| The pancreas is a unique organ of vertebrates originating from the endoderm,which is divided into endocrine and exocrine glands.The exocrine gland of the pancreas is composed of an acinar tissue that produces digestive enzymes,a ductal system for transporting these enzymes,and participates in food digestion.Insufficient secretion of enzymes can affect the digestion and absorption of food,leading to various diseases such as hypoproteinemia,fatty diarrhea,and endocrine disorders.Although there are few cases of congenital exocrine pancreatic dysplasia,studying the early developmental mechanisms of exocrine pancreas can help understand the pathogenesis of pancreatic related diseases and provide a theoretical basis for developing treatment strategies for these diseases.The Wnt/β-catenin pathway is an important signaling pathway that regulates stem cell proliferation and differentiation,which plays an important role in the development of various tissues and organs,and is conserved in evolution.Wnt/β-catenin signaling is the basis for endoderm induction and formation of the hindgut endoderm,and is involved in the specification and pattern formation of the pancreas.The specification and differentiation of exocrine pancreas depend on Wnt/β-catenin inhibition in many different species.Abnormal Wnt/β-catenin activation or closure of the catenin pathway can lead to pancreatic developmental defects and various developmental related diseases.Transmembrane p24 trafficking protein 10(TMED10)is a conserved vesicular transport protein located in the endoplasmic reticulum,Golgi apparatus,and cell membrane.It has been found to be dysregulated in Alzheimer’s disease,Sjogren’s syndrome,and osteoarthritis,implying its important role in the pathogenesis.TMED10 is commonly expressed in multiple tissues of mammals.In mice,the expression of TMED10 is concentrated in the exocrine pancreas;However,its biological functions and potential mechanisms are still largely unknown.Firstly,we aligned the amino acid sequence of zebrafish Tmed10 with that of humans and mice,and confirmed that the gene is conserved among the three species.Then we analyzed the expression pattern of tmed10 m RNA in early zebrafish embryos using in situ hybridization technology.At 24 hours after fertilization(hpf),tmed10 was expressed throughout the body.At 48 hpf,tmed10 began to be expressed in ptf1 a positive cells.At 72 hpf,tmed10 is highly expressed in acinar cells.To investigate the effect of tmed10 on the development of exocrine pancreas,we used CRISPR/Cas9 technology to knock out tmed10 in zebrafish and obtained a Tmed10 protein partially deficient mutant cq144.The mutant lost 6 bases on exon 4,resulting in the loss of 2 amino acids at the N-terminus of the coiled-coil(CC)domain.The other mutation types are frameshift mutations,and no homozygotes were detected among the48 embryos genotyped at 24 hpf.It implies the complete loss of Tmed10 protein caused embryonic death.There are literatures reported that the CC domain is necessary for the cell membrane localization of Tmed10 protein,so we detected the cell distribution of mutated Tmed10 protein.By labeling membrane protein as reference,we found that the mutated Tmed10 protein was almost non-existent on the cell membrane,while the wildtype Tmed10 protein and membrane protein were co localized.Therefore,we speculate that the mutated protein has lost its ability to locate on the cell membrane.Compared with wild-type embryos,The cq144 mutant showed relatively normal body shapes,except for a slightly shorter body length at 72 hpf.While the exocrine pancreas of the cq144 mutants was significantly smaller than that of the wild-type embryos,and in situ hybridization results showed a significant decrease in the expression level of marker cpa1 and mist1 during acinar maturation.Meanwhile,no obvious defect in pancreatic duct,intestine,liver,and β cell defect was detected,indicating that the influence of Tmed10 defect is relatively specifically to exocrine pancreas.To determine whether the phenotype of the cq144 mutant is caused by the tmed10 mutation,we injected morpholine oligonucleotides into wild-type embryos to inhibit Tmed10 protein translation.The tmed10 morpholine mutants exhibited the same phenotypes as the cq144 mutants.The phenotypes of the cq144 mutant are caused by the TMED10 deficiency,which is crucial for the development of pancreatic exocrine glands in zebrafish juveniles.Previous studies have shown that TMED10 is a γ-secretase complex component and has an inhibitory effect on γ-secretase.We speculated that the lack of Tmed10 in the mutant would lead to increased γ-secretase activity.Through antibody staining,we found that substrate of γ-secretase,membrane-bound β-catenin significantly decreased in Tmed10-deficient embryos.Meanwhile,there was no significant change in the transcription level of β-catenin.To verify that the decrease of membrane-bound β-catenin was caused by increased γ-secretase activity,we injected psen1 and psenen m RNA into WT embryos to increase the active form of γ-secretase.Similarly,injection of psen1 and psenen m RNA leads to a pancreatic exocrine gland phenotype similar to that of tmed10 mutant.Furthermore,we attempted to rescue the phenotype of the tmed10 mutant by using γ-secretase inhibitor N-[N-(3,5-difluorophenacetyl)-l-alanyl]-s-phenylglycinetbutyl ester(DAPT).After DAPT treatment,the phenotypes cause by Tmed10-deficient were rescued.The above results indicate that Tmed10 regulates the differentiation of exocrine cells through γ-secretase.We predicted that the decreased membrane-bound β-catenin may influence signal transduction.It has been reported that increased β-catenin signaling could affect the differentiation of exocrine cells.Therefore,we checked the expression of β-catenin signaling target genes ccnd1 and met by in situ hybridization.The results showed that these two genes were upregulated in the tmed10 mutant.To confirm the effect of enhancedβ-catenin signaling on the pancreatic exocrine gland in zebrafish,we used β-catenin signal activator Li Cl to promote β-catenin activation in wild-type embryos.The treated embryos exhibited reduced pancreatic size and downregulated acinar maturation markers,consistent with the phenotype of the tmed10 mutants.Furthermore,we attempted to overexpress β-catenin signaling suppressor protein DKK-1 to rescue the phenotype of the tmed10 mutant by using Tg(hsp:dkk1-GFP).After overexpression of DKK-1,the phenotype of exocrine pancreas defect was rescued in most of the tmed10 mutant embryos,including the size of exocrine pancreas and the expression of acinar maturation markers.The above results indicate that increased β-catenin signaling is an important cause of exocrine pancreas defects in the tmed10 mutant.In summary,our study found that Tmed10 deficiency leads to developmental defects in exocrine pancreas,demonstrating that Tmed10 plays an important role in the differentiation of pancreatic acinar cells.Tmed10 affects the differentiation of pancreatic acinar cells through regulating γ-secretase activity.Decrease of membrane-bound β-catenin,accompanied with increased β-catenin signaling is an important reason for the defects of embryonic pancreatic exocrine gland in Tmed10-deficient embryos.In addition,we found that high expression of Tmed10 was accompanied with excessive accumulation of membrane-bound β-catenin,which suggested the role of Tmed10 in regulating membrane-bound β-catenin.These findings may provide a theoretical basis for developing treatment strategies for TMED10 related diseases. |
