这项工作是由部分从GTEC（2731336和1411318），美国国立卫生研究院（K01DK081351）和美国国家科学基金会（1354837），以CHS补助感谢阿莱姆·乔治斯的手稿的批判性阅读的支持。

斑马鱼中提出和使用符合国家卫生研究院和科技机构动物护理和使用委员会乔治亚理工学院获得批准的标准，一个标准协议孕育。 1.溶液的制备<ol><li>准备20升蛋水（与&#39;胚胎中期"交替使用），以维持胚胎/幼虫斑马鱼。溶解1.5克卡索4和6克250毫升蒸馏水中即时海洋海盐。倒入填充有20升蒸馏水和搅动一个大玻璃瓶。 </li><li>制备1升1-苯基-2-硫脲（PTU）原液（20倍）。在1升蒸馏水溶于水0.6克PTU粉末。将1ml每次20毫升胚胎培养基原液添加到0.003％作为工作溶液中的最终浓度。 注意:PTU可能会导致以下吸入或皮肤接触全身毒性。准备并根据适当的处理准则中使用。 </li><li>制备1升3- aminobenzoate甲磺酸酯（三卡因）原液（20倍）。溶解在蒸馏水4克三卡因粉末。通过加入1M的Tris缓冲液（pH 9）调节pH至7，并且使终体积到1L蒸馏水。分装于50ml并储存在-20°C。使用前解冻。 注意:三卡因可诱发感觉的损失。准备并根据适当的处理准则中使用。 </li><li>制备100ml幼虫甲硝唑（MTZ）的工作方案。通过在100毫升胚胎中期的0.1％二甲基亚砜溶解0.25克MTZ粉（DMSO）做出新的15毫米MTZ解决方案。通过剧烈摇晃完全溶解MTZ粉末。让新鲜MTZ解决方案，并用铝箔，以防止MTZ的光催化降解。 注意:MTZ可能引起刺激。准备并根据适当的处理准则中使用。 </li><li>制备100ml幼虫乙醇/甲硝唑（乙醇/ MTZ）的工作方案。让新鲜MTZ解决方案，并用铝箔，以防止光德MTZ的层次。刚好在使用之前添加1.5毫升100％乙醇入100ml MTZ溶液至1.5％的最终浓度。 </li><li>制成500毫升之成人MTZ工作溶液。通过在500毫升系统溶于水0.83克MTZ粉在0.1％DMSO做出新的10毫米MTZ解决方案。通过剧烈摇晃完全溶解MTZ粉末。让新鲜MTZ解决方案，并用铝箔，以防止MTZ的光催化降解。 </li><li>准备1升PEM缓冲。溶解30.2克PIPES，0.74克EGTA，室温下，用MgSO 4在蒸馏水中。调节pH至7，用NaOH。带来终体积为1升，用蒸馏水。 </li></ol> 2.幼虫斑马鱼的制备<ol><li> [ 的Tg（fabp10a:CFP-NTR）的设置配合GT1; TG（TP1:mCherry）jh11] 15,17与TG（HAND2:EGFP）成年斑马鱼PD24交配坦克13成年斑马鱼。使用分频器进行定时交配。 </li><li>取出Ðivider第二天早上，让鱼不中断交配。收获的胚胎后2小时由紧张系统的水和胚胎转移到百毫米培养皿胚胎中期。 </li><li>保持每培养皿不超过100的胚胎。在立体显微镜，删除使用玻璃吸管未受精的卵子。在28℃生长的胚胎和10小时后，受精后（HPF）添加苯基硫脲（PTU）至0.003％的终浓度，以抑制颜料的发展。 </li></ol> 3.乙醇，甲硝唑治疗与肝再生的斑马鱼幼体<ol><li>加入乙醇到胚胎培养基中的1.5％的终浓度制备新鲜的EtOH溶液。不要添加PTU。 </li><li>对待每个培养皿20毫升乙醇溶液56〜80 HPF胚胎。盖培养皿用保鲜膜，以防止乙醇蒸发。 </li><li>理清[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11 </SUP&gt;; TG（HAND2:EGFP）PD24]幼虫具有类似肝脏大小，由CFP表达式决定，在80 HPF。排序乙醇处理幼虫时，不要使用三卡因，因为这将导致高死亡率与随后乙醇/ MTZ治疗。保持排序的幼虫以每培养皿30的胚胎的密度。 </li><li>在0.1％DMSO准备新鲜的15毫米MTZ胚胎网上平台。添加乙醇适量到MTZ溶液，使1.5％的最终浓度。在28℃培养，每培养皿20ml的EtOH / MTZ溶液中24小时的幼虫。盖培养皿用保鲜膜来维持乙醇浓度，用铝箔避光。 </li><li>在治疗结束后，通过与新鲜胚胎中期幼虫转移到新的培养皿中除去乙醇/ MTZ解决方案。与胚胎中期幼虫洗3次，并保持在20毫升胚胎中期无PTU。 </li><li>要理清与肝细胞几乎完全消融的幼虫，观察荧光与80X的放大倍数在CFP滤波器的落射荧光显微镜下。成功消融导致在肝脏最小CFP荧光和显著降低肝脏体积。 </li><li>为了避免乙醇/ MTZ处理幼虫死亡，不使用三卡因排序。修正了免疫幼虫（参见第5节），或在28℃下保持有序的幼虫在培养皿中作进一步分析。 </li></ol> 4.化学屏幕在乙醇/ MTZ处理斑马鱼幼体<ol><li>带来含有10mM在DMSO化学股96孔板（参考材料清单用于与商业化学文库详情）从-80℃冷冻机中。用铝箔盖，防止化学品的光催化降解。用温和的摇摆解冻储备液在室温。 </li><li>通过稀释的10mM储备溶液与胚胎培养基中至50μM的（在96孔板中的终浓度制备化学溶液:初始小号creen;在1.5ml管:重新测试）。控制幼虫在胚胎中期同等浓度的DMSO处理。 </li><li>近乎完整的肝切除，这与1.5％乙醇/ 15毫米MTZ处理和分类如前所述，要么96孔（初始屏幕）或24孔转移幼虫（复检）板胚培养基的密度预充每孔5幼虫。使用重复的井每一种化学品。 </li><li>除去胚胎培养基和添加或250微升（96孔板）或500微升（24孔平板）的化学溶液每个孔中。盖板用铝箔和治疗幼虫在28℃50小时。每天检查化学浸泡幼虫并从单井任何死幼虫。 </li><li>在化学处理的结束时，在80X放大倍数观察与CFP滤波器的落射荧光显微镜下的CFP表达肝细胞的数目和/或强度。现场照片显示幼虫增强或降低D号码和/或用数码相机对记录CFP表达肝细胞的强度。 </li><li>在第5节保留通过甲醛固定的共焦成像幼虫。 </li><li>要重新测试便于在初始屏幕肝细胞再生的化学药品，检查它们的效力在更高和更低的浓度，以找到在4.1-4.5（&#39;复检&#39;）中描述的方法是最有效的浓度。 </li></ol> 5.斑马鱼幼体固定，免疫染色和共焦成像<ol><li>通过加入三卡因至0.02％的最终浓度麻醉幼虫。传送10-15幼虫到1.5ml管中，用磷酸缓冲盐水（PBS）洗一次。删除PBS和加入1 ml新鲜配制的2％甲醛的PEM缓冲修复O / N在4℃。 小心:甲醛会导致刺激和已知为人类致癌物。处理在化学通风橱。 </li><li>正确丢弃，然后固定解决方案WASH 3次PBS在室温。固定幼虫可以保持在PBS中在4℃下长达数天。在进行下一步骤及时提供了更好的免疫染色的结果。 </li><li>小心地取出蛋黄和皮肤覆盖使用立体显微镜下＃55钳肝区。 </li><li>通透并且在块缓冲块幼虫（4％牛血清白蛋白和0.3％的Triton X-100的PBS）O / N在4℃。 </li><li>用兔抗胶原蛋白I抗体孵育以1:1的稀释度:100在块缓冲器O / N在4℃。洗5次为用洗涤缓冲液（0.3％的Triton X-100的PBS），每次15分钟。 </li><li>在缓冲块Ø200 / N在4°C:用AlexaFluor 647孵育共轭驴抗兔抗体以1稀释。洗5次与洗涤液各15分钟。然后用PBS洗一次。 </li><li>轻轻幼虫转移到利用玻璃吸管，并与左外侧面朝上定向固定幼虫载玻片上。删除多余的PBS使用的Kimwipes。添加安装媒体一滴和密封玻璃盖用指甲油。在进行共焦成像，立即大大建议。 </li><li>使用配有40X / 1.3油透镜捕捉图像共焦系统。在20-50％使用的激光强度。在1微米的间隔拍摄ž堆栈的图像。 </li></ol> 6.准备成年斑马鱼的<ol><li> [ 的Tg（fabp10a:CFP-NTR）的设置配合GT1; TG（TP1:mCherry）jh11] 15,17成年斑马鱼交配坦克。嘉实由紧张系统的水和胚胎转移到100毫米培养皿胚胎中期第二天早上胚胎。 </li><li>保持每培养皿不超过100的胚胎。在立体显微镜，删除使用玻璃吸管未受精的卵子。在28℃生长的胚胎和后10 HPF添加PTU至0.003％的终浓度，以抑制颜料的发展。 </li><li>在4 DPF，使用三卡因麻醉幼虫和梳理[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11]幼虫。通过改变新鲜胚胎培养基几次洗出三卡因。允许，直到他们达到每分钟18次120-180正常的心脏率的幼虫在28°C恢复。 </li><li>提高排序的幼虫基于标准程序19 6-12个月大。 </li></ol> 7.乙醇，甲硝唑治疗和肝再生的成年斑马鱼<ol><li>传输6-12个月[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11]成年斑马鱼使用网交配坦克。保持鱼以每罐不超过10鱼的密度。 </li><li>通过在系统加水EtOH中的1％的终浓度制备新鲜的EtOH溶液。治疗成人鱼在每个水箱500毫升乙醇溶液中，并保持它们在28℃的孵化器。 </li><li>转成鱼新鲜的乙醇溶液每天丢弃死鱼道具erly作为一个生物危害。连续处理动物MTZ治疗前72小时。 </li><li>准备系统新鲜水10毫米MTZ解决方案在0.1％DMSO。预温在28℃培养箱的MTZ解决方案。治疗与1升油箱交叉500毫升MTZ解决8小时的鱼在28℃下，用铝箔避光。 </li><li>在治疗期间每小时观察并立即删除死鱼。正确丢弃死鱼作为一个生物危害。在MTZ治疗结束后，由动物转移到新鲜制水两次洗出MTZ。 </li><li>让动物在孵化器在28℃，恢复和维护。饲料，日用鱼转移到新鲜制水，直至用于分析的时间点。 </li></ol> 8.成年斑马鱼肝固定，免疫染色和共焦成像<ol><li>麻醉成年鱼用0.02％三卡因和在冰水安乐死15分钟。帕特在纸巾上的干鱼，并把它放在解剖垫。 </li><li>通过除去皮肤和肌肉如先前露出胃肠器官描述20。用剪刀从臀鳍切割沿腹部皮肤和下层肌肉厣，然后沿鱼的侧后方回臀鳍。 </li><li>把鱼在15ml锥形管中，并用PBS洗一次。删除PBS和添加4毫升新制备的2％甲醛的PEM缓冲修复O / N在4℃。 </li><li>丢弃正确定影溶液，并在室温用PBS洗3次。固定的样品可以保持在PBS中在4℃下数天。在进行下一步骤立即给出更好的免疫染色的结果。 </li><li>通过切割食道解剖与来自鱼的体腔肝整个肠。嵌入用4％的低熔琼脂糖在切片模具胃肠器官。 </li><li>使用vibratome在50微米的间隔在冰冷PBS切割样品成横截面，从前端开始。转移秒系统蒸发散到用PBS 6孔板用＃1漆刷。 </li><li>通透及缓冲块O /块切片ñ4℃。 </li><li>用兔抗胶原蛋白I抗体孵育以1:1的稀释度:100在块缓冲器O / N在4℃。洗5次，每次用洗涤缓冲液15分钟。 </li><li>在缓冲块Ø200 / N在4°C:用AlexaFluor 647孵育共轭驴抗兔抗体以1稀释。洗5次，15分钟，每用洗涤缓冲液并用PBS洗涤一次。 </li><li>轻轻的部分传送到使用＃1油漆刷载玻片上。删除多余的PBS使用的Kimwipes，加安装媒体的下降，封盖玻璃指甲油。在进行共焦成像立即强烈建议。 </li><li>使用配有40X / 1.3油透镜捕捉图像共焦系统。在20-50％使用的激光强度。在1微米的间隔拍摄ž堆栈的图像。 </li></ol>

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	<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.

我们在乙醇/ MTZ处理回收肝观察HPC介导的肝细胞的再生，这表明即使是在ECM蛋白，包括纤维状I型胶原的显着量的存在下，在高性能计算机保留他们的能力来再生为肝细胞。该MTZ唯一治疗没有增加细胞外基质蛋白沉积显著，而乙醇只能处理没有诱导激活HPC 15。通过利用合并的EtOH / MTZ处理，我们能够调查HPC驱动再生的肝纤维化。由于肝细胞几乎完全消除引起HPC驱动的肝细胞的再生，它是由集群资源公司进行梳理的基础上不存在肝细胞特异性荧光的标准MTZ处理过的幼虫和显著降低肝脏体积的关键。在鱼，鳃和皮肤的血管吸收醇24，所以，没有必要让动物饮随意或需要intragastric输液作为哺乳动物模型。我们住EtOH-和独立坦克随后MTZ处理成鱼无循环水。它每天的鱼转移到新的乙醇溶液中，不断减少乙醇蒸发盖子是必不可少的。虽然48-72小时的EtOH / MTZ治疗有效诱导的ECM蛋白合成中的协议，既不晚期纤维化也不肝硬化已经开发了这些鱼。因此，乙醇长时间接触，如治疗12周14 MTZ的组合，特别是成鱼，可能诱发更严重的肝损伤正常模拟和查询慢性肝功能衰竭HPC驱动的再生。 斑马鱼作为一个优秀的动物模型体内复合测试，由于其体积小，大的后代，透明度和渗透性小分子25。使用斑马鱼的肝纤维化模型中，我们确定了一些已知的和新颖的Wnt ACTI的vators和加速肝细胞的再生缺口15抑制剂。我们目前基于荧光的化学筛选协议包括几个步骤的所有执行手动包括制备化学物质，转印EtOH中处理/肝烧蚀幼虫到24孔板中，治疗化学品，并分析对肝细胞再生的化学品的影响。它专门使用落射荧光和/或共聚焦显微镜来捕获个别动物，这是费力和耗时的图像。如果与确定化学浓度范围自动化药物给药系统组合，可自动处理和获得的斑马鱼的蜂窝分辨率成像与量化数据收集将促进大规模筛选26,27。这些特征的组合的高通量平台将加强28，其可以给最小毒性增强肝细胞再生最大功效。尽管有这些limita肝脏的操作，因为许多关键球员和主要类型的细胞斑马鱼和哺乳动物29之间保守的，采用活体为主的小分子使用斑马鱼肝纤维化模型将促进对慢性肝病新的治疗策略有效的发现筛选。 另外两个小组独立已经表明，肝纤维化无侮辱几乎完全丢失后，转分化能源效益守则向肝细胞分化，通过30,31与假设这一步骤胆管细胞，完全分化的BEC，主要包括在斑马鱼国家资源/能源效益守则。尽管转可以是驱动肝细胞再生的作用机制之一，我们的结果表明，高性能计算机，这是众所周知的占多数的BEC在斑马鱼肝脏32，下调的Notch活性以分化成肝细胞。同时，我们有理由推测，日Ë完全分化的BEC，胆管，保持较高水平的Notch活性无再生过程中转化为肝细胞。有趣的是，在EtOH的存在下，如先前报道13，我们发现，造血干细胞的数目与改变的形态学增加从一个星形配置，以与丢失细胞质的处理的肌成纤维细胞状。活化的HSC特征在于ECM蛋白合成是在肝脏修复4的集成过程负责伤口愈合异常的原理罪魁祸首。虽然慢性损伤往往会覆盖肝脏的再生能力显着，移植成功的高性能计算机重新填充纤维化/肝硬化大鼠肝33。因此，我们的斑马鱼肝纤维化模型将阐明调解持续纤维化对HPC介导的肝细胞的再生效果的分子和细胞机制的重要工具。此外，限定多细胞Çrosstalk，通过利用我们的斑马鱼肝纤维化模型平衡再生和纤维化，将进一步推动设计可行的治疗策略体内慢性肝功能衰竭。

尽管肝细胞1，它是在肝脏的主要实质细胞类型的显着的再生能力，慢性肝衰竭损害这种能力，导致肝祖细胞（HPC）依赖性再生2。 慢性肝损伤主要由酗酒，慢性丙型肝炎病毒（HCV）感染3和非酒精性脂肪肝病（NAFLD）4而得。它导致持续的肝纤维化，这是与细胞外基质（ECM）蛋白的积累相关联。持续ECM积聚通过形成纤维疤痕组织5，随后形成具有高发病率和死亡率肝硬化扭曲完好肝架构。已经进行了许多尝试来通过注重抑制纤维化的细胞因子和活化的肌成纤维细胞6主要减轻纤维化反应。后者主要由肝星状细胞衍生（HSCS）负责肝瘢痕形成4原理肝非实质细胞。尽管如此，刺激内源性细胞来源，包括高性能计算机再生肝细胞纤维化的持续侮辱的存在再生疗法等待进一步的调查。 肝纤维化的许多实验模型已在哺乳动物中已经描述。四氯化碳（CCL 4）的重复注射已广泛用于诱导肝纤维化的小鼠和大鼠模型7。当与高脂肪（HF）饮食组合，酒精导致纤维化基因表达和肝纤维化8的相当上调。虽然脂肪变性（脂肪蓄积）急性酒精暴露的结果，它使肝脏容易受到更严重的肝损伤9。 斑马鱼， 斑马鱼已成为研究为再生一个宝贵的脊椎动物模型系统。虽然其他低等脊椎动物如蝾螈和蝾螈有一个显着的能力，再生，斑马鱼比其他模型系统的优势在问候操纵潜在的再生所需要的基因操作和可视化战略的因素10。斑马鱼也代表了由简单地将乙醇（EtOH中）至其水研究酒精性肝病（ALD）一个有吸引力的脊椎动物模型。急性乙醇暴露在幼虫和成年斑马鱼引起的脂肪肝11-13。当成年斑马鱼接收扩展EtOH中曝光，用的纤维化相关基因14上调观察胶原沉积。然而，需要一种用于开发模型来研究肝脏再生响应于EtOH中作为纤维化刺激。 最近，我们制定了斑马鱼的15乙醇诱导肝纤维化模型。我们幼虫和adul结合乙醇治疗肝细胞特异性基因消融系统Ť斑马鱼。我们生成了两个转基因株系，TG（fabp10a:CFP-NTR）GT1和Tg的（fabp10a:mCherry-NTR）GT2，其中大肠杆菌硝基还原酶（NTR）分别熔接到青色和mCherry荧光蛋白，的控制下的肝细胞特异性脂肪酸结合蛋白10a中，肝基本 （fabp10a）启动子。在这个系统中，NTR转换无毒的前药甲硝唑（MTZ）插入的DNA间链交联剂16，诱导肝细胞的明确的死亡。利用该模型，我们表明，肝细胞，这是响应于Notch信号群，换算成肝细胞，在不久的缺乏肝细胞，并在过量的ECM。我们指定这些细胞为高性能计算机。此外，通过化学的屏幕，我们确定了Wnt信号的小分子活化剂和Notch信号抑制剂增加在肝纤维化肝细胞的再生。 Therefo再次，我们在斑马鱼肝纤维化模型相比，细胞的文化 - 或基于哺乳动物筛选系统代表了一个极好的化学筛选系统。它是与显著成本和节省时间的益处在体内系统中。在这里，我们描述了建立乙醇诱导肝纤维化模型，并用于执行使用斑马鱼这种模式化学屏幕的详细过程。此外，进行了时间过程分析来调查再生如何肝细胞中的肝纤维化发生。该协议将提供一个宝贵的工具来研究机制，并在肝纤维化增强肝细胞的再生战略。

<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.

图1示出的幼虫斑马鱼的乙醇诱导的肝纤维化模型的发展。要优化斑马鱼暴露乙醇的协议，我们首先评估乙醇的毒性。 2.5天-受精后（dpf）的幼虫暴露在乙醇浓度为1％，1.5％或2％为24小时，随后由并发24小时的EtOH / MTZ治疗。暴露于2％的乙醇造成的死亡率高，而几乎所有的幼虫暴露于1％的乙醇或更少表现出与细胞外基质蛋白胶原蛋白的沉积罕见纤维化很小的变化。基于这些结果，幼虫，用1.5％乙醇预处理24小时（2.5-3.5旦），然后同时MTZ处理24小时（3.5-4.5旦）（ 图1A）。在乙醇/ MTZ处理的幼虫形态显示异常，包括躯干和尾巴向上弯曲，心包水肿，失败膨胀鱼鳔。 （ 图1B）。我们使用了三个转基因株系来分析在乙醇/ MTZ处理幼虫肝纤维化的变化:1） 的Tg（fabp10a:CFP-NTR）GT1系表达的肝细胞特异性fabp10a子15根据与青色荧光蛋白融合的NTR基因; 2） 的Tg（TP1:mCherry）jh11线标志着主动Notch信号（Notch的效应细胞，国家资源公司）或胆管上皮细胞（BEC中）核mCherry 17，它表达的是TP1模块包含多个RBP-控制下的细胞JK-结合位点; 3） 的Tg（HAND2:EGFP）PD24线，标签肝星状细胞（HSC）13。 DMSO处理的对照[ 的Tg（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11; TG（HAND2:EGFP）PD24]肝脏未见纤维状I型胶原沉积4,6,9（ 图1C，C&#39;），而乙醇/ MTZ-治疗肝显示高架I型在25和50小时，后消融（HPA）（ 图1D，D&#39;，E，E&#39;）胶原蛋白的积累。此外，相对于DMSO处理的对照肝脏（ 图1C&#39;&#39;），造血干细胞的数量增加了与改变的形态从一个星形配置在肌成纤维细胞状与乙醇/ MTZ处理再生肝（丢失胞质突起图1D&#39;，E&#39;&#39;）。我们观察到mCherry表达对国家资源公司的两个分立的人口在该乙醇/ MTZ处理再生肝25 HPA:昏暗的红色资源公司（ 图1D&#39;&#39;&#39;，插图，白色箭头 ）和鲜红的国家资源（ 图1D&#39;&#39;&#39;，插图，黄色箭头 ）。在50 HPA，肝细胞特异性CFP开始共表达在昏暗mCherry表达国家资源在整个再生肝脏（ 图1E&#39;&#39;&#39;，插图，白色箭头 ）。这些CFP和昏暗mCherry-coexpressing国家资源公司从明亮的红色资源公司是阴性CFP（ 图1E&#39;&#39;&#39;，插图，黄色箭头 ）明显区别的。以往的研究表明，当肝细胞增殖肝部分切除（PHX）后抑制，高性能计算机扩大了与造血干细胞21的伴随增殖。此外，高性能计算机和的BEC共享组织化学标记物22。因此，我们的结果表明，在CFP和暗淡mCherry共表达国家资源描绘，通过在遇到的Notch下调分化成肝细胞高性能计算机的斑马鱼-对应。维持较高水平的Notch信号传导的国家资源可以保持作为胆管，它们是有区别的积案23。 图2示出了在成年斑马鱼的乙醇诱导的肝纤维化模型的发展。首先，我们暴露成年斑马鱼至1％，1.5％，或2％的乙醇，以评估生存能力。大多数成年斑马鱼做ñOT生存暴露于EtOH中的浓度大于1％（未发表的数据）的超过72小时。因此，对于成年斑马鱼中，我们预处理鱼用1％乙醇72小时，随后用MTZ处理（ 图2A）。通过进行时间过程的分析，我们发现显著升高的I型在2,3胶原沉积在乙醇/ MTZ处理再生肝，和4天后消融（DPA）（ 图2C&#39;，D&#39;，E&#39;）相比DMSO处理肝脏（ 图2B&#39;）。类似于在幼虫观察到的，在12个月大的[Tg的（fabp10a:CFP-NTR）的乙醇/ MTZ处理的再生肝中检测到在CFP和暗淡mCherry共表达细胞GT1; TG（TP1:mCherry）jh11]成鱼在第3和4 DPA（ 图2D，E，镶石，白色箭头 ）。这些数据表明，高性能计算机，响应于Notch信号传导，保持它们的再生为在肝细胞中的能力纤维化侮辱的成年斑马鱼的存在。 图3显示了使用我们的乙醇诱导肝纤维化模型在斑马鱼幼体代表的化学筛选结果。要找准能够加速肝细胞的再生的肝纤维化生物活性化合物，我们进行了化学遗传筛选。我们筛选的75小分子具有良好的特点生物医药活动（干细胞信号复合图书馆，Selleckchem），并使用[ 的Tg（fabp10a 1000不太表征小分子（ActiProbe-1K图书馆，TimTec）一库一库:CFP -NTR）GT1; TG（TP1:mCherry）jh11]幼虫。我们烧蚀肝细胞在1.5％的EtOH / 15毫MTZ的存在下，然后暴露幼虫的化合物的50μM的50小时（ 图 3A）。许多的Wnt激动剂如SB 415286和CHIR-99021，糖原合酶激酶-3，促进肝细胞的再生（ 图3C，D）相比的DMSO（ 图3B）的抑制剂。此外，[4-（1H-1,2,3,4- tetraazol -5-基）-1,2,5-恶二唑-3-基胺]，缩写为HTOA，一种新型的Wnt途径激活剂，增强肝细胞的再生中通过更大的再生肝（ 图3E）指示的肝纤维化。这些数据表明，我们的持续纤维化模型提供了一个宝贵的工具来发现小分子可增强肝细胞的再生。 <img alt="图1" src="/files/ftp_upload/54002/54002fig1.jpg" /> 图1.发展斑马鱼幼体乙醇诱导肝纤维化模型。（A）方案说明在幼虫建立肝纤维化模型乙醇和乙醇/ MTZ治疗时期。 （B）在50值班后消融（HPA），乙醇/ MTZ处理的L-arval斑马鱼发展形态异常，包括躯干和尾巴向上弯曲，心包水肿和失败膨胀鱼鳔。 （CC&#39;&#39;&#39;）在[Tg为肝脏（fabp10a:CFP-NTR）GT1; TG（TP1:mCherry）jh11; TG（HAND2:EGFP）PD24]幼虫用DMSO处理，ECM胶原蛋白1A几乎检测不到（C，C&#39;）和静态造血干细胞表现出明星般的配置（C&#39;&#39;），而国家资源肝细胞（C，C&#39;&#39;&#39;，插图 ）之间分配。 （DE&#39;&#39;&#39;）在EtOH / MTZ处理幼虫的肝脏中，观察到升高的胶原1a中沉积（D，D&#39;，E，E&#39;）。造血干细胞的数目增加，失去了复杂的胞浆突起（D&#39;，E&#39;&#39;）。在25百帕，mCherry表达国家资源公司是由两个不同的种群。黄色箭头指示鲜红资源公司（D&#39;&#39;&#39;，插图 ），而白色箭头指示暗红资源公司（D&#39;&#39;&#39;，插图 ）。在50百帕，TG（fabp10a:CFP-NTR）和TG（TP1:mCherry）共同表达细胞开始出现在整个再生肝脏（E&#39;&#39;&#39;，插图，白色箭头 ），而鲜红的国家资源没有CFP表达（E&#39;&#39;&#39;，插图，黄色箭头 ）。所有共焦图象是除C&#39;&#39;，D&#39;，E&#39;&#39;单平面图像，这是投影图像。比例尺:B，100微米; CE&#39;&#39;&#39;，20微米。乙醇，乙醇; MTZ，甲硝唑; HPA，小时，消融后; DPF，天受精后。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> 图2.发展成年斑马鱼乙醇诱导肝纤维化模型。（A）方案说明在成年建立肝纤维化模型乙醇和MTZ治疗时期。 （BE&#39;） 的Tg（fabp10a:CFP-NTR），TG（TP1:mCherry）和胶原1A表达DMSO-（B和B vibratome节"）和乙醇/ MTZ处理（CE&#39;）成年斑马鱼的肝脏。 （BB&#39;）在DMSO处理的对照，胶原1a中几乎检测不到（B&#39;）中 ，没有Tg的（fabp10a:CFP-NTR）到Tg（TP1:mCherry）共表达细胞（B）。 （CE"）（，D&#39;，E&#39;用在乙醇/ MTZ处理再生肝，胶原1a中沉积明显在2，3和4 DPA℃）升高&#39;和TG（TP1:mCherry）的Tg（CFP-NTR fabp10a）的人口共表达细胞在第3和4 DPA（D，E，镶石，白色箭头 ）。明亮的红色资源公司没有CFP表达（D，E，镶石，黄色箭头 ）。BE，共聚焦单一平面图像。B&#39;-E"，共焦投影图像。比例尺，20微米。乙醇，乙醇; MTZ，甲硝唑; DPA，天后消融。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig2large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/54002/54002fig3.jpg" /> 图使用斑马鱼幼体对乙醇诱导肝纤维化模型代表性的化学筛选结果。（A）计划在肝纤维化的肝细胞的再生屏幕。 （BE）已知和新颖的Wnt信号激活增强肝细胞的再生的肝纤维化。在[:GT1; 的Tg（TP1:mCherry） 的Tg（CFP-NTR fabp10a）jh11]的乙醇/ MTZ治疗肝脏共聚焦预测幼虫用DMSO（B），SB415286（C），CHIR-99021（管理D）中 ，或一种新的Wnt激活[4-（1H-1,2,3,4- tetraazol -5-基）-1,2,5-恶二唑-3-基胺]（缩写为HTOA）（E）。 SB415286和CHIR-99021是糖原合酶激酶-3抑制剂。所有图像都焦投影图像。比例尺，20微米。乙醇，乙醇; MTZ，甲硝唑; HPA，小时，消融后; DPF，天受精后。 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54002/54002fig3large.jpg" target="_blank">请点击此处查看该图的放大版本。</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

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

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 次观看.  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. 

视频: 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.

使用提顿体系的研究斑马鱼再生的分子机制

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

科研

发育生物学

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.

肝细胞特异性消融在斑马鱼研究胆驱动肝再生

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.

鼠标子宫内膜上皮细胞和基质细胞的隔离<em&gt;体外</em&gt;蜕膜

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.

斑马鱼急性酒精性肝损伤的组织学分析

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.

斑马鱼幼虫动物模型的建立研究锥虫运动性体内

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.

定量造血干和祖细胞 (HSPCs) 在斑马鱼胚胎发育中的体外试验研究

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.

部分小叶切除术: 形态学肝再生的外科模型

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.

斑马鱼胚胎全山原位杂交和管状测定在iPSC-ECs中研究恩多林在血管发育中的作用

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.

为梭状体开发拉瓦尔斑马鱼感染模型

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.

检查斑马鱼肌肉疾病模型中的肌肉再生

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

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的​​肝细胞的再生

乙醇引起的肝纤维化模型的斑马鱼发展研究祖细胞介导的肝细胞的再生