| The liver is a crucial digestive organ with a central role in metabolic homeostasis.Hepatocytes make up the majority of a liver and carry out several critical functions including production of bile,storage of glycogen,detoxification,as well as regulation of blood homeostasis.Therefore,the study of liver physiology and pathology should also be one of the main research objects in the field of modern basic medicine and biology.Degenerative disease is a result of continuous degenerative cell changes process,which affect tissues or organs function and increasingly deteriorate over time.Reduced liver function carries severe consequences and,if not reversed,can be a primary cause of death.The genes and molecular regulatory networks underlying organ degeneration therefore are of great research and clinical interest,are incompletely characterized.So better understanding the molecular mechanisms involved in the liver degeneration processes is essential for development of novel drug therapies and cure the disease.Inflammation is triggered by microbial pathogens or danger signals derived from the host.And antigen-presenting cells express receptors that can sense microbial products and endogenous signals released by damaged cells,and that activation of these receptors initiates an inflammatory response.Some reports have showed that inflammation is a central component of most chronic liver diseases,inhibition the activation of inflammasome which are multiprotein complexes that can sense danger signals from damaged cells and pathogens could cure the diseases.The hepatic macrophage-derived pro-inflammatory cytokine TNF-α is a major mediator of both acute and chronic inflammatory responses in liver diseases.NF-κB pathway is wildly reported have important functions in liver physiology and pathology.Inactive NF-κB is located in the cytoplasm.Once it becomes phosphorylated and activated,it is translocated to the nucleus where it binds to promoters of specific target genes and can upregulate the transcription of cytokines and chemokines.The genetic interaction of TNF-α and NF-κB pathway has been demonstrated as an important signaling that regulates immune response as well as homeostasis of liver.The zinc finger proteins contain a large superfamily,and most of them are CCHH or CCCC type.The CCCH type zinc finger-containing proteins are only represent 0.8% of all zinc finger-containing proteins,but they are very unusual in mammalian genomes.Several CCCH type zinc finger proteins have reported function as destabilizing the messenger RNA of TNF-α or ubiquitin ligase to repress inflammatory response and autoimmunity.Zebrafish has been recognized as a powerful model organism for the study of liver development and regeneration owing to its embryological and genetic advantages.In this study,we used the forward genetic screening to identify genes that regulate liver development and regeneration in zebrafish.And,we found a mutant,cq5,that exhibited a severe liver degeneration phenotype in embryos at 5 days post fertilization,the developmental stage when a functional liver has developed.And also the mutant fish exhibit a shorter life span at later stage(after 5 dpf)comparing with the wild-type ones.Carefully analysis the mutant phenotype,we found the cq5 mutant embryos develop normally and having no defect in the digestive organs(liver,intestine and pancreas)before 4 dpf,however,after 4 dpf the mutant larvae exhibit digestive organs gradually loss and degradation,especially in the liver.In order to check the earlier development of cq5 mutant embryos for the endoderm organs,we cross the mutant with Tg(sox17:GFP),and found no obvious difference for the mutant and WT.Several transgenic lines which separately marked the hepatocytes(Tg(fabp10a:Dendra2-NTR)),intestine(Tg(cdh17:GFP))and pancreas(Tg(p48:GFP))were used tracing different organs degeneration process.And we found the intestine and exocrine pancreas were also showing same degeneration as hepatocytes in the cq5 mutant embryos.Mutant gene positional cloning was wildly used to map ENU mutation genes.With the completion of the zebrafish genome sequencing,we could effectively locate the mutant gene in the chromosome and accurately find out its mutation site.Luckily,we identified a typical zinc finger protein Zc3h8(zinc finger CCCH-type containing 8)which located at chromosome 13 was the cq5 mutation gene and contribution to the mutant degeneration phenotype.The Zc3h8 protein contain three repeat and conserved CCCH-type zinc finger domains,the mutation of cq5 result the last amino acid of the second domain changing from histidine to glutamine.By using the genome editing technique to knock out the candidate gene,we found the knockout homozygous of zc3h8 embryos showing same phenotypic characteristics consistent with cq5 mutant.This finally suggested zc3h8 was the cq5 mutant gene.In order to know the expression patterns of zc3h8,we detected its expression by in situ hybridization.And found zc3h8 was highly expressed in the hepatocytes at 5 dpf,but no obvious expression was found in the intestine and pancreas.Transgenic overexpression of zc3h8 gene in wild type embryos,and found that the Zc3h8 protein was localized in cell nucleus expression,suggesting that Zc3h8 might perform its role as a transcription factors,regulate the transcription level of some specific genes.Cell proliferation and apoptosis are two major cellular behaviors for organs homeostasis maintaining.We assessed the proliferation and cell apoptosis for the cq5,and noticed lower proliferation(EdU staining)and higher apoptosis(TUNEL staining)which could be considered as the major contributor to organic degeneration in the mutant liver compared with WT at degeneration later stage(5.5 dpf).Crossing cq5 with the macrophage transgenic fish line(Tg(mpeg1:eGFP)),showed obviously phagocytosis of dead cell in the mutant liver and gut.At the same time,transcriptome sequencing and bioinformatics analysis of sorting hepatocytes for the cq5 mutant embryos during the process of degeneration,we found liver cell specific marker genes were significantly down-regulated in the degeneration process.NF-κB signal related genes,as well as pro-inflammatory related factors,are markedly up-regulated in the mutant degenerated liver and gut.Further checking by real-time PCR and in situ hybridization showed that these genes and pro-inflammatory cytokines were up-regulated in the mutant liver during the entire degeneration.Anti-body staining of phosphorylated p65(RelA),which translocated into the nucleus indicated activation of NF-κB pathways,also showed the NF-κB pathway was just activated in the hepatocytes of mutant liver.These observations suggested that NF-κB signal indeed have function for the liver degeneration.And this also expand the zc3h8 gene function relationship with NF-κB pathway although we have no direct evidence to prove it.Chemical inhibited the inflammatory response or NF-κB signal activity by Dexamethasone and JSH-23)could reduce the liver degradation caused by the mutation.This indicated active NF-κB signal and inflammatory contribute to the mutant degeneration process,but didn?t know which one is the primary problem.These findings suggest that the high level of inflammatory reaction and related NF-κB pathway activation were the major reason of mutant liver degeneration at developmental later stage,and consistent with such a role for the CCCH-type zinc finger proteins in human and mouse,and providing a new sight for research zebrafish zc3h8 gene function in liver homeostasis and negative regulation of inflammatory response.Studies have shown that proinflammatory cytokines may induce activation of the NF-kappa B signal,and activation of the NF-kappa B signal can also induce the release of a series of inflammatory factors and the occurrence of inflammatory responses.In order to distinguish the cq5 mutant phenotype in degradation of causality,we conducted a series of experiments show that,suppressed the NF-κB signaling activity could reduce the inflammatory cytokines expression in intestinal mutants(il1b and mpx).At the same time,this reduced rescue effect of the apoptotic cells also showed in the mutant individuals.Tracing the time point of activation of the NF NF-κB signal in the liver,we found the NF-κB signal was activated in the hepatocytes prior to the degeneration of the liver(4.5 dpf).This is earlier than the time at which liver apoptotic cells appear in large numbers(5 dpf).All of these observations indicate the importance roles of Zc3h8 in maintain metabolism and defend degeneration for the liver.In summary,our work found Zc3h8 playing important role for maintain hepatocytes homeostasis,once it was mutated may cause the digestive organ degeneration.And this process was dependent on the inflammation responds and its related molecules and pathways.Under the normal physiological conditions,Zc3h8 may play an important role in maintaining homeostasis of the liver and other digestive organs(pancreas and intestine)in zebrafish.Although,as a transcription factor expressed in the nucleus,the Zc3h8 whether direct interaction with NF-κB signal was still can not completely understand.But its logic relationship with pro-inflammatory factor was clear,and these findings coincident with the function reported in mice and human for the other type CCCH zinc finger proteins.The research about the Zc3h8,help us to better understand the effect of degeneration related genes mutation and their function for maintain organ development in zebrafish.And also,these finding broad our understanding of the zinc finger protein in repressing inflammation response and provide a new sight for treat digestive organs diseases in clinical. |
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