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>

<ol>
	<li>Michalopoulos, G. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+regeneration.">Liver regeneration.</a> J Cell Physiol. 213 (2), 286-300 (2007).</li><li>Duncan, A. W., Dorrell, C., Grompe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+and+liver+regeneration.">Stem cells and liver regeneration.</a> Gastroenterology. 137 (2), 466-481 (2009).</li><li>Shepard, C. W., Finelli, L., Alter, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+epidemiology+of+hepatitis+C+virus+infection.">Global epidemiology of hepatitis C virus infection.</a> Lancet Infect Dis. 5 (9), 558-567 (2005).</li><li>Hernandez-Gea, V., Friedman, S. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pathogenesis+of+liver+fibrosis.">Pathogenesis of liver fibrosis.</a> Annu Rev Pathol. 6, 425-456 (2011).</li><li>Bataller, R., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+fibrosis.">Liver fibrosis.</a> J Clin Invest. 115 (2), 209-218 (2005).</li><li>Kisseleva, T., Brenner, D. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anti-fibrogenic+strategies+and+the+regression+of+fibrosis.">Anti-fibrogenic strategies and the regression of fibrosis.</a> Best Pract Res Clin Gastroenterol. 25 (2), 305-317 (2011).</li><li>Constandinou, C., Henderson, N., Iredale, J. 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. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alcoholic+fatty+liver:+its+pathogenesis+and+mechanism+of+progression+to+inflammation+and+fibrosis.">Alcoholic fatty liver: its pathogenesis and mechanism of progression to inflammation and fibrosis.</a> Alcohol. 34 (1), 9-19 (2004).</li><li>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=Advances+in+understanding+tissue+regenerative+capacity+and+mechanisms+in+animals.">Advances in understanding tissue regenerative capacity and mechanisms in animals.</a> Nat Rev Genet. 11 (10), 710-722 (2010).</li><li>Jang, Z. 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=Metabolic+profiling+of+an+alcoholic+fatty+liver+in+zebrafish+(Danio+rerio).">Metabolic profiling of an alcoholic fatty liver in zebrafish (Danio rerio).</a> Mol Biosyst. 8 (7), 2001-2009 (2012).</li><li>Passeri, M. J., Cinaroglu, A., Gao, C., Sadler, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hepatic+steatosis+in+response+to+acute+alcohol+exposure+in+zebrafish+requires+sterol+regulatory+element+binding+protein+activation.">Hepatic steatosis in response to acute alcohol exposure in zebrafish requires sterol regulatory element binding protein activation.</a> Hepatology. 49 (2), 443-452 (2009).</li><li>Yin, C., Evason, K. J., Maher, J. J., Stainier, D. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+basic+helix-loop-helix+transcription+factor,+heart+and+neural+crest+derivatives+expressed+transcript+2,+marks+hepatic+stellate+cells+in+zebrafish:+analysis+of+stellate+cell+entry+into+the+developing+liver.">The basic helix-loop-helix transcription factor, heart and neural crest derivatives expressed transcript 2, marks hepatic stellate cells in zebrafish: analysis of stellate cell entry into the developing liver.</a> Hepatology. 56 (5), 1958-1970 (2012).</li><li>Lin, J. N., 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+animal+model+for+alcoholic+liver+disease+in+zebrafish.">Development of an animal model for alcoholic liver disease in zebrafish.</a> Zebrafish. 12 (4), 271-280 (2015).</li><li>Huang, M., 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=Antagonistic+interaction+between+Wnt+and+Notch+activity+modulates+the+regenerative+capacity+of+a+zebrafish+fibrotic+liver+model.">Antagonistic interaction between Wnt and Notch activity modulates the regenerative capacity of a zebrafish fibrotic liver model.</a> Hepatology. 60 (5), 1753-1766 (2014).</li><li>Curado, S., Stainier, D. Y., Anderson, R. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nitroreductase-mediated+cell/tissue+ablation+in+zebrafish:+a+spatially+and+temporally+controlled+ablation+method+with+applications+in+developmental+and+regeneration+studies.">Nitroreductase-mediated cell/tissue ablation in zebrafish: a spatially and temporally controlled ablation method with applications in developmental and regeneration studies.</a> Nat Protoc. 3 (6), 948-954 (2008).</li><li>Parsons, M. J., 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=Notch-responsive+cells+initiate+the+secondary+transition+in+larval+zebrafish+pancreas.">Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas.</a> Mech Dev. 126 (10), 898-912 (2009).</li><li>Baker, K., Warren, K. S., Yellen, G., Fishman, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defective+'pacemaker'+current+(Ih)+in+a+zebrafish+mutant+with+a+slow+heart+rate.">Defective 'pacemaker' current (Ih) in a zebrafish mutant with a slow heart rate.</a> Proc Natl Acad Sci U S A. 94 (9), 4554-4559 (1997).</li><li>Avdesh, A., 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=Regular+care+and+maintenance+of+a+zebrafish+(Danio+rerio)+laboratory:+an+introduction.">Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction.</a> J Vis Exp. (69), e4196 (2012).</li><li>Gupta, T., Mullins, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dissection+of+organs+from+the+adult+zebrafish.">Dissection of organs from the adult zebrafish.</a> J Vis Exp. (37), (2010).</li><li>Paku, S., Schnur, J., Nagy, P., Thorgeirsson, S. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Origin+and+structural+evolution+of+the+early+proliferating+oval+cells+in+rat+liver.">Origin and structural evolution of the early proliferating oval cells in rat liver.</a> Am J Pathol. 158 (4), 1313-1323 (2001).</li><li>Turner, R., 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=Human+hepatic+stem+cell+and+maturational+liver+lineage+biology.">Human hepatic stem cell and maturational liver lineage biology.</a> Hepatology. 53 (3), 1035-1045 (2011).</li><li>Kodama, Y., Hijikata, M., Kageyama, R., Shimotohno, K., Chiba, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+notch+signaling+in+the+development+of+intrahepatic+bile+ducts.">The role of notch signaling in the development of intrahepatic bile ducts.</a> Gastroenterology. 127 (6), 1775-1786 (2004).</li><li>Ryback, R., Percarpio, B., Vitale, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Equilibration+and+metabolism+of+ethanol+in+the+goldfish.">Equilibration and metabolism of ethanol in the goldfish.</a> Nature. 222 (5198), 1068-1070 (1969).</li><li>Mathias, J. 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. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+school+in+liver+development:+lessons+from+zebrafish.">New school in liver development: lessons from zebrafish.</a> Hepatology. 50 (5), 1656-1663 (2009).</li><li>Choi, T. Y., Ninov, N., Stainier, D. Y., Shin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extensive+conversion+of+hepatic+biliary+epithelial+cells+to+hepatocytes+after+near+total+loss+of+hepatocytes+in+zebrafish.">Extensive conversion of hepatic biliary epithelial cells to hepatocytes after near total loss of hepatocytes in zebrafish.</a> Gastroenterology. 146 (3), 776-788 (2014).</li><li>He, J., Lu, H., Zou, Q., Luo, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+liver+after+extreme+hepatocyte+loss+occurs+mainly+via+biliary+transdifferentiation+in+zebrafish.">Regeneration of liver after extreme hepatocyte loss occurs mainly via biliary transdifferentiation in zebrafish.</a> Gastroenterology. 146 (3), 789-800 (2014).</li><li>Yao, Y., 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=Fine+structure,+enzyme+histochemistry,+and+immunohistochemistry+of+liver+in+zebrafish.">Fine structure, enzyme histochemistry, and immunohistochemistry of liver in zebrafish.</a> Anat Rec (Hoboken). 295 (4), 567-576 (2012).</li><li>Yovchev, M. 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 the fibrotic liver. As near-complete elimination of hepatocyte elicits HPC-driven hepatocyte regeneration, it is critical to sort out MTZ-treated larvae based on the criteria of absence of hepatocyte-specific fluorescence and significantly reduced liver volume by clustered NRCs. In fish, the blood vessels of the gill and skin absorb the alcohol24, so that there is no need to allow animals to drink ad libitum or require intragastric infusion as in mammalian models. We housed EtOH- and subsequent MTZ-treated adult fish in separate tanks without circulating the water. It is essential to transfer fish to fresh EtOH solution daily and keep the lid on to minimize the evaporation of EtOH. Although 48-72 hr EtOH/MTZ treatment efficiently induces ECM protein synthesis in our protocols, neither advanced fibrosis nor cirrhosis has been developed in these fish. Therefore, combination of MTZ with prolonged exposure of ethanol such as 12 weeks treatment14, specifically in adult fish, may induce more severe liver damage to properly simulate and interrogate HPC-driven regeneration in chronic liver failure.
Zebrafish serves as an outstanding animal model for in vivo compound testing due to its small size, large progeny, transparency, and permeability to small molecules25. Using the fibrotic liver model in zebrafish, we identified a number of known and novel Wnt activators and Notch inhibitors that accelerated hepatocyte regeneration15. Our current fluorescence-based chemical screening protocol consists of several steps all performed manually including preparation of chemicals, transferring EtOH-treated/hepatocyte-ablated larvae to 24-well plates, treating chemicals, and analysis of effects of chemicals on hepatocyte regeneration. It specifically employs epifluorescence and/or confocal microscope to capture images for individual animal, which is laborious and time consuming. High-throughput platforms that automatically handle and acquire cellular-resolution imaging of zebrafish in combination with quantitative data collection will facilitate large-scale screening26,27.These features will be strengthened if combined with automated drug dosing system that determines the range of chemical concentrations28, which can give minimum toxicity with maximum efficacy for enhancing hepatocyte regeneration. Despite these limitations, as many critical players and main cell types of the liver are conserved between zebrafish and mammals29, employing the in vivo-based small molecule screening using the zebrafish fibrotic liver model will catalyze valid discovery of novel therapeutic strategies for chronic liver disease.
Two additional groups independently have demonstrated that after near total loss of hepatocytes without fibrogenic insults, BECs transdifferentiated into hepatocyte through a step of dedifferentiation30,31 with the assumption that cholangiocytes, fully differentiated BECs, primarily comprise NRCs/BECs in zebrafish. Although transdifferentiation can be one of the mechanisms of driving hepatocyte regeneration, our results indicate that the HPCs, which are known to constitute the majority of BECs in the zebrafish liver32, downregulate Notch activity to differentiate into hepatocytes. Meanwhile, it is plausible to speculate that the fully differentiated BECs, cholangiocytes, maintain higher levels of Notch activity without converting to hepatocytes during regeneration. Intriguingly, in the presence of EtOH, as previously reported13, we found that HSCs increased in number with the altered morphology from a star-like configuration to a myofibroblast-like shape with lost cytoplasmic processes. The activated HSCs characterized by synthesis of ECM proteins are the principle culprit responsible for abnormal wound healing during the integrated process of liver repair4. Although chronic injury often overrides the liver&#39;s remarkable capacity for regeneration, transplanted HPCs successfully repopulated the fibrotic/cirrhotic rat liver33. Hence, our zebrafish fibrotic liver model will be an essential tool for elucidating the molecular and cellular mechanisms that mediate the effects of sustained fibrosis on HPC-mediated hepatocyte regeneration. Furthermore, defining the multi-cellular crosstalk that balances regeneration and fibrosis by utilizing our zebrafish fibrotic liver model will further facilitate to design viable therapeutic strategies for chronic liver failure in vivo.

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 rare deposition of extracellular matrix proteins like collagen. Based on these results, the larvae were pretreated with 1.5% EtOH for 24 hr (2.5-3.5 dpf) and then simultaneously treated with MTZ for 24 hours (3.5-4.5 dpf) (Figure 1A). The EtOH/MTZ-treated larvae showed morphological abnormalities including upward curvature of the trunk and tail, pericardial edema, and failure to inflate swim bladder. (Figure 1B). We used three transgenic lines to analyze fibrogenic changes in the EtOH/MTZ-treated larval livers: 1) Tg(fabp10a:CFP-NTR)gt1 line expresses the NTR gene fused with cyan fluorescence protein under the hepatocyte-specific fabp10a promoter15; 2) Tg(Tp1:mCherry)jh11 line marks cells with active Notch signaling (Notch-responsive cells, NRCs) or biliary epithelial cells (BECs) with nuclear mCherry17, which expression is under the control of the TP1 module containing multiple RBP-Jk-binding sites; and 3) Tg(hand2:EGFP)pd24 line, which labels hepatic stellate cells (HSCs)13. DMSO-treated control [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24 ] livers showed no fibrillar type I collagen deposition4,6,9 (Figure 1C, C&#39;), whereas EtOH/MTZ-treated livers displayed elevated type I collagen accumulation at 25 and 50 hr-post-ablation (hpa) (Figure 1D, D&#39;, E, E&#39;). Additionally, compared to DMSO-treated control livers (Figure 1C&#39;&#39;), HSCs increased in number with the altered morphology from a star-like configuration to a myofibroblast-like shape with lost cytoplasmic processes in the EtOH/MTZ-treated regenerating livers (Figure 1D&#39;&#39;, E&#39;&#39;). We observed two discrete populations of mCherry-expressing NRCs at 25 hpa in the EtOH/MTZ-treated regenerating livers: dim red NRCs (Figure 1D&#39;&#39;&#39;, inset, white arrows) and bright red NRCs (Figure 1D&#39;&#39;&#39;, inset, yellow arrowheads). At 50 hpa, hepatocyte-specific CFP began to co-express in NRCs with dim mCherry expression throughout the regenerating livers (Figure 1E&#39;&#39;&#39;, inset, white arrows). These CFP and dim mCherry-coexpressing NRCs are evidently distinguishable from bright red NRCs that are negative for CFP (Figure 1E&#39;&#39;&#39;, inset, yellow arrowheads). Previous studies showed that when hepatocyte proliferation was suppressed after partial hepatectomy (PHx), HPCs expanded with concomitant proliferation of HSCs21. Additionally, the HPCs and BECs shared histochemical markers22. Hence, our results indicate that the CFP and dim mCherry co-expressing NRCs portray a zebrafish-counterpart of HPCs that differentiate into hepatocytes by encountering Notch downregulation. The NRCs maintaining higher levels of Notch signaling may remain as cholangiocytes, which are differentiated BECs23.
Figure 2 shows the development of an EtOH-induced fibrotic liver model in adult zebrafish. First, we exposed adult zebrafish to 1%, 1.5%, or 2% EtOH to assess viability. Most adult zebrafish did not survive more than 72 hr of exposure to EtOH concentrations greater than 1% (unpublished data). Therefore, for adult zebrafish, we pretreated the fish with 1% EtOH for 72 hr and followed with MTZ treatment (Figure 2A). By performing time-course analyses, we showed significantly elevated type I collagen deposition in the EtOH/MTZ-treated regenerating livers at 2, 3, and 4 days-post-ablation (dpa) (Figure 2C&#39;, D&#39;, E&#39;) compared to DMSO-treated livers (Figure 2B&#39;). Similar to that observed in the larvae, the CFP and dim mCherry co-expressing cells were detected in the EtOH/MTZ-treated regenerating livers of 12-month-old [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] adult fish at 3 and 4 dpa (Figure 2D, E, insets, white arrows). These data indicate that the HPCs, responsive to Notch signaling, maintain their capacity to regenerate as hepatocytes in the presence of fibrogenic insult in adult zebrafish.
Figure 3 shows the representative chemical screening results using our ethanol-induced fibrotic liver model in larval zebrafish. To pinpoint bioactive compounds that accelerate hepatocyte regeneration in the fibrotic liver, we performed chemical genetic screens. We screened a library of 75 small molecules with well-characterized biological and pharmaceutical activities (Stem Cell Signaling Compound Library, Selleckchem) and a library of 1,000 less-characterized small molecules (ActiProbe-1K Library, TimTec) using [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] larvae. We ablated the hepatocytes in the presence of 1.5% EtOH/15 mM MTZ and then exposed the larvae to 50 &#181;M of the compounds for 50 hr (Figure 3A). A number of Wnt agonists such as SB 415286 and CHIR-99021, inhibitors of glycogen synthase kinase-3, promoted hepatocyte regeneration (Figure 3C, D) compared to DMSO (Figure 3B). Furthermore, [4-(1H-1,2,3,4-tetraazol-5-yl)-1,2,5-oxadiazole-3-ylamine], abbreviated as HTOA, a novel Wnt pathway activator, enhanced hepatocyte regeneration in the fibrotic liver as indicated by bigger regenerated liver (Figure 3E). These data suggest that our sustained fibrotic model provides an invaluable tool to discover small molecules that enhance hepatocyte regeneration.
<img alt="Figure 1" src="/files/ftp_upload/54002/54002fig1.jpg" /> 
Figure 1. Development of an ethanol-induced fibrotic liver model in larval zebrafish. (A) Scheme illustrating the periods of EtOH and EtOH/MTZ treatment for establishing a fibrotic liver model in larvae. (B) At 50 hours-post-ablation (hpa), EtOH/MTZ-treated larval zebrafish developed morphological abnormalities including upward curvature of the trunk and tail, pericardial edema, and failure to inflate the swim bladder. (C-C&#39;&#39;&#39;) In the livers of [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11; Tg(hand2:EGFP)pd24 ] larvae treated with DMSO, ECM protein collagen 1a was almost undetectable (C, C&#39;) and quiescent HSCs showed a star-like configuration (C&#39;&#39;), whereas NRCs distributed among hepatocytes (C, C&#39;&#39;&#39;, inset). (D-E&#39;&#39;&#39;) In the livers of EtOH/MTZ-treated larvae, elevated collagen 1a deposition was observed (D, D&#39;, E, E&#39;). HSCs increased in number and lost complex cytoplasmic processes (D&#39;&#39;, E&#39;&#39;). At 25 hpa, mCherry-expressing NRCs are composed of two distinct populations. Yellow arrowheads indicate bright red NRCs (D&#39;&#39;&#39;, inset), whereas white arrows indicate dim red NRCs (D&#39;&#39;&#39;, inset). At 50 hpa, Tg(fabp10a:CFP-NTR) and Tg(Tp1:mCherry) co-expressing cells started emerging throughout the regenerating livers (E&#39;&#39;&#39;, inset, white arrows), while the bright red NRCs had no CFP expression (E&#39;&#39;&#39;, inset, yellow arrowheads). All confocal images are single plane images except C&#39;&#39;, D&#39;&#39;, E&#39;&#39;, which are projection images. Scale bars: B, 100 &#181;m; C-E&#39;&#39;&#39;, 20 &#181;m. EtOH, ethanol; MTZ, metronidazole; hpa, hours-post-ablation; dpf, days-post-fertilization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/54002/54002fig2.jpg" /> 
Figure 2. Development of an ethanol-induced fibrotic liver model in adult zebrafish. (A) Scheme illustrating the periods of EtOH and MTZ treatment for establishing a fibrotic liver model in adult. (B-E&#39;) Tg(fabp10a:CFP-NTR), Tg(Tp1:mCherry), and collagen 1a expression in vibratome sections of DMSO- (B and B&#39;) and EtOH/MTZ-treated (C-E&#39;) adult zebrafish livers.(B-B&#39;) In the DMSO-treated controls, collagen 1a was almost undetectable (B&#39;) with no Tg(fabp10a:CFP-NTR) and Tg(Tp1:mCherry) co-expressing cells (B). (C-E&#39;) In the EtOH/MTZ-treated regenerating livers, collagen 1a deposition was markedly elevated at 2, 3, and 4 dpa (C&#39;, D&#39;, E&#39;) with a population of Tg(fabp10a:CFP-NTR) and Tg(Tp1:mCherry) co-expressing cells at 3 and 4 dpa (D, E, insets, white arrows). The bright red NRCs had no CFP expression (D, E, insets, yellow arrowheads). B-E, confocal single plane images. B&#39;-E&#39;, confocal projection images. Scale bar, 20 &#181;m. EtOH, ethanol; MTZ, metronidazole; dpa, days-post-ablation. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/54002/54002fig3.jpg" /> 
Figure 3. Representative chemical screening results using the ethanol-induced fibrotic liver model in larval zebrafish. (A) Scheme for the hepatocyte regeneration screen in the fibrotic liver. (B-E) Known and novel Wnt activators augment hepatocyte regeneration in the fibrotic liver. Confocal projections of the EtOH/MTZ-treated livers in the [Tg(fabp10a:CFP-NTR)gt1; Tg(Tp1:mCherry)jh11] larvae that were administered with DMSO (B), SB415286 (C), CHIR-99021 (D), or a novel Wnt activator [4-(1H-1,2,3,4-tetraazol-5-yl)-1,2,5-oxadiazole-3-ylamine] (abbreviated as HTOA) (E). SB415286 and CHIR-99021 are glycogen synthase kinase-3 inhibitors. All images are confocal projection images. Scale bar, 20 &#181;m. EtOH, ethanol; MTZ, metronidazole; hpa, hours-post-ablation; dpf, days-post-fertilization. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

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 ...

development-an-ethanol-induced-fibrotic-liver-model-zebrafish-to

Nanyang Technological University

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

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, ...