| The liver is an important digestive organ of the human body.It is involved in various functions of human life activities,including glycogen metabolism,fat metabolism,amino acid metabolism,bile acid metabolism,detoxification and internal waste metabolism to maintain homeostasis.Hepatocytes comprise the majority of the liver and are involved in most liver functions,such as the synthesis of endogenous and exogenous blood proteins and coagulation factors,the storage of glycogen,amino acids,fats and iron,and the secretion of bile.Liver diseases,including hepatitis B,cirrhosis and hepatocellular carcinoma(HCC),are important health problems worldwide,with a very high number of deaths from different liver diseases each year,posing a great threat to human life and social development.Therefore,it is of great significance to elucidate liver development and its cellular and molecular mechanisms.The liver originates from the anterior enteroderm of the abdomen.After liver specialization,liver progenitor cells exfoliate from the endoderm and invade the septum and cross the interlobe to form liver buds.The growth and development of hepatic buds were followed by cell proliferation and volume expansion.Hepatic progenitor cells differentiate into two types of epithelial cells,each of which is arranged into a unique structure and has a unique function.The two main types of epithelial cells in the liver are hepatocytes and bile duct epithelial cells,derived from progenitor cells in liver formation known as hepatocytes.Hepatocytes make up most of the cellular structure of the liver,while bile duct epithelial cells form the bile duct network.The two cells are spatially separated in the liver,where the bile duct adjoins the portal vein to the hepatic artery and is located in the portal vein area of the liver,also known as the "portal triplet".Hepatocytes are widely distributed in the hepatic "lobule",the functional unit of the liver.The bile duct system in mammals consists of intrahepatic and extrahepatic bile ducts,whose main function is to transport bile from the liver to the intestine.Bile duct dysfunction can lead to the accumulation of bile in the liver,impedes the excretion of liver metabolites,and ultimately leads to liver damage,such as Alagille syndrome,biliary atresia and other congenital biliary diseases.The formation of biliary ducts requires the coordination of cell interaction so as to regulate the differentiation and morphogenesis of bile duct epithelial cells.Zebrafish are bony fish whose liver has different histological characteristics from that of mammals.The portal vein,hepatic artery,and biliary tract are randomly distributed in the liver parenchyma,but not orderly arranged in the portal tract as in mammals.The zebrafish hepatocytes surround the timid tubes,rather than the double array of hepatocyte plates found in mammals.The intrahepatic bile duct consists of the bile ducts and the bile duct forming a biliary network.Bile is collected in the gallbladder through large ducts and the extrahepatic biliary system.The structure of the zebrafish liver is somewhat different from that of mammals,but the most basic cell types are the same,such as liver cells and bile duct epithelial cells.Zebrafish liver has a very strong ability of regeneration,which can quickly rebuild a new liver to meet the needs of the body under the condition of severe acute injury.In the whole process of regeneration,the bile duct system,as the source of new liver cells,achieves the regeneration of the liver through transdifferentiation.Zebrafish bile duct system includes two parts,respectively is the intrahepatic bile ducts and extrahepatic duct,the source of development is not the same: intrahepatic bile ducts is by hepatic progenitor cell differentiation,extrahepatic duct is produced by hepatic progenitor cells of the pancreatic duct,both a progenitor cells derived from the endoderm,together constitute a complete network of bile duct of bile duct of liver inside and outside.Thanks to the model animal advantage of zebrafish,large-scale screening of ENU chemically-induced mutants can be carried out,thus enabling us to obtain the desired tissue and organ mutants.In our lab,NAP mutants were selected as the mutants related to bile duct development phenotypes.The bile duct transgenic background was obtained through passage,which was convenient for phenotypic observation and mechanism research in vitro.We detected the NAP mutant by antibody staining of labeled bile duct epithelial cell ALCAM and in situ hybridization of molecular marker genes ANXA4 and CLDN15A1 B for intrahepatic bile ducts.We focused our attention on the development of intrahepatic bile ducts in NAP mutants.Such mutations related to the loss of bile duct differentiation are relatively rare.By positional cloning technology,so as to locate the nap of mutations to 14 chromosome,chromosome walking after further,the mutations of the fragment size shrunk to only five candidate gene,and then by sequencing of exon we found frmpd3 gene code,show the child on the bases of 16 point mutation,causing the encoded amino acids have also changed,by injecting the Morpholino frmpd3 gene observing embryonic bile duct phenotype to determine frmpd3 gene mutations cause nap mutation type surface.The reasons for the loss of intrahepatic bile duct in NAP mutant can be roughly divided into two parts.One is that the bile duct has been differentiated and exists only due to the apoptosis of bile duct cells,which leads to the loss of bile duct in later development.TUNEL staining of the cells denied this explanation.Another is in the liver bile duct cells differentiation of progenitor cells to the bile duct epithelial cells blocked leading to hepatic progenitor cells can't to the bile duct epithelial cell differentiation,after we test confirmed that this kind of situation,that is to say,nap is the absence of bile duct of mutant phenotypes due to hepatic progenitor cells to the result of bile duct epithelial cells differentiation.So what signal caused by the lack of bile duct epithelial cells cannot be differentiated,normal we tested the most direct impact on the liver bile duct divided the notch signal,found that the notch signal receptor ligands jagged1 b,notch1a hes5a/b and target gene are cut,there has been a marked that nap mutant bile duct epithelial cell differentiation is due to lower notch signal in the mutant.In addition,jagged1 B mutates in humans and zebrafish and also causes a decrease in the number of intrahepatic bile ducts similar to the phenotype of NAP mutants,so we speculate that there may be some relationship between the two.We synthesized the coding sequence of the extracellular protein of JAGged1 B through in vitro transcription.After the mutant injected with m RNA,we found that the bile duct deletion phenotype of NAP mutant could be partially rescued,and the number of bile ducts in the normal liver was still reduced compared with that in normal liver.Therefore,the bile duct deletion phenotype of NAP mutants is caused by the blocking of liver progenitor cells to bile duct epithelial cells due to the downregulation of Jagged1b/Notch signal,which leads to the bile duct deletion phenotype in nap mutants at the later stage of development.In this study,large-scale chemical mutagenesis screening was carried out to obtain NAP mutant associated with intrahepatic bile duct deletion.The mutant fr MPD3 was found through localization cloning and gene sequencing,and the endogenous knockdown was performed.The phenotype generated was consistent with the NAP mutant body surface type,which confirmed that FRMPD3 was the mutant gene of NAP mutant.Further study of NAP phenotype confirmed that bile duct deletion was caused by blocked differentiation of bile duct epithelial cells.Because of the effect of notch signal is the most direct signal,the differentiation of intrahepatic bile duct by detecting notch signaling pathways changes in genes for notch signal in the mutant genes were significantly lowered,by using jagged1 b back to save experiment found that can save nap back part of its phenotype,shows through jagged1 b frmpd3 / notch signal to regulate the liver progenitor cells to the bile duct epithelial cell differentiation,at the same time resulting from jagged1 b mutations in human phenotype of Allah's syndrome and nap similar phenotypes,The study of the developmental molecular mechanisms of NAP mutants may also provide theoretical and clinical assistance for the disease. |
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