Department of Biochemistry and Molecular Biology, Nanjing Medical University, Nanjing 210029, Jiangsu Province, China.
World J Gastroenterol. 2013 Jan 14;19(2):227-34. doi: 10.3748/wjg.v19.i2.227.
To investigate the role of inositol-requiring enzyme 1α (IRE1α) in gut development of Xenopus lavies embryos.
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.
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.
IRE1α is not required for germ layer formation but for gut development in Xenopus lavies and it may function via XBP1-dependent pathway.
研究肌醇需求酶 1α（IRE1α）在非洲爪蟾胚胎肠道发育中的作用。
采用体外受精法获得非洲爪蟾胚胎，并在 0.1×MBSH 中培养。在 4 细胞期，将 1ng 的 IRE1α-GR mRNA、1ng 的 IRE1αΔC-GR mRNA、50ng 的 IRE1α 或 XBP1（C）MO 注射到四个卵裂球中，以评分表型和分析标记基因。为了挽救 IRE1α MO 的作用，将 1ng 的 IRE1α-GR mRNA 与 50ng 的 MO 共注射。为了激活 GR 融合蛋白，将地塞米松配制成 5mmol/L 的乙醇储备液，在 0.1×MBSH 中以 10μmol/L 的浓度应用于所需阶段的 mRNA 注射胚胎中。胚胎在地塞米松中培养至 41 期。采用全胚胎原位杂交法检测 IRE1α、IRE1β、Xbra 和 Xsox17α 等特定基因的表达。采用 Western blot 检测非洲爪蟾胚胎发育过程中 IRE1α 蛋白的表达。
在全胚胎原位杂交分析中，非洲爪蟾 IRE1α 和 IRE1β 在蝌蚪期表现出截然不同的表达模式。在胰腺中观察到较高的 IRE1α 表达，在肝脏中发现显著的 IRE1β 转录。IRE1α 蛋白可在从 1 期到 42 期分析的所有发育阶段中检测到。功能获得试验表明，尾部芽期注射 IRE1α mRNA 的胚胎几乎正常，注射 IRE1α mRNA 的胚胎在 10.5 期的 pan-mesodermal 标记基因 Xbra 和内胚层基因 Xsox17α 的表达没有明显变化与未注射对照胚胎相比。在蝌蚪期，注射 IRE1α-GR mRNA 的胚胎没有表现出明显的表型，如肠道卷曲缺陷。功能丧失试验表明，IRE1α MO 注射胚胎在尾部芽期之前形态正常。我们没有观察到中胚层和内胚层标记基因表达的显著变化，而在 40 期之后，大约 80%的 MO 注射胚胎表现出明显的肠道缺陷，肠道没有卷曲，而胃肠道以外的其他结构相对正常。为了测试表型是否是由 IRE1α 的敲低引起的，通过共注射 IRE1α-GR mRMA 进行了挽救实验。获得的数据表明，肠道卷曲缺陷得到了挽救。构建了 IRE1α 的缺失突变体，由没有 C 端激酶和 RNA 酶结构域的 N 端组成，命名为 IRE1αΔC，以研究 IRE1α 的功能结构域。注射 IRE1αΔC-GR mRNA 会导致与内源性 xIRE1α 功能干扰引起的肠道畸形相似的形态改变。为了研究 IRE1α/XBP1 途径是否参与肠道发育，注射了 50ng 的 XBP1 MO，结果表明，XBP1 的敲低导致在蝌蚪期出现类似的形态改变，肠道卷曲缺陷。
IRE1α 对于胚胎原基的形成不是必需的，但对于非洲爪蟾的肠道发育是必需的，它可能通过 XBP1 依赖性途径发挥作用。
