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Maintenance of endoplasmic reticulum (ER) homeostasis is essential for correct protein targeting and secretion. ER stress caused by accumulation of unfolded or misfolded proteins leads to disruption of cellular functions. We have investigated the effect of ER stress on Xenopus embryogenesis. ER stress induced by tunicamycin (TM) treatment of embryos resulted in defects affecting germ layer formation. We observed up-regulation of ER stress response genes, enhanced cytoplasmic splicing of xXBP1 RNA, and increased rate of apoptosis. In animal cap assays, TM treatment inhibited mesoderm formation induced by overexpression of activin/nodal RNA but did not affect mesoderm formation induced by functional activin protein, suggesting that dysfunction of ER caused a failure in activin/nodal processing and/or secretion. The observation that activin protein renders mesoderm formation under ER stress strengthens the role of activin/nodal for mesoderm induction. The results underline the functional significance of ER homeostasis in germ layer formation during Xenopus embryogenesis.
Figure 1. Embryos treated with TM from stage 2 until stage 8.5 showed severe developmental defects. A: Control embryos at stage 18. B: At stage 18, embryos treated with 2.5 μg/ml TM developed with widely open blastopore, displayed no neural fold formation and died soon after neurulation. C: At stage 18, embryos treated with 2 μg/ml showed less severe phenotypes but still displayed open neural folds. D: Control embryos at stage 30. E: At stage 30, embryos treated with 2 μg/ml TM developed with open neural fold and body axis defects. Right panels show dorsal views of embryos. Anterior is to the right.Download figure to PowerPoint
Figure 2. Regulation of ER stress-mediated genes. A: Real time RT-PCR demonstrated that expression of BiP did not change significantly in TM-treated embryos at stage11, but increased at stages 19 and 30. B: Expression of CHOP showed strong up-regulation at all three stages examined. C: Cytoplasmic splicing of xXBP1 was affected by TM treatment. RT-PCR detected stronger bands representing the cytoplasmic splice variant of xXBP1 (xXBP1(C)) in TM-treated embryos (+TM) than in control embryos (−TM) at stages indicated. ODC (ornithine decarboxylase) served as a loading control. Photographic gel documentation is shown in the top panel; result obtained by a Bioanalyzer 2100 instrument (Agilent Biotechnologies) is shown in the bottom panel. It represents the migration of DNA bands run in a mini electrophoresis chip. Strength of bands (measurement of fluorescence signals) reflects the amount of DNA. xXBP1(U), unspliced xXBP1; xXBP1(N), nuclear spliced xXBP1.Download figure to PowerPoint
Figure 3. TM treatment induced apoptosis in embryos. A: At stage 18, embryos without TM treatment showed very few signals representing apoptotic cells. B: The number of cells undergoing apoptosis increased dramatically in TM-treated embryos.Download figure to PowerPoint
Figure 4. Gene expression analysis on embryos treated with TM. Treated and untreated embryos were collected at stage 11 and subjected to real time RT-PCR. Expression of mesodermal, endodermal, and neuroectodermal genes were inhibited in TM-treated embryos.Download figure to PowerPoint
Figure 5. TM treatment did not affect mesoderm differentiation induced by mature activin A. A: Naïve caps without treatment. B: Caps treated with activin A at 10 ng/ml. C: Caps from embryos treated with TM showing no difference from those in A. D: Caps from embryos treated with TM still showed an elongation induced by activin A. E: Real time RT-PCR confirmed no significant effect of previous TM treatment on expression of mesodermal genes in animal caps.Download figure to PowerPoint
Figure 6. TM treatment affected mesoderm differentiation in vegetal-equatorial explants. A: Untreated vegetal-equatorial explants at stage 18 showed elongation. B: Vegetal-equatorial explants from embryos treated with TM did not elongate. C: Real time RT-PCR showed inhibition of mesodermal gene expression in VE explants from embryos treated with TM.Download figure to PowerPoint
Figure 7. TM treatment inhibited mesoderm formation in animal caps of embryos injected with Xnr5/activin mRNA. A: Control embryos. B: Control caps without RNA injection and TM treatment. C: Caps from embryos injected with Xnr5 mRNA showing significant elongation. D: Caps from embryos injected with Xnr5 mRNA and treated with TM showing no elongation anymore. E: Caps from embryos injected with activin mRNA elongate. F: Caps from embryos injected with activin mRNA and treated with TM do not elongate. G: Real-time RT-PCR revealed that TM treatment inhibited mesoderm formation induced by Xnr5 injection.Download figure to PowerPoint
