in+vivo+bioluminescence+imaging+of+stem+cells+and+regeneration+in+adult+zebrafish.">zebraflash transgenic lines for in vivo bioluminescence imaging of stem cells and regeneration in adult zebrafish.</a> Development. 140 (24), 4988-4997 (2013).</li><li>Westhoff, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+an+automated+imaging+pipeline+for+the+analysis+of+the+zebrafish+larval+kidney.">Development of an automated imaging pipeline for the analysis of the zebrafish larval kidney.</a> PLoS One. 8 (12), e82137 (2013).</li><li>Perlman, Z. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multidimensional+drug+profiling+by+automated+microscopy.">Multidimensional drug profiling by automated microscopy.</a> Science. 306 (5699), 1194-1198 (2004).</li><li>Chu, J., Sadler, K. 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We observed HPC-mediated hepatocyte regeneration in the EtOH/MTZ-treated recovering livers, suggesting that even in the presence of substantial amount of ECM proteins including fibrillar type I collagen, the HPCs retain their competency to regenerate as hepatocytes. The MTZ only-treatment did not increase deposition of ECM proteins significantly, whereas the EtOH only-treatment did not induce HPC activation15. By utilizing the combined EtOH/MTZ treatment, we were able to investigate HPC-driven regeneration in ...

Despite the remarkable regeneration capacity of hepatocytes1, which are the major parenchymal cell type of the liver, chronic liver failure impairs this ability, leading to hepatic progenitor cell (HPC)-dependent regeneration2.
Chronic liver damage is mainly derived from alcohol abuse, chronic hepatitis C virus (HCV) infection3 and non-alcoholic fatty liver disease (NAFLD)4. It leads to sustained liver fibrosis, which is associated with the accumulation of extracellular matrix (ECM) proteins. Persisting ECM accumulation distorts intact hepatic architecture by forming a fibrous scar tissue5, subsequently resulting in cirrhosis with high morbidity and mortality. Many attempts have been made to mitigate the fibrotic response mainly by focusing on inhibiting profibrogenic cytokines and activated myofibroblasts6. The latter is primarily derived from hepatic stellate cells (HSCs), the principle hepatic non-parenchymal cells responsible for liver scar formation4. Nevertheless, regenerative therapies that stimulate endogenous cellular sources including HPCs to regenerate hepatocytes in the presence of sustained fibrogenic insults await further investigation.
Many experimental models of hepatic fibrosis have been described in mammals. Repetitive injection of carbon tetrachloride (CCl4) has been widely used to induce liver fibrosis in murine and rat models7. When combined with a high-fat (HF) diet, alcohol led to a substantial upregulation of profibrogenic gene expression and hepatic fibrosis8. While steatosis (lipid accumulation) results from acute alcohol exposure, it makes the liver susceptible to more severe hepatic injury9.
The zebrafish, Danio rerio, has emerged as an invaluable vertebrate model system for studying regeneration. Though other lower vertebrates such as newts and axolotls have a remarkable capacity for regeneration, the zebrafish has advantages over other model systems in regards to the gene manipulation and visualization strategies needed to manipulate potential regenerative factors10. The zebrafish also represents an attractive vertebrate model for studying alcoholic liver disease (ALD) by simply adding ethanol (EtOH) to their water. Acute EtOH exposure to larval and adult zebrafish caused hepatic steatosis11-13. When adult zebrafish received extended EtOH exposure, collagen deposition was observed with upregulation of fibrosis-related genes14. However, a need exists for developing models to study liver regeneration in response to EtOH as a fibrogenic stimulus.
Recently, we developed an EtOH-induced fibrotic liver model in zebrafish15. We combined a hepatocyte-specific genetic ablation system with EtOH treatment in larval and adult zebrafish. We generated two transgenic lines, Tg(fabp10a:CFP-NTR)gt1 and Tg(fabp10a:mCherry-NTR)gt2, in which E.coli nitroreductase (NTR) are fused to the cyan and mCherry fluorescent protein, respectively, under the control of the hepatocyte-specific fatty acid binding protein 10a, liver basic (fabp10a) promoter. In this system, NTR converts a nontoxic prodrug metronidazole (MTZ) into a DNA inter-strand cross-linking agent16, inducing explicit death of hepatocytes. Using this model, we demonstrated that a population of hepatic cells, which are responsive to Notch signaling, converted into hepatocytes in the near absence of hepatocytes and in the excess of ECM. We designated these cells as HPCs. Furthermore, through chemical screens, we identified small molecule activators of Wnt signaling and inhibitors of Notch signaling that augment hepatocyte regeneration in the fibrotic liver. Therefore, our fibrotic liver model in zebrafish represents a superb chemical screening system compared to cell culture- or mammalian-based screening system. It is an in vivo system with significant cost- and time-saving benefits. Here we describe the detailed procedures for establishing an EtOH-induced fibrotic liver model and for performing chemical screens using this model in zebrafish. Furthermore, time-course analyses were performed to investigate how hepatocyte regeneration occurs in the fibrotic liver. This protocol will provide an invaluable tool to study the mechanisms and strategies of enhancing hepatocyte regeneration in the fibrotic liver.

<table>
	<tbody>
		<tr>
			<td>Calcium sulfate hemihydrate (CaSO4)</td>
			<td>Acros</td>
			<td>AC385355000</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium sulfate (MgSO4)</td>
			<td>EMD</td>
			<td>MX0075</td>
			<td></td>
		</tr>
		<tr>
			<td>1,4-Piperazinediethanesulfonic acid (PIPES)</td>
			<td>Sigma-Aldrich</td>
			<td>P6757</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylene glycol-bis(2-aminoethylether)-N,N,N&#8242;,N&#8242;-tetraacetic acid (EGTA)</td>
			<td>Sigma-Aldrich</td>
			<td>E3889</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>E7023</td>
			<td>200 proof</td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Fisher Scientific</td>
			<td>F79-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole (MTZ)</td>
			<td>Sigma-Aldrich</td>
			<td>M3761</td>
			<td></td>
		</tr>
		<tr>
			<td>1-phenyl-2-thiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine)</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphate-buffered saline (PBS) tablet</td>
			<td>Amresco</td>
			<td>E404</td>
			<td>Dissolve one tablet with 100 ml distilled water</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D2438</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine serum albumin</td>
			<td>Fisher Scientific</td>
			<td>BP1600</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher Scientific</td>
			<td>BP151</td>
			<td></td>
		</tr>
		<tr>
			<td>Low-melting agarose&#160;</td>
			<td>Amresco</td>
			<td>BP165</td>
			<td></td>
		</tr>
		<tr>
			<td>Stem Cell Signaling Compound Library</td>
			<td>Selleck Chemicals</td>
			<td>L2100</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>ActiProbe-1K Library</td>
			<td>Timtec</td>
			<td>ActiProbe-1K</td>
			<td>10mM stock in DMSO</td>
		</tr>
		<tr>
			<td>SB 415286</td>
			<td>Selleck Chemicals</td>
			<td>S2729</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>CHIR-99021</td>
			<td>Selleck Chemicals</td>
			<td>S2924</td>
			<td>Dissolve with DMSO to 10mM</td>
		</tr>
		<tr>
			<td>Anti-Collagen I antibody</td>
			<td>Abcam</td>
			<td>ab23730</td>
			<td>Use at 1:100 for immunostaining, reacts with fish</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 Donkey anti-rabbit IgG (H+L)</td>
			<td>Molecular Probes</td>
			<td>A31573</td>
			<td>Use at 1:200 for immunostaining</td>
		</tr>
		<tr>
			<td>Mounting media (Vectorshield)</td>
			<td>Vector Laboratories</td>
			<td>H-1400</td>
			<td></td>
		</tr>
		<tr>
			<td>100 mm petri dish</td>
			<td>VWR</td>
			<td>25384-088</td>
			<td></td>
		</tr>
		<tr>
			<td>24-well plate</td>
			<td>VWR</td>
			<td>10062-896</td>
			<td></td>
		</tr>
		<tr>
			<td>Forceps</td>
			<td>Fine Science Tools</td>
			<td>11255-20</td>
			<td>Dumont #55</td>
		</tr>
		<tr>
			<td>Glass slide</td>
			<td>VWR</td>
			<td>48312-003</td>
			<td>75x25 mm</td>
		</tr>
		<tr>
			<td>Cover glass</td>
			<td>VWR</td>
			<td>48366-045</td>
			<td>18 mm</td>
		</tr>
		<tr>
			<td>Plastic wrap</td>
			<td>Fisher Scientific</td>
			<td>22305654</td>
			<td></td>
		</tr>
		<tr>
			<td>Aluminum foil</td>
			<td>Fisher Scientific</td>
			<td>1213100</td>
			<td></td>
		</tr>
		<tr>
			<td>Kimwipes</td>
			<td>Kimberly-Clark</td>
			<td>34155</td>
			<td></td>
		</tr>
		<tr>
			<td>Vibrotome</td>
			<td>Leica</td>
			<td>VT1000 S</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereo microscope</td>
			<td>Leica</td>
			<td>M80</td>
			<td></td>
		</tr>
		<tr>
			<td>Epifluoresent microscope</td>
			<td>Leica</td>
			<td>M205 FA</td>
			<td></td>
		</tr>
		<tr>
			<td>Confocol microscope</td>
			<td>Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

Figure 1 shows the development of an EtOH-induced fibrotic liver model in larval zebrafish. To optimize a protocol for exposing zebrafish larvae to EtOH, we first assessed EtOH toxicity. 2.5 days-post-fertilization (dpf) larvae were exposed to EtOH concentration 1%, 1.5%, or 2% for 24 hr followed by a concurrent 24 hr EtOH/MTZ treatment. Exposure to 2% EtOH caused high mortality, while nearly all larvae exposed to 1% EtOH or less showed minimal fibrogenic changes with rar...

Watch this Scientific Journal Video about Development of an Ethanol-induced Fibrotic Liver Model in Zebrafish to Study Progenitor Cell-mediated Hepatocyte Regeneration at JoVE.com

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

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.

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

The overall goal of this protocol is to establish an Ethanol-induced Fibrotic Liver Model in Zebrafish and to perform chemical screens using this model. This method can help answer key questions in the liver regeneration field. Such as the molecular and cellular mechanisms of how hepatocytes regenerate in the fibrotic liver.The main advantage of this technique, is that it is a cost effective and time saving in vevo chemical screening. Furthermore, a detailed time course analysis can be performed through visualization of a single cell. Demonstrating the procedure will be Mianbo Huang, a postdoc, and Jin Xu, a graduate student from the laboratory.After preparing reagents according to the text protocol, set up mating tanks with dividers using adult transgenic Zebrafish containing fatty acid binding protein 10a:CFP-NTR with Tp1:mCherry and Tg(hand2:EGFP)Zebrafish. The following morning, two hours after removing the dividers, harvest the embryos by straining the system water and transferring the embryos into 100 millimeter petri dishes containing embryo medium. At 56 hours post fertilization, or HPF, add 1.5 percent ethanol to the embryos.Use plastic wrap to cover the plates to prevent an ethanol evaporation. Then incubate at 28 degrees Celsius for 24 hours. Prepare fresh 15 millimolar MTZ and embryo medium with 0.1 percent DMSO and add ethanol to a final concentration of 1.5 percent.Then, at 80 HPF, treat the sorted embryos with 20 milliliters of ethanol MTZ solution per petri dish. Use plastic wrap to cover the plates to prevent ethanol evaporation and cover with foil to protect from light. Then incubate at 28 degrees Celsius for 24 hours.The following day, observe the embryos under an epifluorescent microscope using a CFP filter and a magnification of 80x to sort out the larvae with near complete ablation of hepatocytes. To carry out chemical screens, remove the 96 well plates containing ten millimolar chemical stocks in DMSO from the negative 80 degree freezer. Use aluminum foil to cover the plates to prevent photocatalytic degradation of the chemicals and thaw with gentle rocking at room temperature.In 1.5 milliliter tubes, prepare chemical solutions by diluting five microliters of ten millimolar stock solutions with one milliliter of embryo medium to a final concentration of 50 micromolar. Next, transfer five larvae per well with near complete hapatocyte ablation to 24 well plates filled with embryo medium. Then, remove the embryo medium and add 500 microliters of the chemical solutions to each well.With aluminum foil, cover the plates and incubate the larvae at 28 degrees Celsius for 50 hours. After the incubation, observe the number and/or intensity of CFP-expressing hepatocytes under an epifluoesent microscope at 80x magnification. Image live larvae with enhanced or reduced numbers and/or intensity of CFP-expressing hapatocytes.Set up matings of transgenic Zebrafish containing fatty acid binding protein 10a:CFP-NTR with TP1:mCherry and raise the sorted larvae to six to 12 months old. Use a net to transfer the adult transgenic Zebrafish to mating tanks. Prepare fresh one percent ethanol in system water then use 500 milliliters of the solution to replace the system water in the mating tanks.Incubate the tanks at 28 degrees Celsius for 72 hours, transferring fish into fresh ethanol solution and removing any dead fish daily during the incubation. Prepare fresh ten millimolar MTZ solution in system water with 0.1 percent DMSO, then add 500 milliliters of pre-warmed MTZ solution to fish in one liter mating tanks. Use aluminum foil to cover the tanks.Incubate at 28 degrees Celsius for eight hours and analyze according to the text protocol. After anesthetizing according to the text protocol, expose the gastrointestinal organs by using scissors to cut the skin and underlining muscle along the belly from the anal fin to the operculum. Then cut posteriorly along the side of the fish back to the anal fin.Place the fish into a fifteen milliliter conical tube and use PBS to wash once. Then remove the PBS and add four milliliters of freshly prepared three percent formaldehyde in PEM buffer. Incubate at four degrees Celsius overnight to fix the fish.The following day, after washing the samples according to the text protocol, cut the esophagus to dissect the whole gut with the liver from the body cavity. Then place the gastrointestinal organs in a sectioning mold and use four percent low melting agarose to embed the tissue. Next, trim the block into a trapezoid shape and glue it to the base.Then use a vibratome and starting from the interior end of the tissue, cut samples into transverse sections at fifty micrometer intervals to ice-cold PBS. Use a number one paintbrush to transfer the sections to a six well plate with PBS. After immunostaining the sections according to the text protocol, use a number one paintbrush to gently transfer the sections to glass slides.Use kimwipes to remove excess PBS before adding a drop of mounting medium and a cover slip. Then use nail polish to seal the cover glass. Finally, using a confocal system equipped with a 40x 1.3 NA oil lens, capture Z-Stack images at 20 to 50 percent strength and one microammeter intervals.This figure shows that ethanol MTZ treated larvae develop upward curvature of the trunk and tail, paricardialidema, and failure to inflate the swim bladder. As seen here, DMSO treated control livers showed no fibular type one collagen deposition. Whereas ethanol MTZ treated livers displayed elevated type one collagen accumulation at 25 and 50 hours post ablation.In addition, compared to DMSO treated controlled livers, HSC's increased in number and adopted a myofibrillblast-like shape lacking cytoplasmic processes in the ethanol MTZ treated regenerating livers. This figure shows the development of an ethanol induced fibrotic liver model in adult Zebrafish treated with one percent ethanol followed by MTZ. From these time course analyses, significantly elevated type one collagen deposition in the ethanol MTZ treated regenerating livers at two, three, and four days post ablation was observed, as compared to DSMO treated livers.In this chemical screening experiment, hepatocytes were ablated in the presence of 1.5 percent ethanol and 15 millimolar MTZ. And the larvae were then exposed to chemicals for 50 hours. A number of wint agonists, such as inhibitors of glycogen synthase kinase-3 and a wint activator, promoted hepatocyte regeneration compared to DMSO.These data suggest that this sustained fibrotic model provides an invaluable tool to discover small molecules that enhance hepatocyte regeneration. Once mastered, this technique can be done in one week if it is performed properly. While attempting this procedure, it's important to remember to achieve near complete ablation of hepatocytes and to maintain the ethanol concentration throughout the procedure.Following this procedure, the efficiency of the identified chemical compounds can be tested in mammalian liver injury models. After its development, this technique can open up new avenues in the field of cellular regeneration therapy to explore the potential discovery of novel therapeutic strategies for chronic liver failure patients. After watching this video, you should have a good understanding of how to develop an ethanol induced fibrotic liver model in Zebrafish and how to perform chemical screens.Don't forget that working with formaldehyde can be extremely hazardous and precautions, such as wearing gloves and handling the reagent in a chemical fume hood, should always been taken while performing this procedure.

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Research

JoVE Journal

Developmental Biology

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.

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

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

The liver has a great capacity to regenerate. Hepatocytes, the parenchymal cells of the liver, can regenerate in one of two ways: hepatocyte- or biliary-driven liver regeneration. In hepatocyte-driven liver regeneration, regenerating hepatocytes are derived from preexisting hepatocytes, whereas, in biliary-driven regeneration, regenerating hepatocytes are derived from biliary epithelial cells (BECs). For hepatocyte-driven liver regeneration, there are excellent rodent models that have significantly contributed to the current understanding of liver regeneration. However, no such rodent model exists for biliary-driven liver regeneration. We recently reported on a zebrafish liver injury model in which BECs extensively give rise to hepatocytes upon severe hepatocyte loss. In this model, hepatocytes are specifically ablated by a pharmacogenetic means. Here we present in detail the methods to ablate hepatocytes and to analyze the BEC-driven liver regeneration process. This hepatocyte-specific ablation model can be further used to discover the underlying molecular and cellular mechanisms of biliary-driven liver regeneration. Moreover, these methods can be applied to chemical screens to identify small molecules that augment or suppress 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.

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

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. Although many efforts have been made to reveal the underlying mechanisms of decidualization, the exact signaling between the epithelial cells that are in contact with the embryo and the underlying stromal cells remains poorly understood. Therefore, studying decidualization in a way that takes both the epithelial and stromal cells into account could improve our knowledge about the molecular details of decidualization. For this purpose, in vivo models of artificial decidualization are physiologically the most relevant; however, manipulation of intercellular communication is limited. Currently, in vitro cultures of endometrial stromal cells are being used to investigate the modulation of decidualization by several signaling molecules. Conventionally, human or mouse endometrial stromal cells are used. However, the availability of human samples is very often limited. Furthermore, the use of murine tissues is accompanied with variety in the method of culturing. This study presents a validated and standardized method to obtain pure Endometrial Epithelial Cell (EEC) and Stromal Cell (ESC) cultures using adult intact mice treated with estrogen for three consecutive days. The protocol is optimized to improve the yield, viability, and purity of the cells and was further extended in order to study decidualization in a coculture of EEC and ESC. This model may be suitable to exploit the importance of both cell types in decidualization and to evaluate the contribution of significant signaling molecules secreted by EEC or ESC during the intercellular communication.

Isolation of Mouse Endometrial Epithelial and Stromal Cells for In Vitro Decidualization

This study presents a standardized and validated method for the isolation and culture of primary mouse endometrial stromal and epithelial cells, which can be used in a coculture system to study in vitro decidualization.

Decidualization is a progesterone-dependent differentiation process of endometrial stromal cells and is a prerequisite for successful embryo implantation. ...

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.

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 ...

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding and invasion. Current animal models for the study of T. cruzi allow limited observation of parasites in vivo, representing a challenge for understanding parasite behavior during the initial stages of infection in humans. This protozoan has a flagellar stage in both vector and mammalian hosts, but there are no studies describing its motility in vivo.The objective of this project was to establish a live vertebrate zebrafish model to evaluate T. cruzi motility in the vascular system. Transparent zebrafish larvae were injected with fluorescently labeled trypomastigotes and observed using light sheet fluorescence microscopy (LSFM), a noninvasive method to visualize live organisms with high optical resolution. The parasites could be visualized for extended periods of time due to this technique&#39;s relatively low risk of photodamage compared to confocal or epifluorescence microscopy. T. cruzi parasites were observed traveling in the circulatory system of live zebrafish in different-sized blood vessels and the yolk. They could also be seen attached to the yolk sac wall and to the atrioventricular valve despite the strong forces associated with heart contractions. LSFM of T. cruzi-inoculated zebrafish larvae is a valuable method that can be used to visualize circulating parasites and evaluate their tropism, migration patterns, and motility in the dynamic environment of the cardiovascular system of a live animal.

Establishment of Larval Zebrafish as an Animal Model to Investigate Trypanosoma cruzi Motility In Vivo

In this protocol, fluorescently labeled T. cruzi&#160;were injected into transparent zebrafish larvae, and parasite motility was observed in vivo using light sheet fluorescence microscopy.

Chagas disease is a parasitic infection caused by Trypanosoma cruzi, whose motility is not only important for localization, but also for cellular binding ...

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells (HSPCs) in Developing Zebrafish Embryos

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell lineages that comprise mature blood. Isolation and identification of these HSPCs is difficult because they are defined ex post facto; they can only be defined after their differentiation into specific cell lineages. Over the past few decades, the zebrafish (Danio rerio) has become a model organism to study hematopoiesis. Zebrafish embryos develop ex utero, and by 48 h post-fertilization (hpf) have generated definitive HSPCs. Assays to assess HSPC differentiation and proliferation capabilities have been developed, utilizing transplantation and subsequent reconstitution of the hematopoietic system in addition to visualizing specialized transgenic lines with confocal microscopy. However, these assays are cost prohibitive, technically difficult, and time consuming for many laboratories. Development of an in vitro model to assess HSPCs would be cost effective, quicker, and present fewer difficulties compared to previously described methods, allowing laboratories to quickly assess mutagenesis and drug screens that affect HSPC biology. This novel in vitro assay to assess HSPCs is performed by plating dissociated whole zebrafish embryos and adding exogenous factors that promote only HSPC differentiation and proliferation. Embryos are dissociated into single cells and plated with HSPC-supportive colony stimulating factors that cause them to generate colony forming units (CFUs) that arise from a single progenitor cell. These assays should allow more careful examination of the molecular pathways responsible for HSPC proliferation, differentiation, and regulation, which will allow researchers to understand the underpinnings of vertebrate hematopoiesis and its dysregulation during disease.

Development of an In Vitro Assay to Quantitate Hematopoietic Stem and Progenitor Cells HSPCs in Developing Zebrafish Embryos

Here, we present a simple method to quantitate hematopoietic stem and progenitor cells (HSPCs) in embryonic zebrafish. HSPCs from dissociated zebrafish are plated in methylcellulose with supportive factors, differentiating into mature blood. This allows the detection of blood defects and allows drug screening to be easily conducted.

Hematopoiesis is an essential cellular process in which hematopoietic stem and progenitor cells (HSPCs) differentiate into the multitude of different cell ...

Partial Lobular Hepatectomy: A Surgical Model for Morphologic Liver Regeneration

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult liver regeneration after 70% partial hepatectomy results in hepatocyte hypertrophy with some replication in remaining lobes with restoration of metabolic activity, but with permanent loss of the injured lobe&#39;s morphology and architecture. Here, we detail a new surgical method in the neonate that leaves a physiologic environment conducive to regeneration. This model involves amputation of the left lobe apex and a subsequent conservative management regimen, and lacks the necessity for ligation of major liver vessels or chemical injury, leaving a physiologic environment where regeneration may occur. We extend this protocol to amputations on juvenile (P7-14) mice, during which the injured liver transitions from organ regeneration to compensatory growth by hypertrophy. The presented, brief 30 min protocol provides a framework to study the mechanisms of regeneration, its age-associated decline in mammals, and the characterization of putative hepatic stem or progenitors.

Here, we present a new method for partial resection of the left hepatic lobe in neonatal (day 0) mice. This new protocol is suitable for studying acute liver injury and injury response in the neonatal setting.

Morphological organ regeneration following acute tissue loss is common among lower vertebrates, but is rarely observed in mammalian postnatal life. Adult ...

Whole-Mount In Situ Hybridization in Zebrafish Embryos and Tube Formation Assay in iPSC-ECs to Study the Role of Endoglin in Vascular Development

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in zebrafish during different developmental stages. To investigate the role of endoglin(eng) in vessel formation during the development of hereditary hemorrhagic telangiectasia&#160;(HHT), morpholino-mediated targeted knockdown of eng in zebrafish are used to study its temporal expression and associated functions. Here, whole-mount in situ RNA hybridization (WISH) is employed for the analysis of eng and its downstream genes in zebrafish embryos. Also, tube formation assays are performed in HHT patient-derived induced pluripotent stem cell-differentiated&#160;endothelial cells (iPSC-ECs; with eng mutations). A specific signal amplifying system using the whole amount In Situ Hybridization &#8211; WISH provides higher resolution and lower background results compared to traditional methods. To obtain a better signal, the post-fixation time is adjusted to 30 min after probe hybridization. Because fluorescence staining is not sensitive in zebrafish embryos, it is replaced with diaminobezidine (DAB) staining here. In this protocol, HHT&#160;patient-derived iPSC lines containing an eng mutation are differentiated into endothelial cells. After coating a plate with basement membrane matrix for 30 min at 37 &#176;C, iPSC-ECs are seeded as a monolayer into wells and kept at 37 &#176;C for 3 h. Then, the tube length and number of branches are calculated using microscopic images. Thus, with this improved WISH protocol, it is shown that reduced eng expression affects endothelial progenitor formation in zebrafish embryos. This is further confirmed by tube formation assays using iPSC-ECs derived from a patient with HHT. These assays confirm the role for eng in early vascular development.

Presented here is a protocol for whole-mount in situ RNA hybridization analysis in zebrafish and tube formation assay in patient-derived induced pluripotent stem cell-derived endothelial cells to study the role of endoglin in vascular formation.

Vascular development is determined by the sequential expression of specific genes, which can be studied by performing in situ hybridization assays in ...

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United States. The innate immune response against C. difficile has been described, but the exact roles of neutrophils and macrophages in CDI are less understood. In the current study, Danio rerio (zebrafish) larvae are used to establish a C. difficile infection model for imaging the behavior and cooperation of these innate immune cells in vivo. To monitor C. difficile, a labeling protocol using a fluorescent dye has been established. A localized infection is achieved by microinjecting labeled C. difficile, which actively grows in the zebrafish intestinal tract and mimics the intestinal epithelial damage in CDI. However, this direct infection protocol is invasive and causes microscopic wounds, which can affect experimental results. Hence, a more noninvasive microgavage protocol is described here. The method involves delivery of C. difficile cells directly into the intestine of zebrafish larvae by intubation through the open mouth. This infection method closely mimics the natural infection route of C. difficile.

Development of a Larval Zebrafish Infection Model for Clostridioides difficile

Presented here is a safe and effective method to infect zebrafish larvae with fluorescently labeled anaerobic C. difficile by microinjection and noninvasive microgavage.

Clostridioides difficile infection (CDI) is considered to be one of the most common healthcare-associated gastrointestinal infections in the United ...

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.

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