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figure 2. Timing of Slug rescue of Slug MO phenotypes:
To analyze the timing of Slug activity in the early embryo, we injected one cell of two cell embryos with Slug MO (10 ng/embryo) together with RNA (650 pg/embryo) encoding the chimeric GR-Slug-GFP protein. A: In the absence of the activating drug dexamethasone, the Slug MO phenotype, i.e. suppression of Xbra expression in stage 11 embryos (A)(arrow) and suppression of Sox9 expression in stage 16 embryos (D), was unaltered. When embryos were treated with dexamethasone (20 µM) beginning at stage 8, there as an essentially complete rescue of Xbra (B,C) and Sox9 expression (E,H). Treatment of embryos with dexamethasone at stage 11 (early gastrulation) was also effective at rescuing Sox9 expression (F,H), while addition of dexamethasone at stage 13 (late gastrulation/early neurulation) produced at most a partial and inefficient rescue of Sox9 expression (G,H).
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Rescue of Slug MO effects by Bcl-xL
A: Injection of the Slug MO leads to an increase in TUNEL staining. B: This increase is blocked by the injection of Bcl-xL RNA (600 pg/embryo and co-injected with LacZ RNA). Injected sides of embryos are marked by an “*” and red staining; line marks midline of the embryo, with anterior (“An”) and posterior (“Ps”) indicated. Injection of Bcl-xL RNA (600 pg/embryo) rescues Apod (C- Slug MO injected, D-Slug MO+Bcl-xl RNA injected), Xbra (ε- Slug MO injected, F-Slug MO+Bcl-xl RNA injected), and Sox9 expression (G- Slug MO injected, H-Slug MO+Bcl-xL RNA injected). I: Injection of Bcl-xL RNA into one cell of a two-cell embryo led to a dramatic increase in the intensity and extent of Slug expression at stage 16; the region of Slug expression on the uninjected (control) side of the embryo is indicated by the dashed circle. J: Injection of Bcl-xL RNA produced an increase in Slug RNA levels in animal caps prepared at stage 8/9 and analyzed by QRT-PCR when uninjected embryos reached stage 11. Ornithine decarboxylase (ODC) was used as normalization control. RNA levels in the control case were set to 100%. |
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Figure 6. NF-κB regulation of mesodermal and neural markers:
The Slug MO induced loss of Xbra (A,B), Apod (C,D) and Sox9 (E,F) expression was rescued by injection of RelA RNA (600 pg/embryo)(A, C, E-Slug MO alone, B, D, F-Slug MO+RelA RNA). G: Treatment of early embryos with AKBA (50 µM from the 4-cell stage on) lead to a decrease in Xbra staining (control and AKBA-treated embryos marked). Injection of RNA encoding IκBsa (H,I) or RelAδSP (J–L) had effects similar to that seen in Slug MO injected embryos; that is, both induced the reduction of Xbra (H,J), Apod (K) and Sox9 (I,L) RNA staining. AKBA treatment had no reproducible effect on Sox9 expression (data not shown). Arrows mark affected regions. |
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Slug morpholino effects:
Panel A is a comparison of the Slug morpholino sequence (“MO”) with X. laevis SlugA, SlugB, and Snail RNA sequences; start codons are underlined. B–G: Injection of the Slug MO (10 ng/embryo) blocks the expression of Xbra (B-uninjected, C-Slug MO injected), Xmenf (D-uninjected, E-injected), and Antipodean (Apod)(F-uninjected, G-injected). Arrows (C,E,G) point to region of suppression; vegetal pole (“VP”) is indicated. In G, the red staining is due to a β-galactosidase lineage marker. H: Injection of the Slug MO into one cell of a two cell embryo blocks the expression of Sox9 on the injected side (arrow); “*” marks otic placode domain of Sox9 expression. I: Sox9 expression in Slug MO injected embryos is rescued by co-injection of mycGFP-Slug RNA (650 pg/embryo). In analogous studies, the effects of the Slug MO (10 ng/embryo) on Xbra (J), Apod (K) and Sox9 (L) expression were rescued by injection of Snail RNA (500 pg/embryo). In H, I and L, the line marks midline of the embryo, with anterior (“An”) and posterior (“Ps”) indicated.
doi:10.1371/journal.pone.0000106.g001 |
