Molecular Mechanisms Of Tel2 Governing Zebrafish Biliary-driven Liver Regeneration

The liver is an essential organ with remarkable regenerative capacities in vertebrates.Upon acute and mild liver injury,the high-rate proliferation of hepatocytes and biliary epithelial cells(BECs)restores its mass and function.Although the normal liver has a fantastic regenerative capacity,following acute injury or resection,the regenerative ability becomes overwhelmed in two essential scenarios:in the setting of severe acute liver injury or when there is a severe chronic liver injury with aberrant liver architecture and marked liver fibrosis.These scenarios are clinically relevant and often result in severe morbidity and mortality.While there have been decades of research into understanding the signals that control regeneration of the normal liver,the mechanisms at play when the abnormal liver attempts regeneration are less well described.Understanding how regeneration fails or is impaired in the severely damaged liver is an important goal.Lessons learned from relevant animal models might have importance in the clinical setting and aid the development of new therapies to either promote regeneration or prevent defective restoration that arise during liver regeneration.Currently,a unique cell type called bipotential progenitor cells(BP-PCs)with intermediate features between hepatocytes and BECs was found in severe liver diseases.Recent studies uncovered that BECs could firstly dedifferentiate into BP-PCs after extreme hepatocyte ablation,then redifferentiate into hepatocytes and BECs to accomplish liver regeneration in zebrafish.This transdifferentiation from BECs into hepatocytes is conserved in mice chronic liver injury models.Moreover,given that conversion of BECs to hepatocytes called BEC-driven liver regeneration plays a crucial part in the regeneration of severe liver damage,the profound elucidation on mechanisms underlying BEC-driven liver regeneration is necessary.In terms of the conserved features of the liver regeneration process and regulatory mechanisms across different species in vertebrates,zebrafish as an ideal animal model has been applied to liver regeneration research.The liver functions in zebrafish larval of bile production,serum protein secretion,and lipogenesis are fully operational at 5 days post fertilization(dpf)as in adults.Thus,zebrafish larvae are suitable for the study of liver regeneration after 5 dpf.Based on the conversion of the prodrug Metronidazole(Mtz)to a cytotoxic DNA cross-linking agent by the bacterial Nitroreductase(NTR)enzyme,the NTR/Mtz system has been successfully used to establish conditional targeted cell ablation and regeneration models in zebrafish.Using the NTR/Mtz system,we and others independently ablated near-total hepatocytes in zebrafish to study BEC-driven liver regeneration.Previous reports revealed the essential role of BMP signaling,Notch signaling,mTORCl signaling,BET proteins,Hdacl,and FXR in BEC-driven liver regeneration.On the other hand,we and others found that multiple specific transcription factors of liver development were reused in liver regeneration,including Hhex,Foxa3,and Sox9b.However,the molecules and mechanisms on the specific regulation of liver regeneration,the re-activation of developmental regulators,and how these regulators affect the BEC-driven liver regeneration remain unknown.Tel2 was originally isolated in a mutant yeast S.cerevisiae strain with short telomeres.Then several orthologs among different organisms:nematode Rad-5/Clk-2,Drosophila LqfR,mammalian hClk2/Telo2,and plant Tel2 were recognized.These Tel2 orthologs have the evolutionally conserved protein sequence:telomere length regulation domain.Initially,the functions of Tel2 on telomere length regulation and telomeric DNA localization were identified both in yeast and nematode.However,mammalian Tel2 was verified to exhibit no obvious telomere function.Tel2 also performs a conserved function in all eukaryotic organisms examined so far:promoting assembly,stabilizing,and maintaining the proteins of the phosphatidylinositol 3-kinase-related protein kinase(PIKK)family as a cochaperone.The PIKK family comprises ATM,ATR,DNA-PK,and all proteins involved in DNA damage response;SMG1,a protein involved in nonsensemediated mRNA decay;TRRAP,a protein required for transcriptional control;and mTOR,a regulator of cell metabolism and proliferation in response to environmental cues and stress.Although Tel2 as a collaborator of PIKKs could potentially illustrate its spatiotemporal and tissue-specific functions in cellular activities in eukaryotes,including in zebrafish,little is known about its role in BEC-driven liver regeneration.In this study,zebrafish as a model organism was used to investigate the molecular mechanisms of liver regeneration.Using the NTR/Mtz system,we specifically induced zebrafish hepatocyte ablation and constructed a zebrafish model of BEC-driven liver regeneration.With a large-scale N-ethylnitrosourea(ENU)-mediated forward genetic screen,we explored essential genes that could specifically affect liver regeneration in zebrafish.We found that a stably inherited liver logan(lvl)cq122 mutant exhibited immature swim bladders during development.But early development of the liver,intestine,and exocrine pancreas of the lvl mutant was normal before 5 dpf,which was checked by in situ hybridizations of cp,fabp10a,fabp2,and trypsin.Nevertheless,liver regeneration in lvl mutant exhibited severe defects,and we considered that lvl was a specifically liver regeneration defective mutant.To comprehend the molecular nature of the phenotype in lvl mutant,we identified the target gene of lvl mutant by genetic mapping and located the mutation site to tel2 genomic loci,which caused the prematurely terminated translation of Tel2 protein.We found that tel2 was consistently highly expressed in BECs and BECderived cells during liver regeneration by whole-mount in situ hybridization(WISH)and fluorescent in situ hybridization(FISH)coupled antibody staining.Another tel2 mutant line tel2cq123,constructed using the CRISPR/Cas9 system,similarly exhibited severe defects of liver regeneration.We performed rescue experiments using the Tg(hsp70l:Tel2GFP)cq125 line to overexpress WT zebrafish Tel2 upon heat shock and found that overexpression of Tel2 could essentially rescue the deficiency of regenerating liver size.Therefore,loss-of-function mutation of tel2 provokes the lvl mutant phenotype of impaired liver regeneration.By testing Proxl and Hnf4a,we found that the tel2 mutation did not affect the dedifferentiation of BECs during liver regeneration.In contrast,BODIPY FL C5,in situ hybridizations of cp and gc,and antibody staining of Bhmt and Alcam indicated that the tel2 mutation severely repressed the redifferentiation of BP-PCs during zebrafish liver regeneration.Using ChIP-PCR assay and zebrafish telomere length assay,we found that zebrafish Tel2 lost the ability of telomeric localization.Moreover,tel2 mutation did not affect the stability of zebrafish telomere length,indicating that Tel2 might perform capacities independent of telomeric function to affect liver regeneration in zebrafish.After detecting the re-activation of key regulators of liver development during BECdriven liver regeneration in zebrafish by WISH and FISH coupled antibody,we found that tel2 mutation did not affect the transcription of foxa3 and sox9b but severely inhibited the re-activation of hhex transcription.Using the Tg(soxl7:GFP)s870 transgenic line,we found little expression of tel2 in early endoderm,indicating that tel2 could specifically affect the re-activation of hhex transcription during liver regeneration.The heterozygous mutant of hhexcq124 constructed by the CRISPR/Cas9 system exhibited significantly smaller regenerating livers.WISH showed that the expression of cp,gc,and bhmt was severely decreased in hhexcq124 heterozygous mutant,and antibody staining of Bhmt was also suppressed in BP-PCs.The hhex heterozygous mutant exhibited a similar defect in liver regeneration as the tel2 mutant.Using the Tg(hsp70l:Hhex-GFP)cq127 line to overexpress Hhex upon heat shock,we found that overexpression of Hhex partially rescued the defective liver regeneration in the tel2 mutant,and the expression of mature hepatocyte markers during liver regeneration was rescued as well.Furthermore,we enhanced the expression of hhex exclusively in BECs using Cre/loxP-based genetic lineage with Tg(tpl:CreERT2)jh12 and Tg(hsp70l:loxP-mCherry-STOP-loxP-Hhex-Myc)cq128 line.We found that BEC-specific overexpression of Hhex alleviated the deficiency of the regenerating liver in the tel2 mutant,suggesting that Tel2 governed liver regeneration with a cell-autonomous process through Hhex.Meanwhile,we revealed that the proliferation of BEC-derived cells was reduced at ROh and hardly could be detected at R24h and R48h in tel2 mutant as measured by antibody staining of PCNA.Whereas the tel2 mutation did not induce apoptosis as measured by TUNEL labeling and yH2AX.Moreover,the proliferation of BECs and BEC-derived cells was partially rescued in tel2 mutant from R0h to R48h by Hhex overexpression.Altogether,we identified a novel mutation in tel2 that can specifically lead to defective liver regeneration in zebrafish.Further investigations indicate that tel2 modifies BECdriven liver regeneration by regulating the redifferentiation and proliferation of BP-PCs.We also uncover that the deficiency of liver regeneration in tel2 mutant is partially related to the blockade of hhex transcription.This study suggests Tel2/Hhex pathway is necessary for BEC-driven liver regeneration.