AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One a...AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One and half nanogram of IRE1α,1 ng of IRE1α-GR mRNA,1 ng of IRE1αΔC-GR mRNA,and 50 ng of IRE1α morpholino oligonucleotide(MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis.To rescue the effect of IRE1α MO,1 ng of IRE1α-GR mRNA was coinjected with 50 ng of MO.For the activation of the GR-fusion proteins,dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH.Embryos were kept in dexamethasone up to stage 41.Whole-mount in situ hybridization was used to determine specific gene expression,such as IRE1α,IRE1β,Xbra and Xsox17α.IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS:In the whole-mount in situ hybridization analysis,xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage.The relatively higher expression of IRE1α was observed in the pancreas,and significant transcription of IRE1β was found in the liver.IRE1α protein could be detected at all developmental stages analyzed,from stage 1 to stage 42.Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos.And at tadpole stage,the embryos injected with IRE1α-GR mRNA did not display overt phenotype,such as gut-coiling defect.Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages.We did not observe a significant change of mesodermal and endodermal marker gene ex展开更多
基金Supported by The National Natural Science Foundation of China,No.30971680A Project Funded by the Priority Academic Program Development of Jiangsu Higher Education Institutions, No.JX10131801101
文摘AIM:To investigate the role of inositol-requiring enzyme 1α(IRE1α) in gut development of Xenopus lavies embryos.METHODS:Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 × MBSH.One and half nanogram of IRE1α,1 ng of IRE1α-GR mRNA,1 ng of IRE1αΔC-GR mRNA,and 50 ng of IRE1α morpholino oligonucleotide(MO) or XBP1(C)MO were injected into four blastomeres at 4-cell stage for scoring the phenotype and marker gene analysis.To rescue the effect of IRE1α MO,1 ng of IRE1α-GR mRNA was coinjected with 50 ng of MO.For the activation of the GR-fusion proteins,dexamethasone was prepared as 5 mmol/L stock solutions in 100% ethanol and applied to the mRNA injected embryos at desired stages in a concentration of 10 μmol/L in 0.1 × MBSH.Embryos were kept in dexamethasone up to stage 41.Whole-mount in situ hybridization was used to determine specific gene expression,such as IRE1α,IRE1β,Xbra and Xsox17α.IRE1α protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS:In the whole-mount in situ hybridization analysis,xenopus IRE1α and IRE1β showed quite different expression pattern during tadpole stage.The relatively higher expression of IRE1α was observed in the pancreas,and significant transcription of IRE1β was found in the liver.IRE1α protein could be detected at all developmental stages analyzed,from stage 1 to stage 42.Gain-of-function assay showed that IRE1α mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17α at stage 10.5 was not significantly changed in embryos injected with IRE1α mRNA as compared to uninjected control embryos.And at tadpole stage,the embryos injected with IRE1α-GR mRNA did not display overt phenotype,such as gut-coiling defect.Loss-of-function assay demonstrated that the IRE1α MO injected embryos were morphologically normal before the tailbud stages.We did not observe a significant change of mesodermal and endodermal marker gene ex
