Inositol-Requiring Enzyme 1 Alpha Is Required For Gut Development In Xenopus Lavies Embryos

AIM: To investigate the role of inositol-requiring enzyme 1 alpha (IRE1 alpha) in gut development of Xenopus lavies embryos.METHODS: Xenopus embryos were obtained with in vitro fertilization and cultured in 0.1 x MBSH. One and half nanogram of IRE1 alpha, 1 ng of IRE1 alpha-GR mRNA, 1 ng of IRE1 alpha Delta C-GR mRNA, and 50 ng of IRE1 alpha 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 IRE1 alpha MO, 1 ng of IRE1 alpha-GR mRNA was co-injected 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 mu mol/L in 0.1 x MBSH. Embryos were kept in dexamethasone up to stage 41. Whole-mount in situ hybridization was used to determine specific gene expression, such as IRE1 alpha, IRE1 beta, Xbra and Xsox17 alpha. IRE1 alpha protein expression during Xenopus embryogenesis was detected by Western blotting.RESULTS: In the whole-mount in situ hybridization analysis, xenopus IRE1 alpha and IRE1 beta showed quite different expression pattern during tadpole stage. The relatively higher expression of IRE1 alpha was observed in the pancreas, and significant transcription of IRE1 beta was found in the liver. IRE1 alpha protein could be detected at all developmental stages analyzed, from stage 1 to stage 42. Gain-of-function assay showed that IRE1 alpha mRNA injected embryos at tailbud stage were nearly normal and the expression of the pan-mesodermal marker gene Xbra and the endodermal gene Xsox17 alpha at stage 10.5 was not significantly changed in embryos injected with IRE1 alpha mRNA as compared to uninjected control embryos. And at tadpole stage, the embryos injected with IRE1 alpha-GR mRNA did not display overt phenotype, such as gut-coiling defect. Loss-of-function assay demonstrated that the IRE1 alpha MO injected embryos were morphologically normal before the tailbud stages. We did not observe a significant change of mesodermal and endodermal marker gene expression, while after stage 40, about 80% of the MO injected embryos exhibited dramatic gut defects in which the guts did not coil, but other structures outside the gastrointestinal tract were relatively normal. To test if the phenotypes were specifically caused by the knockdown of IRE1 alpha, a rescue experiment was performed by co-injection of IRE1 alpha-GR mRMA with IRE1 alpha MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1 alpha was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1 alpha Delta C, to investigate the functional domain of IRE1 alpha. Injection of IRE1 alpha Delta C-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1 alpha. In order to investigate if IRE1 alpha/XBP1 pathway was involved in gut development, 50 ng of XBP1 MO was injected and the results showed that knockdown of XBP1 resulted in similar morphological alterations with gut-coiling defect at tadpole stage.CONCLUSION: IRE1 alpha is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway. (C) 2013 Baishideng. All rights reserved.