Functional Study Of Gspt1l In Liver Development Of Zebrafish

Liver only exists in vertebrates,which plays an important role in glucose metabolism,lipid metabolism,protein metabolism and toxin metabolism.The development of liver is strictly regulated in space and time.In recent years,the research on liver development has enabled people to have a deeper understanding of the fate determination of liver cells and the molecular mechanism of liver cell differentiation,providing an important theoretical basis for the treatment of liver diseases.Zebrafish has the advantages of in vitro development,transparent embryo,high embryo production and short development span,and has became an ideal model animal to study early embryonic development.Moreover,the molecular mechanism regulating liver development is conservative between human and zebrafish.The liver development of zebrafish can be roughly classified into such periods as Specification,Budding,and Outgrowth.Although numerous studies have revealed the molecular mechanisms that regulate liver specialization and differentiation,the molecular mechanism of the liver growth and differentiation process are not been established.Zebrafish gspt1l?G1 to S phase transition 1,like?is a homologous gene of human GSPT1/eRF3??Eukaryotic peptide chain release factor GTP-binding subunit eRF3??,belonging to the small GTPase family.GSPT1 facilitates termination of peptide chain translation by activating Eukaryotic translation termination factor 1 eRF1 in combination with GTP.Studies have shown that GSPT1 can also induce cell apoptosis through the dissociation of 14-3-3 protein and apoptosis signals-regulating Kinase 1?ASK1?complex.GSPT1 deletion resulted in cell cycle G1?Gap 1 phase?arrest and increased free ribosomal subunits in the cytoplasm.The mutation of gspt1l in zebrafish leads to embryonic death and angiogenesis during development.Foreward genetic screening is an important method to find novel genes.In previous studies,ENU?N-ethyl-N-nitrosourea?was used to randomly induce gene mutations and a series of families with hepatic developmental defects were screened.Among them,the specification and budding of the liver,exocrine pancreas and intestinal progenitor cells in the v28 mutant were normal,but their growth was inhibited.In addition,the delayed expression of the liver differentiation gene lfabp and gc in the v28 mutant suggested that hepatocytes were defective in differentiation.We found that there is a nonsense mutation of gspt1l gene in v28 mutants through positional cloning.In addition,gspt1l wild-type mRNA injection can partially rescue the liver phenotype of v28 mutant,indicating that gspt1l is the mutation gene of v28.gspt1l is expressed in liver,exocrine pancreas and intestinal tract,which is consistent with the phenotype of digestive organ defects in v28mutant.In order to study the role of gspt1l in the development of digestive organs,we first proved that gspt11^v28 mutation affected the growth of liver by reducing the rate of cell proliferation through cell proliferation and apoptosis experiments.It has been reported that the mutation of gspt1l in zebrafish activates the unfolded protein response?UPR?to induce the brain vessels malformation.UPR mainly regulates the stress response of cells under ER stress through ATF6?Activating Transcription Factor 6?,PERK?Double-stranded RNA-Activated Protein Kinase?PKR?–like ER Kinase?and IRE1?Inositol Requiring Enzyme 1?pathways.In the early stage of ER stress,UPR mainly promotes cell survival.When ER stress continues to damage cells,UPR turns to promote apoptosis.In vitro studies found that the cell survival promoting activity of ATF6 and IRE1pathways will gradually decline in the second phase of ER stress,while we found that the activity of IRE1 pathway will not decline in gspt11^v28 mutant.Moreover,inhibition of ER stress by TUDCA?Sodium Tauroursodeoxycholate?,an ER stress inhibitor,can reduce the activity of IRE1 pathway in gspt11^v28 mutants and partially rescue the phenotype of delayed hepatocyte differentiation,but it has no effect on the proliferation defects of liver and other digestive organs.In order to further prove whether the activity of the IRE1 pathway is the cause affecting hepatocyte differentiation,we further used the specific inhibitor of Ire1?,STF-083010,but we found that inhibiting STF-083010 could not rescue the phenotype of delayed hepatocyte differentiation.These results suggest that gspt1l is necessary for the development of liver and other digestive organs,gspt11^v28mutation hinders liver differentiation in zebrafish via the UPR and maybe in an Ire1?RNase activity independent manner.