The authors thank Drs. Hukriede and Tsang for discussions. M.K. was supported by an NIH training grant (T32EB001026). This work was supported in part by grants from the American Liver Foundation, the March of Dimes Foundation (5-FY12-39), and the NIH (DK101426) to D.S.

Zebrafish were raised and bred according to standard procedure; experiments were approved by the University of Pittsburgh Institutional Animal Care and Use Committee.
1. Preparation of Embryos/larvae
<ol>
	<li>To conduct timed matings, set up adult male and female Tg(fabp10a:CFP-NTR)s931 hemizygous or homozygous fishes O/N and place a divider between them. Remove this divider the following morning when mating is desired. Collect embryos by straining the water using a fine plastic mesh strainer.</li>
	<li>Turn the strainer upside down and rinse the mesh surface with a fine stream of egg water dispensed from a squeeze bottle. Transfer 100 embryos into a 100&#160;mm petri dish with 25 ml of egg water. Keep no more than 100 embryos per petri dish and raise them at 28 &#176;C.</li>
	<li>To inhibit pigmentation, add phenylthiourea (PTU) into the petri dishes prior to 10 hr post-fertilization (hpf), and raise embryos/larvae until 80 hpf. The final concentration of PTU is 0.2 mM. CAUTION: PTU is toxic. Use in accordance with appropriate handling guidelines.</li>
	<li>Anesthetize the larvae by adding 3-amino benzoic acid ethyl ester (Tricaine), at a final concentration of 0.016%, into the petri dishes. Then, using an epifluorescence microscope, sort CFP-positive larvae. Use larvae with a similar liver size and discard those with a smaller or larger liver. 
	NOTE: any CFP-positive larvae, regardless of intensity, can be used because there is no difference in the extent of hepatocyte ablation between the hemizygous and homozygous larvae. CAUTION: tricaine is toxic. Use in accordance with appropriate handling guidelines.</li>
	<li>Remove the egg water containing Tricaine and add egg water supplemented with 0.2 mM PTU. Keep the embryos/larvae at 28 &#176;C.</li>
</ol>
2. Mtz Treatment
<ol>
	<li>Prepare fresh 10 mM Mtz solution by dissolving 0.171 g of Mtz powder in 100 ml egg water supplemented with 0.2% DMSO and 0.2 mM PTU. To completely dissolve the Mtz, mix the solution at RT&#160;with rapid stirring for 20-30 minutes. To help solubilize Mtz and to enhance Mtz permeation into the larvae, add DMSO when preparing Mtz. CAUTION: prolonged exposure or increased concentration of Mtz is toxic. Use in accordance with appropriate handling guidelines.</li>
	<li>Separate the larvae into control and experimental groups by transferring desired number of larvae into two different 100&#160;mm petri dish or multi-well plate. For the experimental group, keep the larvae in 10 mM Mtz solution; for the control group, keep them in egg water containing 0.2% DMSO.</li>
	<li>Cover the plates or petri dishes containing the Mtz solution with aluminum foil to prevent the photo-inactivation of Mtz, and incubate at 28 &#176;C. The duration of Mtz treatment depends on the larval stage and the transgenic line.</li>
	<li>To observe similar severe hepatocyte ablation in the Tg(fabp10a:CFP-NTR) line, treat the larvae with the Mtz solution from either 3.5 days post-fertilization (dpf) (for a duration of 36 hr) or from 5 dpf (for a duration of 24 hr).</li>
</ol>
3. Analysis of Biliary-driven Liver Regeneration
<ol>
	<li>At the end of the ablation period, remove the Mtz solution from the plates. Wash the Mtz-treated larvae by adding 25 ml of egg water. Swirl the plate 2-3 times to wash the larvae before discarding the egg water. Repeat three times and then immerse the larvae once more in egg water. To avoid heart edema and larval death in the Mtz-treated larvae, add a lower concentration of Tricaine, at a final concentration of 0.008%, to immobilize the larvae.</li>
	<li>For liver analysis, examine the liver size of the Mtz-treated larvae under an epifluorescence microscope with the CFP filter. Assess hepatocyte ablation levels based on the relative liver size: large (non-ablated; 0-10% larvae), medium (partially ablated; 10-20%), and very small (fully ablated; 80-90%).</li>
	<li>Remove the larvae with non-ablated or partially ablated livers. Only keep larvae with a tiny liver, which is indicative of severe hepatocyte ablation. For liver analysis at the regeneration 0 hr time point (R0h), harvest the larvae after Mtz washout and CFP sorting. Otherwise, keep the sorted larvae in egg water supplemented with 0.2 mM PTU until ready for harvesting.</li>
	<li>Transfer harvested larvae into a 1.5&#160;ml microcentrifuge tube and replace egg water with the fixation solution of 3% formaldehyde in PEM buffer. Incubate the tube on a rotator O/N at 4 &#176;C.</li>
	<li>Replace the fixation solution with 1 ml of PBSDT and rotate the tube containing the larvae on the rotator for 5 min.</li>
	<li>Transfer the fixed larvae into a 100&#160;mm petri dish containing PBSDT and using forceps, manually remove yolk and pectoral fins under a dissecting microscope. Transfer up tp 20 dissected larvae into a 1.5&#160;ml tube.</li>
	<li>Replace PBSDT solution with 1 ml of blocking solution and rock the tube on a rotator at RT for 2 hr.</li>
	<li>Replace blocking solution with 100 &#181;l of blocking solution containing primary antibodies. Slowly rock the tube on a rotator O/N&#160;at 4&#176;C.</li>
	<li>Remove the primary antibody solution and wash with PBSDT 5 times for 10 min each time. Then add 100 ul of blocking solution containing secondary antibodies and rock it on a rotator at RT for 2hr.</li>
	<li>Wash 5 times with PBSDT for 10 min each time at RT by rocking on a rotator.</li>
	<li>Orient the larvae laterally on a microscope slide and add a drop of mounting media. Carefully cover them with a cover glass and seal the cover glass with a nail polisher. Now, it is ready for confocal microscopy.</li>
</ol>

<ol>
	<li>Riehle, K. J., Dan, Y. Y., Campbell, J. S., Fausto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+concepts+in+liver+regeneration.">New concepts in liver regeneration.</a> J Gastroen Hepatol. 26, 203-212 (2011).</li><li>Fausto, N., Campbell, 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=The+role+of+hepatocytes+and+oval+cells+in+liver+regeneration+and+repopulation.">The role of hepatocytes and oval cells in liver regeneration and repopulation.</a> Mechanisms of Development. 120, 117-130 (2003).</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, 466-481 (2009).</li><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:+Alternative+epithelial+pathways.">Liver regeneration: Alternative epithelial pathways.</a> Int J Biochem Cell B. 43, 173-179 (2011).</li><li>Falkowski, O., 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=Regeneration+of+hepatocyte+'buds'+in+cirrhosis+from+intrabiliary+stem+cells.">Regeneration of hepatocyte 'buds' in cirrhosis from intrabiliary stem cells.</a> Journal of hepatology. 39, 357-364 (2003).</li><li>Yoon, S. 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=Epithelial+Cell+Adhesion+Molecule+(EpCAM)+Marks+Hepatocytes+Newly+Derived+from+Stem/Progenitor+Cells+in+Humans.">Epithelial Cell Adhesion Molecule (EpCAM) Marks Hepatocytes Newly Derived from Stem/Progenitor Cells in Humans.</a> Hepatology. 53, 964-973 (2011).</li><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, 286-300 (2007).</li><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+after+Partial+Hepatectomy+Critical+Analysis+of+Mechanistic+Dilemmas.">Liver Regeneration after Partial Hepatectomy Critical Analysis of Mechanistic Dilemmas.</a> Am J Pathol. 176, 2-13 (2010).</li><li>Yanger, K., 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=Adult+hepatocytes+are+generated+by+self-duplication+rather+than+stem+cell+differentiation.">Adult hepatocytes are generated by self-duplication rather than stem cell differentiation.</a> Cell stem cell. 15, 340-349 (2014).</li><li>Schaub, J. R., Malato, Y., Gormond, C., Willenbring, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evidence+against+a+Stem+Cell+Origin+of+New+Hepatocytes+in+a+Common+Mouse+Model+of+Chronic+Liver+Injury.">Evidence against a Stem Cell Origin of New Hepatocytes in a Common Mouse Model of Chronic Liver Injury.</a> Cell reports. 8, 933-939 (2014).</li><li>Dovey, 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=Topoisomerase+II+alpha+is+required+for+embryonic+development+and+liver+regeneration+in+zebrafish.">Topoisomerase II alpha is required for embryonic development and liver regeneration in zebrafish.</a> Molecular and cellular biology. 29, 3746-3753 (2009).</li><li>Goessling, W., 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=APC+mutant+zebrafish+uncover+a+changing+temporal+requirement+for+wnt+signaling+in+liver+development.">APC mutant zebrafish uncover a changing temporal requirement for wnt signaling in liver development.</a> Developmental Biology. 320, 161-174 (2008).</li><li>Sadler, K. C., Krahn, K. N., Gaur, N. A., Ukomadu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Liver+growth+in+the+embryo+and+during+liver+regeneration+in+zebrafish+requires+the+cell+cycle+regulator,+uhrf1.">Liver growth in the embryo and during liver regeneration in zebrafish requires the cell cycle regulator, uhrf1.</a> Proceedings of the National Academy of Sciences of the United States of America. 104, 1570-1575 (2007).</li><li>Cox, A. G., 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=S-nitrosothiol+signaling+regulates+liver+development+and+improves+outcome+following+toxic+liver+injury.">S-nitrosothiol signaling regulates liver development and improves outcome following toxic liver injury.</a> Cell reports. 6, 56-69 (2014).</li><li>North, T. 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=PGE2-regulated+wnt+signaling+and+N-acetylcysteine+are+synergistically+hepatoprotective+in+zebrafish+acetaminophen+injury.">PGE2-regulated wnt signaling and N-acetylcysteine are synergistically hepatoprotective in zebrafish acetaminophen injury.</a> Proc Natl Acad Sci U S A. 107, 17315-17320 (2010).</li><li>Curado, S., 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=Conditional+targeted+cell+ablation+in+zebrafish:+A+new+tool+for+regeneration+studies.">Conditional targeted cell ablation in zebrafish: A new tool for regeneration studies.</a> Developmental Dynamics. 236, 1025-1035 (2007).</li><li>Pisharath, H., Rhee, J. M., Swanson, M. A., Leach, S. D., Parsons, 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=Targeted+ablation+of+beta+cells+in+the+embryonic+zebrafish+pancreas+using+E.+coli+nitroreductase.">Targeted ablation of beta cells in the embryonic zebrafish pancreas using E. coli nitroreductase.</a> Mech Dev. 124, 218-229 (2007).</li><li>Curado, S., Stainier, D. Y. R., 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, 948-954 (2008).</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, 776-788 (2014).</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. , (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, 789-800 (2014).</li><li>Sakaguchi, T. F., Sadler, K. C., Crosnier, C., 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=Endothelial+signals+modulate+hepatocyte+apicobasal+polarization+in+zebrafish.">Endothelial signals modulate hepatocyte apicobasal polarization in zebrafish.</a> Curr Biol. 18, 1565-1571 (2008).</li><li>Ninov, N., Borius, M., Stainier, D. Y. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+levels+of+Notch+signaling+regulate+quiescence,+renewal+and+differentiation+in+pancreatic+endocrine+progenitors.">Different levels of Notch signaling regulate quiescence, renewal and differentiation in pancreatic endocrine progenitors.</a> Development. 139, 1557-1567 (2012).</li><li>Lorent, K., Moore, J. C., Siekmann, A. F., Lawson, N., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reiterative+use+of+the+notch+signal+during+zebrafish+intrahepatic+biliary+development.">Reiterative use of the notch signal during zebrafish intrahepatic biliary development.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 239, 855-864 (2010).</li><li>Itoh, T., Miyajima, 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+Regeneration+by+Stem/Progenitor+Cells.">Liver Regeneration by Stem/Progenitor Cells.</a> Hepatology. 59, 1617-1626 (2014).</li><li>Mathias, J. R., Zhang, Z., 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=Enhanced+cell-specific+ablation+in+zebrafish+using+a+triple+mutant+of+Escherichia+coli+nitroreductase.">Enhanced cell-specific ablation in zebrafish using a triple mutant of Escherichia coli nitroreductase.</a> Zebrafish. 11, 85-97 (2014).</li><li>Stueck, A. E., Wanless, I. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regression+of+cirrhosis:+The+maturation+sequence+of+buds+arising+from+hepatocyte+progenitor+cells.">Regression of cirrhosis: The maturation sequence of buds arising from hepatocyte progenitor cells.</a> Hepatology. 58, 235A (2013).</li></ol>

The authors declare that they have no competing financial interests.

Severe, but not mild, hepatocyte ablation elicits biliary-driven liver regeneration. Therefore, following Mtz treatment and washout, it is important to examine the liver size of the Mtz-treated larvae, which can be assessed by intrinsic CFP fluorescence from the fabp10a:CFP-NTR transgene. Since a small percentage of the Mtz-treated larvae will display non-ablated (0-5%) or partially ablated (10-20%) livers, it is essential to sort out and only analyze the larvae with a very small liver, which is indicative of se...

The liver is a highly regenerative organ. During liver regeneration, regenerating hepatocytes, the parenchymal cells of the liver, are derived from pre-existing hepatocytes (hepatocyte-driven liver regeneration) or BECs (biliary-driven liver regeneration)1,2. Liver injury usually elicits the proliferation of pre-existing hepatocytes; however, when hepatocyte proliferation is compromised, BECs can contribute to hepatocytes2-4. These two modes of liver regeneration are clinically significant. Upon surgical removal of a portion of the human liver (e.g., because of liver tumors or live liver donors), hepatocytes in the remaining liver proliferate to recover the lost liver mass. By contrast, in patients with severe liver diseases, hepatocyte proliferation is greatly compromised, so that BECs or liver progenitor cells (LPCs) appear to contribute to regenerating hepatocytes5,6. The rodent 2/3 partial hepatectomy model, in which hepatocytes proliferate to recover the lost liver mass, has significantly contributed to the current understanding of hepatocyte-driven liver regeneration7,8. However, there is no valid rodent model in which regenerating hepatocytes are mainly derived from BECs. Although several rodent liver toxin models led to the identification of biliary-driven liver regeneration2-4, recent lineage tracing studies in mice indicate a minimal contribution of BECs to regenerating hepatocytes in these models9,10. Some of the rodent liver injury models, including partial hepatectomy11-13 and acetaminophen-induced liver damage14,15, have been applied to zebrafish and led to the identification of novel genes or pathways implicated in liver regeneration. However, hepatocyte-driven, but not BEC-driven, liver regeneration occurs in these zebrafish liver injury models. Therefore, a novel liver injury model in which BECs extensively contribute to regenerating hepatocytes is needed for a better understanding of BEC-driven liver regeneration.
The overall goals of the hepatocyte ablation model described here are (1) to generate a liver injury model in which BECs extensively contribute to regenerating hepatocytes, and (2) elucidate the molecular and cellular mechanisms underlying BEC-driven liver regeneration. We hypothesized that severity of injury determines the mode of liver regeneration; thus, we predicted that biliary-driven liver regeneration would initiate upon severe hepatocyte injury. To test this hypothesis, we developed a zebrafish liver injury model by generating a transgenic line, Tg(fabp10a:CFP-NTR)s931, that highly expresses bacterial Nitroreductase (NTR) fused with cyan fluorescent protein (CFP) under the hepatocyte-specific fabp10a promoter. Since NTR converts the non-toxic prodrug, metronidazole (Mtz), into a cytotoxic drug, it ablates only the intended NTR-expressing cells16-18, in this case, the hepatocytes. By manipulating the duration of Mtz treatment, the extent of hepatocyte ablation can be controlled. Using this model, we recently reported that upon severe hepatocyte loss, BECs extensively give rise to regenerating hepatocytes19, which was further confirmed by two other independent studies20,21. Therefore, compared to the aforementioned rodent and zebrafish liver injury models, our hepatocyte ablation model is more advantageous for studying BEC-driven liver regeneration.
This protocol describes the procedure for performing liver regeneration experiments using the zebrafish hepatocyte ablation model. This model will be appropriate for determining the mechanisms underlying biliary-driven liver regeneration and for chemical screens to identify small molecules that can repress or augment liver regeneration.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>3-amino benzoic acid ethyl ester (Tricaine) powder</td>
			<td>Sigma-Aldrich</td>
			<td>A5040</td>
			<td>Make 0.4% (15 mM) tricaine stock : Bring 0.4 g tricaine to 100 mL with egg water. Adjust to pH 7.&#160;</td>
		</tr>
		<tr>
			<td>Dimethyl sulfoxide (DMSO)</td>
			<td>Sigma-Aldrich</td>
			<td>D8418</td>
			<td></td>
		</tr>
		<tr>
			<td>Metronidazole</td>
			<td>Sigma-Aldrich</td>
			<td>M1547</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sigma-Aldrich</td>
			<td>T8532</td>
			<td></td>
		</tr>
		<tr>
			<td>Formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium sulfate dihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>C3771</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Phenylthiourea (PTU)</td>
			<td>Sigma-Aldrich</td>
			<td>P7629</td>
			<td></td>
		</tr>
		<tr>
			<td>Mounting media (Vectashield)</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>100 x 15 mm petri dish&#160;</td>
			<td>Denville Scientific Inc.</td>
			<td>M5300</td>
			<td></td>
		</tr>
		<tr>
			<td>clear nail polish</td>
			<td>Fisher Scientific</td>
			<td>50949071</td>
			<td></td>
		</tr>
		<tr>
			<td>fine forceps</td>
			<td>Fine Science Tools</td>
			<td>11251-20</td>
			<td>Dumont #5&#160;</td>
		</tr>
		<tr>
			<td>glass slide</td>
			<td>Fisher Scientific</td>
			<td>12-544-1</td>
			<td></td>
		</tr>
		<tr>
			<td>glass coverslip</td>
			<td>Fisher Scientific</td>
			<td>12-540-A and 12-542-A</td>
			<td>18 x 18 mm</td>
		</tr>
		<tr>
			<td>zn-5 (mouse anti-Alcam)</td>
			<td>ZIRC</td>
			<td>zn-5</td>
			<td>1:10 dilution</td>
		</tr>
		<tr>
			<td>goat anti-Hnf4a</td>
			<td>Santa Cruz</td>
			<td>sc-6556 (C-19)</td>
			<td>1:50 dilution</td>
		</tr>
		<tr>
			<td>rat anti-RFP</td>
			<td>Allele Biotechnology</td>
			<td>ACT-CM-MRRFP10</td>
			<td>1:300 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Donkey Anti-goat IgG (H+L)</td>
			<td>Jackson laboratories</td>
			<td>705-605-147</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>AlexaFluor 647 AffiniPure Goat Anti-mouse IgG (H+L)</td>
			<td>Invitrogen</td>
			<td>A21240</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>Cy3 donkey anti-rat IgG</td>
			<td>Jackson laboratories</td>
			<td>712-165-150</td>
			<td>1:500 dilution</td>
		</tr>
		<tr>
			<td>dissecting microscope</td>
			<td>Leica Microsystems</td>
			<td>S6F</td>
			<td></td>
		</tr>
		<tr>
			<td>epifluorescence microscope</td>
			<td>Leica Microsystems</td>
			<td>M205FA</td>
			<td></td>
		</tr>
		<tr>
			<td>confocal microscope</td>
			<td>Carl Zeiss</td>
			<td>LSM700</td>
			<td></td>
		</tr>
		<tr>
			<td>egg water</td>
			<td></td>
			<td></td>
			<td>0.3 g/L of sea salts &#8216;Instant Ocean&#8217; and 0.5 mM CaSO4 in distilled water.</td>
		</tr>
		<tr>
			<td>10X PBS</td>
			<td></td>
			<td></td>
			<td>25.6 g Na2HPO4&#183;7H2O, 80 g NaCl, 2 g KCl, 2 g KH2PO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.&#160; Autoclave for 20 min at 121&#176;C.</td>
		</tr>
		<tr>
			<td>1X PBSDT</td>
			<td></td>
			<td></td>
			<td>0.2% Triton X-100 and 0.2% DMSO in 1 X PBS.</td>
		</tr>
		<tr>
			<td>PEM buffer</td>
			<td></td>
			<td></td>
			<td>30.2 g PIPES, 761 mg EGTA, 1 mM MgSO4.&#160; Bring to 1 L with distilled water.&#160; Adjust pH 7.</td>
		</tr>
		<tr>
			<td>Blocking solution</td>
			<td></td>
			<td></td>
			<td>Mix 1 ml of heat-inactivated horse serum with 9 ml of 1X PBSDT.</td>
		</tr>
	</tbody>
</table>

hepatocyte-specific ablation in zebrafish to study biliary-driven liver regeneration

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.

Mtz treatment for 36 hr (A36h, A stands for ablation) from 3.5 to 5 dpf dramatically reduced liver size (Figure 1B). After Mtz washout, considered as the beginning of liver regeneration (R), strong fabp10a:CFP-NTR and fabp10a:dsRed expression reappeared within 30 hr (Figure 1B). The intrahepatic biliary network, as assessed by expression of Alcam that is present in the membrane of BECs22, initially collapsed but was re-established at 54 hr post-washout (R54h)...

Watch this Scientific Journal Video about Hepatocyte-specific Ablation in Zebrafish to Study Biliary-driven Liver Regeneration at JoVE.com

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

To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

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

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8.5K Views.  University of Pittsburgh. To help assess the molecular mechanisms underlying zebrafish biliary-driven liver regeneration, we established a liver injury model in which the nitroreductase-expressing hepatocytes are genetically ablated upon metronidazole treatment. In this protocol, we describe how to adeptly manipulate, monitor and analyze hepatocyte ablation and biliary-driven liver regeneration.

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

Kidney Regeneration in Adult Zebrafish by Gentamicin Induced Injury

The kidney is essential for fluid homeostasis, blood pressure regulation and filtration of waste from the body. The fundamental unit of kidney function is the nephron. Mammals are able to repair existing nephrons after injury, but lose the ability to form new nephrons soon after birth. In contrast to mammals, adult fish produce new nephrons (neonephrogenesis) throughout their lives in response to growth requirements or injury. Recently, lhx1a has been shown to mark nephron progenitor cells in the adult zebrafish kidney, however mechanisms controlling the formation of new nephrons after injury remain unknown. Here we show our method for robust and reproducible injury in the adult zebrafish kidney by intraperitoneal (i.p.) injection of gentamicin, which uses a noninvasive visual screening process to select for fish with strong but nonlethal injury. Using this method, we can determine optimal gentamicin dosages for injury and go on to demonstrate the effect of higher temperatures on kidney regeneration in zebrafish.

Here we present a reliable method to study adult kidney regeneration by inducing acute kidney injury by gentamicin injection. We show that injury is dependent on gentamicin dosage and environmental temperature using in situ hybridization to label lhx1a+ developing new nephrons.

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

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 ...

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

Sustained liver fibrosis with continuation of extracellular matrix (ECM) protein build-up results in the loss of cellular competency of the liver, leading to cirrhosis with hepatocellular dysfunction. Among multiple hepatic insults, alcohol abuse can lead to significant health problems including liver failure and hepatocellular carcinoma. Nonetheless, the identity of endogenous cellular sources that regenerate hepatocytes in response to alcohol has not been properly investigated. Moreover, few studies have effectively modeled hepatocyte regeneration upon alcohol-induced injury. We recently reported on establishing an ethanol (EtOH)-induced fibrotic liver model in zebrafish in which hepatic progenitor cells (HPCs) gave rise to hepatocytes upon near-complete hepatocyte loss in the presence of fibrogenic stimulus. Furthermore, through chemical screens using this model, we identified multiple small molecules that enhance hepatocyte regeneration. Here we describe in detail the procedures to develop an EtOH-induced fibrotic liver model and to perform chemical screens using this model in zebrafish. This protocol will be a critical tool to delineate the molecular and cellular mechanisms of how hepatocyte regenerates in the fibrotic liver. Furthermore, these methods will facilitate potential discovery of novel therapeutic strategies for chronic liver disease in vivo.

Sustained fibrosis with deposition of excessive extracellular matrix proteins leads to cirrhosis. Alcohol abuse is one of the main causes of severe liver disease. We established an ethanol-induced zebrafish fibrotic liver model to study the mechanisms and strategies of promoting hepatocyte regeneration upon alcohol-induced injury.

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

Surgical Ablation Assay for Studying Eye Regeneration in Planarians

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their abundant pluripotent stem cell population and ability to regenerate all cell and tissue types after injuries that would be catastrophic for most animals. Recently, planarians have gained popularity as a model for eye regeneration. Their ability to regenerate the entire eye (comprised of two tissue types: pigment cells and photoreceptors) allows for the dissection of the mechanisms regulating visual system regeneration. Eye ablation has several advantages over other techniques (such as decapitation or hole punch) for examining eye-specific pathways and mechanisms, the most important of which is that regeneration is largely restricted to eye tissues alone. The purpose of this video article is to demonstrate how to reliably remove the planarian optic cup without disturbing the brain or surrounding tissues. The handling of worms and maintenance of an established colony is also described. This technique uses a 31 G, 5/16-inch insulin needle to surgically scoop out the optic cup of planarians immobilized on a cold plate. This method encompasses both single and double eye ablation, with eyes regenerating within 1-2 weeks, allowing for a wide range of applications. In particular, this ablation technique can be easily combined with pharmacological and genetic (RNA interference) screens for a better understanding of regenerative mechanisms and their evolution, eye stem cells and their maintenance, and phototaxic behavioral responses and their neurological basis.

This protocol shows how to consistently excise planarian eyes (optic cups) without disturbing surrounding tissues. Using an insulin needle and syringe, either one or both eyes can be ablated to facilitate investigations into the mechanisms regulating eye regeneration, the evolution of visual regeneration, and the neural basis of light-induced behavior.

In the study of adult stem cells and regenerative mechanisms, planarian flatworms are a staple in vivo model system. This is due in large part to their ...

Histological Analyses of Acute Alcoholic Liver Injury in Zebrafish

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related morbidity and mortality and affects more than 2 million people in the United States. A better understanding of the cellular and molecular mechanisms underlying alcohol-induced liver injury is crucial for developing effective treatment for ALD. Zebrafish larvae exhibit hepatic steatosis and fibrogenesis after just 24 h of exposure to 2% ethanol, making them useful for the study of acute alcoholic liver injury. This work describes the procedure for acute ethanol treatment in zebrafish larvae and shows that it causes steatosis and swelling of the hepatic blood vessels. A detailed protocol for Hematoxylin and Eosin (H&#38;E) staining that is optimized for the histological analysis of the zebrafish larval liver, is also described. H&#38;E staining has several unique advantages over immunofluorescence, as it marks all liver cells and extracellular components simultaneously and can readily detect hepatic injury, such as steatosis and fibrosis. Given the increasing usage of zebrafish in modeling toxin and virus-induced liver injury, as well as inherited liver diseases, this protocol serves as a reference for the histological analyses performed in all these studies.

This protocol describes histological analyses of the livers from zebrafish larvae that have been treated with 2% ethanol for 24 h. Such acute ethanol treatment results in hepatic steatosis and swelling of the hepatic vasculature.

Alcoholic Liver Disease (ALD) refers to damage to the liver due to acute or chronic alcohol abuse. It is among the leading causes of alcohol-related ...

Methods for the Study of Regeneration in Stentor

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model organism to study wound healing and subsequent cell regeneration. The Stentor genome became available recently, along with modern molecular biology methods, such as RNAi. These tools make it possible to study single-cell regeneration at the molecular level. The first section of the protocol covers establishing Stentor cell cultures from single cells or cell fragments, along with general guidelines for maintaining Stentor cultures. Culturing Stentor in large quantities allows for the use of valuable tools like biochemistry, sequencing, and mass spectrometry. Subsequent sections of the protocol cover different approaches to inducing regeneration in Stentor. Manually cutting cells with a glass needle allows studying the regeneration of large cell parts, while treating cells with either sucrose or urea allows studying the regeneration of specific structures located at the anterior end of the cell. A method for imaging individual regenerating cells is provided, along with a rubric for staging and analyzing the dynamics of regeneration. The entire process of regeneration is divided in three stages. By visualizing the dynamics of the progression of a population of cells through the stages, the heterogeneity in regeneration timing is demonstrated.

Methods for the Study of Regeneration in Stentor

The giant ciliate, Stentor coeruleus, is an excellent system to study regeneration and wound healing. We present procedures for establishing Stentor cell cultures from single cells or cell fragments, inducing regeneration by cutting cells, chemically inducing the regeneration of membranellar band and oral apparatus, imaging, and analysis of cell regeneration.

Cells need to be able to regenerate their parts to recover from external perturbations. The unicellular ciliate Stentor coeruleus is an excellent model ...

Quantifying Liver Size in Larval Zebrafish Using Brightfield Microscopy

In several transgenic zebrafish models of hepatocellular carcinoma (HCC), hepatomegaly can be observed during early larval stages. Quantifying larval liver size in zebrafish HCC models provides a means to rapidly assess the effects of drugs and other manipulations on an oncogene-related phenotype. Here we show how to fix zebrafish larvae, dissect the tissues surrounding the liver, photograph livers using bright-field microscopy, measure liver area, and analyze results. This protocol enables rapid, precise quantification of liver size. As this method involves measuring liver area, it may underestimate differences in liver volume, and complementary methodologies are required to differentiate between changes in cell size and changes in cell number. The dissection technique described herein is an excellent tool to visualize the liver, gut, and pancreas in their natural positions for myriad downstream applications including immunofluorescence staining and in situ hybridization. The described strategy for quantifying larval liver size is applicable to many aspects of liver development and regeneration.

Here we demonstrate a method for quantifying liver size in larval zebrafish, providing a way to assess the effects of genetic and pharmacologic manipulations on liver growth and development.

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

Confocal Microscope-Based Laser Ablation and Regeneration Assay in Zebrafish Interneuromast Cells

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally replenished in non-mammalian vertebrates such as zebrafish. The zebrafish lateral line system is a useful model for characterizing sensory hair cell regeneration. The lateral line is comprised of hair cell-containing organs called neuromasts, which are linked together by a string of interneuromast cells (INMCs). INMCs act as progenitor cells that give rise to new neuromasts during development. INMCs can repair gaps in the lateral line system created by&#160;cell death. A method is described here for selective INMC ablation using a conventional laser-scanning confocal microscope and transgenic fish that express green fluorescent protein in INMCs. Time-lapse microscopy is then used to monitor INMC regeneration and determine the rate of gap closure. This represents an accessible protocol for cell ablation that does not require specialized equipment, such as a high-powered pulsed ultraviolet laser. The ablation protocol may serve broader interests, as it could be useful for the ablation of additional cell types, employing a tool set that is already available to many users. This technique will further enable the characterization of INMC regeneration under different conditions and from different genetic backgrounds, which will advance the understanding of sensory progenitor cell regeneration.

Laser ablation is a widely applicable technology for studying regeneration in biological systems. The presented protocol describes use of a standard laser-scanning confocal microscope for laser ablation and subsequent time-lapse imaging of regenerating interneuromast cells in the zebrafish lateral line.

Hair cells are mechanosensory cells that mediate the sense of hearing. These cells do not regenerate after damage in humans, but they are naturally ...

Examining Muscle Regeneration in Zebrafish Models of Muscle Disease

Skeletal muscle has a remarkable ability to regenerate following injury, which is driven by obligate tissue resident muscle stem cells. Following injury, the muscle stem cell is activated and undergoes cell proliferation to generate a pool of myoblasts, which subsequently differentiate to form new muscle fibers. In many muscle wasting conditions, including muscular dystrophy and ageing, this process is impaired resulting in the inability of muscle to regenerate. The process of muscle regeneration in zebrafish is highly conserved with mammalian systems providing an excellent system to study muscle stem cell function and regeneration, in muscle wasting conditions such as muscular dystrophy. Here, we present a method to examine muscle regeneration in zebrafish models of muscle disease. The first step involves the use of a genotyping platform that allows the determination of the genotype of the larvae prior to eliciting an injury. Having determined the genotype, the muscle is injured using a needle stab, following which polarizing light microscopy is used to determine the extent of muscle regeneration. We therefore provide a high throughput pipeline which allows the examination of muscle regeneration in zebrafish models of muscle disease.

Skeletal muscle regeneration is driven by tissue resident muscle stem cells, which are impaired in many muscle diseases such as muscular dystrophy, and this results in the inability of muscle to regenerate. Here, we describe a protocol that allows the examination of muscle regeneration in zebrafish models of muscle disease.

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

Differentiation and Mechanical Stimulation of hiPSC-Derived Endothelial Cells

In Vitro Model of Fetal Human Vessel On-chip to Study Developmental Mechanobiology

The heart is the first organ to be functionally established during development, thus initiating blood circulation very early in gestation. Besides transporting oxygen and nutrients to ensure fetal growth, fetal circulation controls many crucial developmental events taking place within the endothelial layer through mechanical cues. Biomechanical signals induce blood vessel structural changes, establish arteriovenous specification, and control the development of hematopoietic stem cells. The inaccessibility of the developing tissues limits the understanding of the role of circulation in early human development; therefore, in vitro models are pivotal tools for the study of vessel mechanobiology. This paper describes a protocol to differentiate endothelial cells from human induced pluripotent stem cells and their subsequent seeding into a fluidic device to study their response to mechanical cues. This approach allows for long-term culture of endothelial cells under mechanical stimulation followed by&#160;retrieval of the endothelial cells for phenotypical and functional characterization. The in vitro model established here will be instrumental to elucidate&#160;the intracellular molecular mechanisms that transduce the signaling mediated by mechanical cues, which ultimately orchestrate vessel development during human fetal life.

Author Spotlight: Studying hiPSC-Derived Endothelial Cells Cultured Under Fluidic-Mediated Mechanical Stimulation 

Described here is a simple workflow to differentiate endothelial cells from human pluripotent stem cells followed by a detailed protocol for their mechanical stimulation. This allows for the study of the developmental mechanobiology of endothelial cells. This approach is compatible with downstream assays of live cells collected from the culture chip after mechanical stimulation.

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