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Inositol requiring enzyme-1 (IRE1) is highly conserved from yeasts to humans. Upon endoplasmic reticulum (ER) stress, IRE1 activates X-box-binding protein 1 (XBP1) by unconventional splicing of XBP1 mRNA, which activates unfolded protein response (UPR) to restore ER homeostasis. In mice, IRE1α plays an essential role in extraembryonic tissues. However, its precise action during the early stage of development is unknown. In this study, the gain and loss-of-function analyses were used to investigate the function of Xenopus IRE1α (xIRE1α). The effects of xIRE1α during embryo development were detected with RT-PCR and whole mount in situ hybridization. ER stress was induced by tunicamycin. The apoptotic cells were measured by TUNNEL assays. Although both gain and loss of xIRE1α function had no significant effect on Xenopus embryogenesis, knockdown of xIRE1α could rescue tunicamycin-induced developmental defects and apoptosis. The finding indicates that xIRE1α is not required for embryogenesis but is required for tunicamycin-induced developmental defects and apoptosis in Xenopus laevis.
Fig. 1. xIRE1α does not affect mesoderm and endoderm formation.A: control MO injected embryo at tailbud stage. B and C: embryo injected with 50 ng xIRE1α MO or 1.5 ng xIRE1α mRNA. Overexpression or knockdown of xIRE1α did not change the expression of Xbra (D–F) and Xsox17a (G–I) at stage 10.5.
Fig. 2. Developmental defects caused by tunicamycin (TM) were rescued with the injection of IRE1αMO and xXBP1(C) MO.A: control MO injected embryos at stage 18. B: embryos treated with 2 μg/mL TM at stage 18. C: embryos treated with 2 μg/mL TM and injected with 50 ng IRE1αMO at stage 18. D: embryos treated with 2 μg/mL TM and injected with 50 ng IRE1βMO at stage 18. E: rescue effect on XBP1 splicing. TM treatment led to an increase of xXBP1(C) while TM treatment together with xIRE1α MO injection rescued the change in embryos at stage 11. F: control MO injected embryos at stage 32. G: embryos treated with 2 μg/mL TM at stage 32. H: embryos treated with 2 μg/mL TM and injected with 50 ng IRE1αMO at stage 32. I: embryos treated with 2 μg/mL TM and injected with 50 ng XBP1(C) MO at stage 32. RT-: no-reverse transcriptase control.
Fig. 3. Rescue effects of xIRE1α knockdown on gene expression.Control, TM treated and rescued embryos were collected at stage 11 or 18 and subjected to RT-PCR. Expression of mesodermal, endodermal, and neuroectodermal genes were inhibited in TM-treated embryos and rescued by xIRE1α knockdown. RT-: no-reverse transcriptase control.
Fig. 4. xIRE1α knockdown rescued the apoptosis in embryos of stage 18 with TM treatment by TUNNEL.A: The control embryos without TM treatment showed few staining indicating apoptosis (arrow). B: The TM-treated embryos showed significant signal for apoptosis (arrows). C: The embryos were injected with 50 ng of IRE1αMO at 4 cell stage, then treated with TM, and the apoptotic signal was decreased (arrows). D: A quantitative presentation is given. E: Expression of CHOP is inhibited in TM-treated embryos and rescued by xIRE1α knockdown. *P < 0.05 compared with control embryos.
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