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: <deleted - database limit> 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 Xsox17a 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)CGR 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.