| The liver is the biggest digestive organ in the body,it plays central roles in metabolic homeostasis.The liver controls the metabolism,synthesis,storage,and redistribution of nutrients,carbohydrates,fats,and vitamins.In addition,the liver functions as the main detoxifying organ,which can remove waste products and harmful xenobiotics through metabolic transformation and bile excretion.80%of the cells in the liver are hepatic parenchyma cells,and the series of liver functions are mainly achieved by the hepatocytes.Therefore,hepatocytes must be maintained in a normal number and health state to ensure the function of the entire liver.When local lesions such as trauma,tumor,or intrahepatic bile duct stones occur,the diseased liver tissue is often surgically removed,ultimately leaving an enough liver size that can maintain normal functions.However,liver resection can lead to reduced liver function and postoperative complications such as infection,bleeding,and liver failure.Thus,promoting liver regeneration after injury can provide new strategies for the therapy of liver diseases.Differ from other organs,the liver is unique for its powerful ability to regenerate after liver injury.A series of evolutionary safeguards guarantee the complex functions of the liver even after severe injury.Deepening the understanding of liver regeneration is very important for the treatment of liver disease and may shed a light on the treatment of cirrhosis.In general,the regeneration of the liver after physical or chemical injury is achieved through the proliferation of residual hepatocytes.However,when hepatocytes are extremely damaged or the proliferation of residual hepatocytes is inhibited,liver regeneration is mainly accomplished by transdifferentiation of biliary cells.This process can be divided into the dedifferentiation of biliary cells and the redifferentiation of bi-potential progenitor cells.In some pathological conditions such as chronic hepatitis or advanced cirrhosis,bi-potential progenitor cells accumulate in the diseased liver,and the number of these progenitor cells in the liver is related to pathogenicity and mortality.Therefore,studying the formation and differentiation of these progenitor cells can provide theoretical basis for the treatment of liver diseases.The biliary-mediated liver regeneration model has been established in recent years.The first one is the zebrafish rapid injury model,which is based on the NTR-Mtz system.The zebrafish model can induce the damage of almost all hepatocytes,and the subsequent liver regeneration process can be observed in a short time.Another model is the mouse drug induced chronic injury model,in which the proliferation of liver cells is inhibited and the whole regeneration process takes a long time to observe.Given that the research models are established recently,although some regulatory factors involved in biliary-derived liver regeneration have been identified in recent years,the understanding of the regulatory mechanisms in this process is far from enough.In this study,we explored the roles of ERK1(extracellular signal-related kinase 1)in biliary-mediated liver regeneration using the zebrafish liver injury model.The Mtz can induce the death of hepatocytes without affecting other cell types such as biliary cells and endothelial cells in the transgenic line Tg(lfabp:DenNTR).By using this model,we first checked the expression of phosphorylated ERK proteins in different regenerative stages after liver injury.The antibody staining of p-Erk1/2 showed that the expressions of phosphorylated ERK proteins were upregulated in biliary-derived cells at 8 hours,24 hours,and 48 hours post-injury,and returned to the level before liver injury.Pharmacological inhibition of the phosphorylation of ERK1/2 led to the reduction of liver size and Dendra2 expression,indicating the requirement of ERK activation in liver regeneration.Then,the results of in situ hybridization showed that the m RNA expression level of ERK2 had no big difference before and after liver injury;however,ERK1 showed increased m RNA expression level at 8 hours,24 hours,and 48hours post-injury,which is similar to the protein expression level of p-Erk1/2.By performing the CRISPR/Cas9 technology,we successfully constructed the mutant lines of ERK1 and ERK2.The liver development showed no obvious defect in the ERK1mutant;however,after Mtz-induced hepatocyte injury,the ERK1 mutant exhibited defective liver regeneration,which is similar to that after ERK1/2 inhibitor treatment.In contrast to the ERK1 mutant,the liver development and regeneration were both normal in the ERK2 mutant,suggesting that ERK signaling pathway exerts its function through ERK1 in the biliary-mediated liver regeneration.To investigate which regenerative stages ERK1 participate in,we checked the dedifferentiation of biliary cells,the proliferation of bi-potential progenitor cells,and the redifferentiation of bi-potential progenitor cells.The biliary cells showed bigger diameter after liver injury in the ERK1 mutant,and the expression of bi-potential progenitor cell markers foxa3,hhex,and sox9b showed normal expression levels in the ERK1 mutant,indicating that the dedifferentiation of biliary cells to bi-potential progenitor cells was unaffected upon ERK1 mutation.In addition,the ERK1 mutants exhibited normal bi-potential cell proliferation and no excessive apoptosis.However,the mature hepatocytes were notably decreased in the regenerating livers of ERK1mutant,and the expressions of hepatocyte markers fabp10a,cp,gc,bhmt,ttr,tfa,and hepatocyte functional marker abcb11b were significantly downregulated in the ERK1mutant.Antibody staining of Dendra2,Alcam,Sox9,and Hnf4?showed that the Alcam~+/Sox9~+,Dendra2~+/Alcam~+,and Alcam~+/Hnf4?~+cells were accumulated in the regenerating livers of ERK1 mutant,indicating the accumulation of bi-potential progenitor cells and the defective redifferentiation of bi-potential progenitor cells to hepatocytes.Surprisingly,by assessing the relative number and bile excretion of nascent biliary cells,we found that the redifferentiation of bi-potential progenitor cells to biliary cells was unaffected upon ERK1 mutation,suggesting the specific regulation of ERK1 in the redifferentiation of bi-potential progenitor cells to hepatocytes.ERK1 is a potential downstream effector of farnesoid X receptor(FXR),and FXR plays significant roles in hepatocyte proliferation-mediated liver regeneration.By characterizing the phenotype of FXR mutant,we found that the dedifferentiation of biliary cells and the proliferation of bi-potential progenitor cells were unaffected,while the redifferentiation of bi-potential progenitor cells was inhibited upon FXR mutation.Thus,the defective regenerative phenotype is similar between FXR and ERK1 mutants.Active ERK signaling was notably reduced,and the transcription of ERK1 was also significantly downregulated in the FXR mutant.After heat-shock induced overexpression of ERK1,the expressions of hepatocyte markers were rescued in the regenerating livers of FXR mutant.However,ERK1 overexpression could not rescue the defective redifferentiation of bi-potential progenitor cells to biliary cells,indicating that FXR regulates the redifferentiation of bi-potential progenitor cells to biliary cells through other effectors.Additionally,we checked if FXR-ERK1 axis regulates liver regeneration in adult zebrafish.The adult FXR mutant also showed defective liver regeneration,and the overexpression of ERK1 could rescue the defective regenerative phenotype,suggesting that the regulation of FXR-ERK1 in liver regeneration is conserved in larval and adult zebrafish.In conclusion,our work demonstrates the essential roles of ERK1 upregulation and activation in liver regeneration after extreme hepatocyte injury,and ERK1 acts as a downstream effector of FXR to control the redifferentiation of bi-potential progenitor cells to hepatocytes. 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