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Xenopus embryo is a useful model for evaluating the adverse effects of any compounds on the cellular processes essential for early development and adult tissue homeostasis. Our chemical library screening with frog embryos identified artesunate (ART) as an inhibitor of the BMP signaling pathway to interfere with the specification of embryonic germ layers. Exposure to ART led to reduction of the anterior-posterior body axis, malformed tail structures and loss of pigment cells in the trunk region of embryos. The severely defective embryos exhibited truncation of posterior structures, resembling the phenotypes of tadpoles depleted of BMPs. Consistent with these morphological deformities, ART exposure inhibited the BMP-dependent transcriptions of target genes and specification of ventral mesoderm. In contrast, the expression of an organizer-specific gene induced by Activin/Nodal signaling remained unchanged in ART-treated cells. ART also enhanced anterior neural differentiation at the expense of epidermal and neural crest cell fates. Unexpectedly, we observed that ART exposure accelerates proteasomal degradation of a BMP transducer Smad1, leading to upregulation of MAP kinase activity. Taken together, these results suggest that ART acts as an inhibitor of BMP signaling pathway, exerting severe adverse effects on the specification of germ layers in vertebrate early development.
Figure 1. Morphological phenotypes of ART-exposed Xenopus embryos. (A) Embryos were exposed or not to DMSO (for control) or ART (400 μM) from the one-cell stage until the untreated sibling embryos reached tadpole stages. All embryos are shown in lateral views with anterior to the left. Scale bar, 500 μm. (B) Experimental design of treatment with ART. Developmental stages are shown from stage 1 to tadpole stage. Embryos were cultured in the presence of ART (400 μM) during the periods represented by the gray bars, and the phenotypes were analyzed at the tadpole stage. (C) Quantification of the phenotypes resulting from the treatment conditions shown in (B). DMSO-treated embryos were grown as in (A). Un, untreated control embryo.
Figure 2. ART inhibits BMP signals. (A-F) Animal cap tissues were excised at stage 8.5 from the whole embryos, cultured in the absence or presence of ART (400 μM) and/or Activin (2 ng/ml) until the sibling control whole embryos reached stage 10.5 (for Ventx2.2, Ventx1.2 and Gsc) or 19 (for E.keratin, Sox2 and MyoD) and then harvested for qPCR experiments. *P < 0.05, **P < 0.01 by unpaired Student’s t-test. n.s., not significant.
Figure 3. Interfering effects of ART on germ layer specification. (A-C) Embryos were treated or not with DMSO or ART (400 μM) as indicated from stage 8.5 until the untreated sibling embryos reached stage 10.5 (for Gsc and Ventx1.2), 16 (for Sox2, E.keratin and Sox9) or 19 (for Otx2, HoxB9 and MyoD) and then subjected to in situ hybridization against these markers. Embryos are shown in vegetal view with dorsal to the top (panels in (A)), in dorsal view with anterior to the top (panels in upper two tiers in (B) and panels in lower two tiers in (C)) or in anterior view with dorsal to the top (panels in bottom tier in (B) and panels in top tier in (C)). Scale bar, 250 μm. The histograms in each figure are presented as the mean ± SD (n, number of analyzed embryos). ***P < 0.001 by unpaired Student’s t-test. n.s., not significant. Un, untreated control embryo.
Figure 4. ART induces ERK activation via Smad1 turnover. (A) Animal caps excised at stage 8.5 from the whole embryos were cultured to stage 12 in the presence or absence of ART (400 μM) and then subjected to western blotting. (B) Embryos were pre-treated with cycloheximide (CHX, 10 μg/ml) for 2 hours before animal cap explants were excised at stage 8.5 from the whole embryos. Dissected animal cap cells were exposed to DMSO or ART for the indicated times and then harvested for western blot analysis. (C-E) Four-cell stage embryos were injected or not in the animal pole region with DN FGFR1 (500 pg) or DN FGFR4 (500 pg) mRNA as indicated, and then animal cap tissues were dissected at stage 8.5, cultured in media containing DMSO or ART with or without MG-132 (MG, 10 µM), chloroquine (CQ, 100 µM), SU5402 (20 µM) or U0126 (20 µM) as denoted and harvested at stage 12 for western blotting. Co AC, untreated control animal cap. (-) in (C-E) indicates animal caps treated with ART only.
