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J Biomed Res
2014 Mar 01;282:123-31. doi: 10.7555/JBR.28.20130076.
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IRE1α is essential for Xenopus pancreas development.
Yuan L, Li X, Feng J, Yin C, Yuan F, Wang X.
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Inositol requiring enzyme-1 (IRE1) is highly conserved from yeasts to humans. Upon the endoplasmic reticulum (ER) stress, IRE1 activates X-box-binding protein 1 (XBP1) by unconventionally splicing XBP1 mRNA, which activates the unfolded protein response (UPR) to restore ER homeostasis. In mice, IRE1α inactivity leads to embryonic death and IRE1α plays an essential role in extraembryonic tissues and the placenta. However, its precise action in the embryo proper is still unknown. In this study, the loss of function analysis was performed to investigate the function of Xenopus IRE1α (xIRE1α) during pancreas development. Firstly, the complete open reading frame of xIRE1α was amplified and the expression pattern was detected. The effects of Xenopus IRE1α and XBP1 during embryo development were detected with whole-mount in situ hybridization. The results demonstrated that xIRE1α was much closer to human IRE1α when compared with their sequence alignment. xIRE1α was expressed strongly in developing pancreas and the knockdown of xIRE1α inhibited the differentiation and specification of the pancreas. xIRE1α, which was required for cytoplasmic splicing of XBP1 pre-mRNA and XBP1MO, also showed inhibitory effects on pancreas development. These results suggest that xIRE1α is essential for pancreas development during embryogenesis and functions via the XBP1 dependent pathway.
Fig. 1. Xenopus IRE1α sequence analysis.A: Alignment of Xenopus xIRE1α, xIRE1β, hIRE1α and hIRE1β animo acid sequences. Identical residues are marked by asterisks. Gaps are introduced to achieve optimum alignment. B: Percentage of identity between IRE1 proteins. C: Phylogenetic tree of IRE1 proteins of different species created by ClustalW (h, Homo sapiens; x, Xenopus laevis).
Fig. 2. IRE1α is expressed in the developing pancreas.Whole-mount in situ hybridization data for IRE1α expression at stage 40 (St 40) (A) and 43 (B). The arrows indicate pancreas. C and D are negative control.
Fig. 3. Inhibition of in vivo translation of a xIRE1α/GFP fusion construct by IRE1α-MO. xIRE1α/GFP RNA or xIRE1αmut/GFP RNA was injected into 4 blastomeres at 4-cell stage alone (A, D), or co-injected with 50 ng control MO (B, E) or 50 ng IRE1α MO (C, F). Embryos were collected at stage 31 and GFP was monitored.
Fig. 4. IRE1α knockdown specifically inhibits the expression of differentiation marker genes. Whole mount in situ hybridization analyses revealed that the expression of insulin (B) and amylase (D) was not detected in IRE1α MO injected embryos at stage 43 compared with control MO injected embryos (A, C). The white arrows point to the positive staining of insulin and amylase.
Fig. 5. IRE1α knockdown inhibits the expression of specification marker genes.Whole mount in situ hybridization analyses revealed that the expression of pdx1 (B) and ptf1α (D) was significantly suppressed in IRE1α MO injected embryos at stage 30 compared with control MO injected embryos (A, C). The white arrows indicate positive staining.
Fig. 6. Effects of xIRE1α on cytoplasmic splicing of xXBP1.RT-PCR (A) detected an increase of cytoplasmic variant xXBP1 (C) in embryos injected with xIRE1α mRNA and a decrease of the xXBP1(C) in embryos injected with IRE1αMO at stage 11 and 18. ODC (ornithine decarboxylase) served as a loading control. Monitoring the xXBP1 splicing by xIRE1α in vivo (B-D). Embryos injected with 500 pg xXBP1(U)-EosFP RNA individually or in combination with 1 ng xIRE1α RNA and/or 50 ng XBP (C) MO. xXBP1 (U), unspliced xXBP1; xXBP1(N), nuclear spliced xXBP1. RT-: no-reverse transcriptase control.
Fig. 7. IRE1α knockdown inhibits pancreas marker gene expression.Whole mount in situ hybridization analyses revealed that the expression of insulin (B) and amylase (D) was not detected in 50 ng XBP1MO injected embryos at stage 43 compared with control MO injected embryos (A,C). The white arrows indicate positive staining.
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