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World J Gastroenterol
2013 Jan 14;192:227-34. doi: 10.3748/wjg.v19.i2.227.
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Inositol-requiring enzyme 1α is required for gut development in Xenopus lavies embryos.
Guo J
,
Li XX
,
Feng JJ
,
Yin CY
,
Wang XJ
,
Wang N
,
Yuan L
.
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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 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 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 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 IRE1α, a rescue experiment was performed by co-injection of IRE1α-GR mRMA with IRE1α MO. The data obtained demonstrated that the gut coiling defect was rescued. The deletion mutant of IRE1α was constructed, consisting of the N-terminal part without the C-terminal kinase and RNase domains named IRE1αΔC, to investigate the functional domain of IRE1α. Injection of IRE1αΔC-GR mRNA caused similar morphological alterations with gut malformation by interfering with the function of endogenous xIRE1α. In order to investigate if IRE1α/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: IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.
Figure 1
Expression pattern of xIRE1 in Xenopus lavies during development. A, B: Whole-mount in situ hybridizations revealed relatively high expression of inositol-requiring enzyme (IRE) 1α in pancreas (white arrow in A) and relatively high expression of IRE1β in liver (white arrow in B); C: Western blotting revealed temporal expression of IRE1α during Xenopus embryogenesis.
Figure 2
Overexpression of inositol-requiring enzyme 1α affected neither mesoderm and endoderm formation nor gut development. A: Overexpression of inositol-requiring enzyme (IRE) 1α did not change the phenotype and the expression of Xbra and Xsox17α at stage 10.5; B: IRE1α mRNA injected embryos at tailbud stage were nearly normal; C: Embryos injected with IRE1α-GR mRNA did not display any defect until tadpole stage.
Figure 3
Inositol-requiring enzyme 1α morpholino oligonucleotide blocks inositol-requiring enzyme 1α translation in an in vitro transcription/translation assay. In this assay, xIRE1α was effectively transcribed/translated from a pCS2+xIRE1α construct. However, translation was dramatically decreased by addition of IRE1α morpholino oligonucleotide (MO). IRE: Inositol-requiring enzyme.
Figure 4
Xenopus inositol-requiring enzyme 1α is required for gut development. A: Knockdown of x IRE1α did not change the phenotype and the expression of Xbra and Xsox17α at stage 10.5; B: The inositol-requiring enzyme (IRE) 1α morpholino oligonucleotide (MO)-injected embryos were morphologically normal before the tailbud stages; C: IRE1α knockdown upon injection of 50 ng of MO resulted in a gut defective phenotype. Surgically-resected guts from embryos were shown in C under panel. Coiled structure of gut was not detected in IRE1α MO-injected embryos at stage 43. MO injection was repeated 5 times in a total of 278 embryos.
Figure 5
Rescue of xIRE1α knockdown with IRE1α-GR mRNA. Gut defective phenotype caused by 50 ng of morpholino oligonucleotide (MO) could be rescued by co-injection of 1 ng of inositol-requiring enzyme (IRE) 1α-GR mRNA. Co-injection was done 3 times in a total of 258 embryos.
Figure 6
Injection of IRE1αÎC-GR mRNA caused morphological alterations with gut malformation. A: Diagram depicting the construction of deletion mutants. S and TM denote the signal peptide and the transmembrane domain; B-D: Gut defects were also observed with a dominant-negative mutant, IRE1αÎC, lacking the cytoplasmic kinase and RNase domains.
Figure 7
XBP1 knockdown affected gut development. Injection of XBP1(C) morpholino oligonucleotide (MO) caused similar phenotype with gut-coiling defect at stage 43.
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