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 distilled water and agitate.</li>
	<li>Prepare 1 L 1-phenyl-2-thiourea (PTU) stock solution (20x). Dissolve 0.6 g PTU powder in 1 L distilled water. Add 1 ml of stock solution per 20 ml embryo medium to a final concentration of 0.003% as a working solution. 
	CAUTION: PTU may cause systemic toxicity following inhalation or dermal exposure. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 1 L ethyl 3-aminobenzoate methanesulfonate (Tricaine) stock solution (20x). Dissolve 4 g Tricaine powder in distilled water. Adjust pH to 7 by adding 1 M Tris buffer (pH 9), and bring the final volume to 1 L with distilled water. Aliquot into 50 ml and store at -20 &#176;C. Thaw before use. 
	CAUTION: Tricaine can induce a loss of sensation. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 100 ml larval metronidazole (MTZ) working solution. Make fresh 15 mM MTZ solution by dissolving 0.25 g MTZ powder in 0.1% dimethyl sulfoxide (DMSO) in 100 ml embryo medium. Dissolve MTZ powder completely by vigorous shaking. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ. 
	CAUTION: MTZ may cause irritation. Prepare and use in accordance with appropriate handling guidelines.</li>
	<li>Prepare 100 ml larval ethanol/metronidazole (EtOH/MTZ) working solution. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ. Add 1.5 ml 100% ethanol into 100 ml MTZ solution to a final concentration of 1.5% just before use.</li>
	<li>Prepare 500 ml adult MTZ working solution. Make fresh 10 mM MTZ solution by dissolving 0.83 g MTZ powder in 0.1% DMSO in 500 ml system water. Dissolve MTZ powder completely by vigorous shaking. Make fresh MTZ solution and use aluminum foil to prevent photocatalytic degradation of MTZ.</li>
	<li>Prepare 1 L PEM buffer. Dissolve 30.2 g PIPES, 0.74 g EGTA, and 0.12 g MgSO4 in distilled water. Adjust pH to 7 with NaOH. Bring final volume to 1 L with distilled water.</li>
</ol>
2. Preparation of Larval Zebrafish
<ol>
	<li>Set up mating of [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11]15,17 adult zebrafish with Tg(hand2:EGFP)pd24 13 adult zebrafish in mating tanks. Use dividers to conduct timed mating.</li>
	<li>Remove the divider the following morning and allow fish to mate without disruption. Harvest embryos 2 hr later by straining system water and transferring the embryos into 100 mm Petri dishes with embryo medium.</li>
	<li>Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 &#176;C and add phenylthiourea (PTU) to a final concentration of 0.003% after 10 hours-post-fertilization (hpf) to inhibit pigment development.</li>
</ol>
3. Ethanol, Metronidazole Treatment and Liver Regeneration in Larval Zebrafish
<ol>
	<li>Prepare fresh EtOH solution by adding ethanol into embryo medium to a final concentration of 1.5%. Do not add PTU.</li>
	<li>Treat embryos with 20 ml ethanol solution per Petri dish from 56 to 80 hpf. Cover Petri dishes with plastic wrap to prevent ethanol evaporation.</li>
	<li>Sort out [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24] larvae with similar liver size, as determined by CFP expression, at 80 hpf. Do not use Tricaine when sorting the EtOH-treated larvae because this will cause high mortality rate with subsequent EtOH/MTZ treatment. Keep sorted larvae at a density of 30 embryos per Petri dish.</li>
	<li>Prepare fresh 15 mM MTZ in embryo medium in 0.1% DMSO. Add proper amount of EtOH into MTZ solution to make a final concentration of 1.5%. Incubate larvae in 20 ml EtOH/MTZ solution per Petri dish for 24 hr at 28 &#176;C. Cover Petri dishes with plastic wrap to maintain EtOH concentration and with aluminum foil to protect from light.</li>
	<li>At the end of the treatment, remove EtOH/MTZ solution by transferring larvae to new Petri dishes with fresh embryo medium. Wash larvae 3 times with embryo medium and keep them in 20 ml embryo medium without PTU.</li>
	<li>To sort out the larvae with near-complete ablation of hepatocytes, observe fluorescence under an epifluorescence microscope with the CFP filter at a magnification of 80X. Successful ablation results in minimal CFP fluorescence in the liver and significantly reduced liver volume.</li>
	<li>To avoid death of the EtOH/MTZ-treated larvae, do not use Tricaine for sorting. Fix the larvae for immunostaining (refer to section 5) or keep sorted larvae in Petri dishes at 28 &#176;C for further analyses.</li>
</ol>
4. Chemical Screens in EtOH/MTZ-treated Larval Zebrafish
<ol>
	<li>Bring the 96-well plates containing 10 mM chemical stocks in DMSO (refer to the Materials List for details regarding the commercial chemical libraries) from the -80 &#176;C freezer. Cover with aluminum foil to prevent photocatalytic degradation of chemicals. Thaw the stock solutions at RT with gentle rocking.</li>
	<li>Prepare chemical solutions by diluting &#160;10 mM stock solutions with&#160;embryo medium to a final concentration of 50 &#181;M&#160;(in 96 well plates: initial screen; in 1.5 ml tubes: retesting). Control larvae were treated with equal concentrations of DMSO in embryo medium.&#160;</li>
	<li>Transfer larvae with near-complete hepatocyte ablation, which were treated with 1.5% EtOH/15 mM MTZ and sorted as aforementioned, to either 96-well (initial screen) or 24-well (retesting)&#160;&#160;plates prefilled with embryo medium at a density of 5 larvae per well. Use duplicate wells for each chemical.</li>
	<li>Remove embryo medium and add either 250 &#181;l (96-well plates) or 500 &#181;l (24-well plates)&#160;&#160;chemical solutions to each well. Cover plates with aluminum foil and treat larvae for 50 hr at 28 &#176;C. Inspect chemical-soaking larvae daily and remove any dead larvae from individual wells.</li>
	<li>At the end of the chemical treatment, observe the number and/or intensity of CFP-expressing hepatocytes under an epifluorescence microscope with the CFP filter at a magnification of 80X. Photograph live larvae showing enhanced or reduced number and/or intensity of CFP-expressing hepatocytes with a digital camera for records.</li>
	<li>Preserve larvae by formaldehyde fixation for confocal imaging as described in section 5.</li>
	<li>To retest the chemicals that facilitate hepatocyte regeneration in the initial screen, examine their potency at higher and lower concentrations to find the most efficacious concentration with the procedures described in 4.1-4.5 (&#39;retesting&#39;).</li>
</ol>
5. Larval Zebrafish Fixation, Immunostaining, and Confocal Imaging
<ol>
	<li>Anesthetize larvae by adding Tricaine to a final concentration of 0.02%. Transfer 10-15 larvae to a 1.5 ml tube and wash once with phosphate-buffered saline (PBS). Remove PBS and add 1 ml of freshly prepared 2% formaldehyde in PEM buffer to fix O/N at 4 &#176;C. 
	CAUTION: Formaldehyde causes irritation and is known to be a human carcinogen. Handle in a chemical fume hood.</li>
	<li>Discard fixing solution properly and then wash 3 times with PBS at RT. Fixed larvae can be kept in PBS at 4 &#176;C for up to several days. Proceeding to the next step promptly gives better immunostaining results.</li>
	<li>Carefully remove yolk and skin covering the liver area using a #55 forceps under a stereomicroscope.</li>
	<li>Permeabilize and block larvae in Block buffer (4% bovine serum albumin and 0.3% Triton X-100 in PBS) O/N at 4 &#176;C.</li>
	<li>Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 &#176;C. Wash 5 times for 15 min each with Wash buffer (0.3% Triton X-100 in PBS).</li>
	<li>Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 &#176;C. Wash 5 times for 15 min each with Wash buffer. Then wash once with PBS.</li>
	<li>Gently transfer larvae to glass slides using a glass pipette, and orient fixed larvae with the left lateral side facing up. Remove excess PBS using Kimwipes. Add a drop of mounting media, and seal cover glass with nail polish. Conducting confocal imaging immediately is greatly recommended.</li>
	<li>Use a confocal system equipped with a 40X/1.3 oil lens to capture images. Use laser strength at 20-50%. Capture Z stack images at 1 &#181;m intervals.</li>
</ol>
6. Preparation of Adult Zebrafish
<ol>
	<li>Set up mating of [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11]15,17 adult zebrafish in mating tanks. Harvest embryos the next morning by straining system water and transferring the embryos into 100 mm Petri dishes with embryo medium.</li>
	<li>Keep no more than 100 embryos per Petri dish. Under a stereomicroscope, remove unfertilized eggs using a glass pipette. Grow embryos at 28 &#176;C and add PTU to a final concentration of 0.003% after 10 hpf to inhibit pigment development.</li>
	<li>At 4 dpf, use Tricaine to anesthetize larvae and sort out [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] larvae. Wash out Tricaine by changing to fresh embryo medium several times. Allow larvae to recover at 28 &#176;C until they achieve normal heart rate of 120-180 beats per minute18.</li>
	<li>Raise sorted larvae to 6-12 months old based on the standard procedure19.</li>
</ol>
7. Ethanol, Metronidazole Treatment, and Liver Regeneration in Adult Zebrafish
<ol>
	<li>Transfer 6-12 months old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adult zebrafish to mating tanks using a net. Keep fish at a density of no more than 10 fish per tank.</li>
	<li>Prepare fresh EtOH solution by adding EtOH in system water to a final concentration of 1%. Treat adult fish with 500 ml EtOH solution in each tank and maintain them in a 28 &#176;C incubator.</li>
	<li>Transfer adult fish to fresh EtOH solution daily, discard dead fish properly as a biohazard. Continuously treat animals for 72 hr before MTZ treatment.</li>
	<li>Prepare fresh 10 mM MTZ solution in system water in 0.1% DMSO. Pre-warm the MTZ solution in a 28 &#176;C incubator. Treat fish with 500 ml MTZ solution in 1 L crossing tank for 8 hr at 28 &#176;C, protect from light using aluminum foil.</li>
	<li>Observe hourly during the treatment and remove dead fish immediately. Discard dead fish properly as a biohazard. At the end of the MTZ treatment, wash out MTZ by transferring animals to fresh system water twice.</li>
	<li>Allow animals to recover and maintain them in an incubator at 28 &#176;C. Feed and transfer fish to fresh system water daily until the time point for analyses.</li>
</ol>
8. Adult Zebrafish Liver Fixation, Immunostaining, and Confocal Imaging&#160;
<ol>
	<li>Anesthetize adult fish with 0.02% Tricaine and euthanize in ice water for 15 min. Pat the fish dry on a paper towel and place it on a dissecting mat.</li>
	<li>Expose gastrointestinal organs by removing skin and muscles as previously described20. Use scissors to cut the skin and underlying muscle along the belly from the anal fin to the operculum, and then posteriorly along the side of the fish back to the anal fin.</li>
	<li>Put the fish in a 15 ml conical tube and wash once with PBS. Remove PBS and add 4 ml of freshly prepared 2% formaldehyde in PEM buffer to fix O/N at 4 &#176;C.</li>
	<li>Discard fixing solution properly, and wash 3 times with PBS at RT. Fixed samples can be kept in PBS at 4 &#176;C for several days. Proceeding to the next step immediately gives better immunostaining results.</li>
	<li>Dissect the whole gut with liver from the body cavity of the fish by cutting the esophagus. Embed the gastrointestinal organs with 4% low-melting agarose in a sectioning mold.</li>
	<li>Use a vibratome to cut samples into transverse sections at 50 &#181;m intervals in ice cold PBS, starting from the anterior end. Transfer sections to 6-well plate with PBS using a #1 paint brush.</li>
	<li>Permeabilize and block sections in Block buffer O/N at 4 &#176;C.</li>
	<li>Incubate with rabbit anti-Collagen I antibody at a dilution of 1:100 in Block buffer O/N at 4 &#176;C. Wash 5 times, 15 min each with Wash buffer.</li>
	<li>Incubate with AlexaFluor 647 conjugated donkey anti-rabbit antibody at a dilution of 1:200 in Block buffer O/N at 4 &#176;C. Wash 5 times, 15 min each with Wash buffer and once with PBS.</li>
	<li>Gently transfer sections to glass slides using a #1 paint brush. Remove excess PBS using Kimwipes, add a drop of mounting media, and seal cover glass with nail polish. Conducting confocal imaging immediately is highly suggested.</li>
	<li>Use a confocal system equipped with a 40X/1.3 oil lens to capture images. Use laser strength at 20-50%. Capture Z stack images at 1 &#181;m intervals.</li>
</ol>

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I., Xue, Y., Shafritz, D. A., Locker, J., Oertel, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Repopulation+of+the+fibrotic/cirrhotic+rat+liver+by+transplanted+hepatic+stem/progenitor+cells+and+mature+hepatocytes.">Repopulation of the fibrotic/cirrhotic rat liver by transplanted hepatic stem/progenitor cells and mature hepatocytes.</a> Hepatology. 59 (1), 284-295 (2014).</li></ol>

The authors declare that they have no competing financial interests.

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 (CaSO&lt;sub&gt;4&lt;/sub&gt;)</td><td>Acros</td><td>AC385355000</td><td></td></tr><tr><td>Magnesium sulfate (MgSO&lt;sub&gt;4&lt;/sub&gt;)</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)-&lt;br /&gt;&lt;em&gt;N,N,N&amp;prime;,N&amp;prime;&lt;/em&gt;-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&amp;nbsp;</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>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>ActiProbe-1K Library</td><td>Timtec</td><td>ActiProbe-1K</td><td>10&amp;nbsp;mM stock in DMSO</td></tr><tr><td>SB 415286</td><td>Selleck Chemicals</td><td>S2729</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</td></tr><tr><td>CHIR-99021</td><td>Selleck Chemicals</td><td>S2924</td><td>Dissolve with DMSO to 10&amp;nbsp;mM</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>Epifluoresence 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 ...

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Research

JoVE Journal

Developmental Biology

9.1K Views.  Georgia Institute of Technology. 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. 

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

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 ...

Assessing Teratogenic Changes in a Zebrafish Model of Fetal Alcohol Exposure

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, including mental retardation due to disordered and damaged brain development. Fetal alcohol spectrum disorder (FASD) is a term used to cover a continuum of birth defects that occur due to maternal alcohol consumption, and occurs in approximately 4% of children born in the United States. With 50% of child-bearing age women reporting consumption of alcohol, and half of all pregnancies being unplanned, unintentional exposure is a continuing issue2. In order to best understand the damage produced by ethanol, plus produce a model with which to test potential interventions, we developed a model of developmental ethanol exposure using the zebrafish embryo. Zebrafish are ideal for this kind of teratogen study3-8. Each pair lays hundreds of eggs, which can then be collected without harming the adult fish. The zebrafish embryo is transparent and can be readily imaged with any number of stains. Analysis of these embryos after exposure to ethanol at different doses and times of duration and application shows that the gross developmental defects produced by ethanol are consistent with the human birth defect. Described here are the basic techniques used to study and manipulate the zebrafish FAS model.

In order to understand the molecular mechanisms of the ethanol-induced developmental damage, we have developed a zebrafish model of ethanol exposure and are exploring the physical, cellular, and genetic alterations that occur after ethanol exposure1. We then seek to find potential interventions and rapidly test them in this animal model.

Fetal alcohol syndrome (FAS) is a severe manifestation of embryonic exposure to ethanol. It presents with characteristic defects to the face and organs, ...

Medicine

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 ...

Analysis of Liver Microenvironment During Early Progression of Non-Alcoholic Fatty Liver Disease-Associated Hepatocellular Carcinoma in Zebrafish

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all liver cancer cases. With the introduction of effective treatments to prevent and treat hepatitis B/C, non-alcoholic fatty liver disease (NAFLD), and the more aggressive form know as non-alcoholic steatohepatitis (NASH), are quickly becoming the number one risk factors to develop HCC in modern societies. To better understand the role NASH has on the development of HCC we designed a NASH-associated HCC zebrafish. The optical clarity and genetic tractability of the zebrafish larvae make them an appealing and powerful model to study the liver microenvironment and immune cell composition using non-invasive fluorescent live imaging. This protocol describes how to use a NASH-associated HCC zebrafish model to investigate the effect of cholesterol surplus in the liver microenvironment and its impact on immune cell composition at early stages of the disease. First, we feed HCC larvae (s704Tg), which express hepatocyte-specific activated beta-catenin, with a 10% high cholesterol diet for 8 days to develop a NASH-associated HCC model. Here we describe how to make use of different transgenic lines to evaluate several early malignancy features in the liver by non-invasive confocal microscopy, such as liver area, cell, and nuclear morphology (hepatocytes area, nuclear area, nuclear:cytoplasmic ratio (N:C ratio), nuclear circularity, micronuclei/nuclear herniation scoring) and angiogenesis. Then, using transgenic lines with tagged immune cells (neutrophils, macrophages, and T cells) we show how to analyze liver immune cell composition in NASH-associated HCC larvae. The described techniques are useful to evaluate liver microenvironment and immune cell composition at early hepatocarcinogenesis stages, but they can also be modified to study such features in other liver disease models.

Here, we present how to generate a non-alcoholic fatty liver disease (NAFLD)-associated Hepatocellular Carcinoma (HCC) zebrafish model to study the impact of cholesterol surplus on liver microenvironment and immune cell landscape.

Liver cancer is currently the third&#160;leading cause of cancer related death worldwide, and Hepatocellular Carcinoma (HCC) accounts for 75-90% of all ...

Cancer Research

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.

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval ...

Quantification of Ethanol Levels in Zebrafish Embryos Using Head Space Gas Chromatography

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies and neurological impairments. With a complex pathology, FASD affects approximately 1 in 100 children born in the United States each year. Due to the highly variable nature of FASD, animal models have proven critical in our current mechanistic understanding of ethanol-induced development defects. An increasing number of laboratories has focused on using zebrafish to examine ethanol-induced developmental defects. Zebrafish produce large numbers of externally fertilized, genetically tractable, translucent embryos. This allows researchers to precisely control timing and dosage of ethanol exposure in multiple genetic contexts and quantify the impact of embryonic ethanol exposure through live imaging techniques. This, combined with the high degree of conservation of both genetics and development with humans, has proven zebrafish to be a powerful model in which to study the mechanistic basis of ethanol teratogenicity. However, ethanol exposure regimens have varied between different zebrafish studies, which has confounded the interpretation of zebrafish data across these studies. Here is a protocol to quantify ethanol concentrations in zebrafish embryos using head space gas chromatography.

This work describes a protocol to quantify ethanol levels in a zebrafish embryo using head space gas chromatography from proper exposure methods to embryo processing and ethanol analysis.

Fetal Alcohol Spectrum Disorders (FASD) describe a highly variable continuum of ethanol-induced developmental defects, including facial dysmorphologies ...

The Zebrafish Tol2 System: A Modular and Flexible Gateway-Based Transgenesis Approach

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to prenatal ethanol exposure. Due to the complex pathology of FASD, animal models have proven critical to our current understanding of ethanol-induced developmental defects. Zebrafish have proven to be a powerful model to examine ethanol-induced developmental defects due to the high degree of conservation of both genetics and development between zebrafish and humans. As a model system, zebrafish possess many attributes that make them ideal for developmental studies, including large numbers of externally fertilized embryos that are genetically tractable and translucent. This allows researchers to precisely control the timing and dosage of ethanol exposure in multiple genetic contexts. One important genetic tool available in zebrafish is transgenesis. However, generating transgenic constructs and establishing transgenic lines can be complex and difficult. To address this issue, zebrafish researchers have established the transposon-based Tol2 transgenesis system. This modular system uses a multisite Gateway cloning approach for the quick assembly of complete Tol2 transposon-based transgenic constructs. Here, we describe the flexible Tol2 system toolbox and a protocol for generating transgenic constructs ready for zebrafish transgenesis and their use in ethanol studies.

This work describes a protocol for the modular Tol2 transgenesis system, a gateway-based cloning method to create and inject transgenic constructs into zebrafish embryos.

Fetal alcohol spectrum disorders (FASD) are characterized by a highly variable set of structural defects and cognitive impairments that arise due to ...

Robust Zebrafish Cell Transplantation for Regeneration and Development Research

High-resolution Cell Transplantation in Embryonic and Larval Zebrafish

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation between animals to track cell fate and to induce mismatches in the genetic, spatial, or temporal properties of donor and host cells is a powerful means of examining the nature of these interactions. Organisms such as chick and amphibians have made crucial contributions to our understanding of development and regeneration, respectively, in large part because of their amenability to transplantation. The power of these models, however, has been limited by low genetic tractability. Likewise, the major genetic model organisms have lower amenability to transplantation. 
The zebrafish is a major genetic model for development and regeneration, and while cell transplantation is common in zebrafish, it is generally limited to the transfer of undifferentiated cells at the early blastula and gastrula stages of development. In this article, we present a simple and robust method that extends the zebrafish transplantation window to any embryonic or larval stage between at least 1 and 7 days post fertilization. The precision of this approach allows for the transplantation of as little as one cell with near-perfect spatial and temporal resolution in both donor and host animals. While we highlight here the transplantation of embryonic and larval neurons for the study of nerve development and regeneration, respectively, this approach is applicable to a wide range of progenitor and differentiated cell types and research questions.

Author Spotlight: Mapping Cellular Connectivity in the Zebrafish Nervous System During Development and Regeneration

Here we present a protocol to transplant cells with high spatial and temporal resolution in zebrafish embryos and larvae at any stage between at least 1 and 7 days post fertilization.

Development and regeneration occur by a process of genetically encoded spatiotemporally dynamic cellular interactions. The use of cell transplantation ...

Acute Kidney Injury Model Induced by Cisplatin in Adult Zebrafish

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney due to its low molecular weight. Such accumulation causes the death of tubular cells and can induce the development of Acute Kidney Injury (AKI), which is characterized by a quick decrease in kidney function, tissue damage, and immune cells infiltration. If administered in specific doses cisplatin can be a useful tool as an AKI inducer in animal models. The zebrafish has appeared as an interesting model to study renal function, kidney regeneration, and injury, as renal structures conserve functional similarities with mammals. Adult zebrafish injected with cisplatin shows decreased survival, kidney cell death, and increased inflammation markers after 24 h post-injection (hpi). In this model, immune cells infiltration and cell death can be assessed by flow cytometry and TUNEL assay. This protocol describes the procedures to induce AKI in adult zebrafish by intraperitoneal cisplatin injection and subsequently demonstrates how to collect the renal tissue for flow cytometry processing and cell death TUNEL assay. These techniques will be useful to understand the effects of cisplatin as a nephrotoxic agent and will contribute to the expansion of AKI models in adult zebrafish. This model can also be used to study kidney regeneration, in the search for compounds that treat or prevent kidney damage and to study inflammation in AKI. Moreover, the methods used in this protocol will improve the characterization of tissue damage and inflammation, which are therapeutic targets in kidney-associated comorbidities.

This protocol describes the procedures to induce Acute Kidney Injury (AKI) in adult zebrafish using cisplatin as a nephrotoxic agent. We detailed the steps to evaluate the reproducibility of the technique and two techniques to analyze inflammation and cell death in the renal tissue, flow cytometry and TUNEL, respectively.

Cisplatin is commonly used as chemotherapy. Although it has positive effects in cancer-treated individuals, cisplatin can easily accumulate in the kidney ...

Partial Hepatectomy in Adult Zebrafish

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy livers are capable of regenerating from toxic damage, but in advanced liver disease, the natural ability of the liver to regenerate is impaired. Zebrafish have emerged as a powerful experimental system for studying regeneration. They are an ideal model for studying liver regeneration from partial hepatectomy, a procedure with direct clinical relevance in which part of the liver is surgically removed, leaving the rest intact. There is no standard protocol for partial hepatectomy; previous studies using this model have used slightly different protocols and reported disparate results. Described here is an efficient, reproducible protocol for performing a partial hepatectomy in adult zebrafish. We use this technique to demonstrate that zebrafish are capable of epimorphic regeneration of the resected lobe. This protocol can be used to further interrogate the mechanisms required for liver regeneration in zebrafish.

This protocol describes the procedure for removing the ventral lobe of the liver in adult zebrafish to enable the study of liver regeneration.

Liver failure is one of the leading causes of death worldwide, and mortality from chronic liver disease is rising sharply in the United States. Healthy ...

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

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