XB-IMG-75518
Xenbase Image ID: 75518
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Fig. 1. Xenopus FoxB1, acting downstream of Oct-25, promotes neural induction and suppresses BMP-dependent epidermal differentiation. (A) The expression of Xenopus FoxB1 is activated by the Oct-25 transcription factor. GR-Oct-25 mRNA (1000 pg) was injected into the animal pole of 4-cell stage embryos. Ectodermal explants were isolated at the blastula stage, treated with or without dexamethazone (DEX) to activate Oct-25, cultured until the early gastrula stage (st. 10.5), and analyzed by semi-quantitative RT-PCR. Emb and –RT indicate whole embryo RNA processed in the presence or absence of reverse transcriptase, respectively. Histone was used as a loading control. (B) Overexpression of FoxB1 promotes neural induction and inhibits epidermal differentiation. GR-FoxB1 mRNA (500 pg and 1000 pg) was injected into the animal pole of 4- to 8-cell stage embryos. Ectodermal explants were isolated at the blastula stage, treated with DEX to activate FoxB1 and cultured until early neurula stage (st. 15). The expression of marker genes was determined by semi-quantitative RT-PCR. FoxB1 induced the expression of neural markers Sox2 and SIP1 with a moderate reduction in the expression of epidermal keratin (lanes 4 and 5). Expression of the mesodermal marker muscle actin was not induced by FoxB1 activation. (C) FoxB1 inhibits epidermal keratin gene expression induced by a constitutively activated BMP type I receptor (CA-BMPR) and promotes Sox2 gene expression. The changes in the gene expression profile were determined by semi-quantitative RT-PCR (upper panel) and further confirmed by quantitative real-time PCR (QPCR) (lower panel) using the same RNA samples shown in the upper panel. Increasing amounts of CA-BMPR mRNA (9, 19, 38, 75 and 150 pg) were injected alone or together with GR-FoxB1 mRNA (1000 pg) into the animal pole of 2-cell stage embryos. Ectodermal explants were isolated at the blastula stage, and dissociated for 6.5 h (lanes 4–15). Cells were treated with DEX to activate FoxB1 during the dissociation, reaggregated and cultured until neurula stages (st. 21). “Intact” indicates intact ectodermal cells obtained from uninjected embryos. The QPCR data are shown as arbitrary units normalized to ornithine decarboxylase (ODC) gene expression. (D) FoxB1 decreases endogenous levels of phosphorylated Smad1/5/8 in Xenopus ectodermal cells. Increasing amounts of GR-FoxB1 mRNA (500 pg and 1000 pg) were injected into the animal pole of 4- to 8-cell stage embryos. Embryos were treated with DEX and ectodermal explants were isolated at the blastula stage, cultured until the early gastrula stage (st. 10.5). Whole cell lysates from explants were immunoblotted with anti-phospho Smad1/5/8, anti-Smad1/5/8, anti-HA and anti-Tubulin antibodies, respectively. GR-FoxB1 was tagged at the C-terminus with the HA epitope. Tubulin was used as a loading control. (E) FoxB1 interacts preferentially with a non-phosphorylatable mutant form of Smad8 (Smad8AAVA) rather than a phospho-mimicking mutant form of Smad8 (Smad8SEVE) and wild-type Smad8. HeLa cells were transfected with the indicated combination of expression constructs (Myc-tagged FoxB1 and different constructs of Flag-tagged Smad8). The Flag-tagged Smad8 constructs were immunoprecipitated (IP) from transfected cells with anti-FLAG antibodies, and the immunoprecipitates were then immunoblotted (IB) with anti-Myc antibodies (top panel). Input extracts were immunoblotted with the indicated antibodies (the lower two panels). (F) Wild-type FoxB1 is localized to the nucleus of HeLa cells. Transfected cells with Myc-FoxB1 DNA were immunostained with anti-Myc antibody (magenta, left panel). Nuclei were visualized with Hoechst 33342 (blue, middle panel). The merged image is shown in the right panel. Scale bar, 10 μm. (G) Effect of FoxB1 on the subcellular localization of Smad8AAVA in Xenopus ectodermal cells. Dissociated ectodermal cells from embryos injected with FLAG-Smad8AAVA mRNA alone (top panel) or both FLAG-Smad8AAVA and Myc-GR-FoxB1 mRNA (lower panel), were immunostained with anti-FLAG (green) and anti-Myc (magenta) antibodies. Nuclei were visualized with Hoechst 33342 (blue). In the absence of FoxB1, Smad8AAVA was localized to both the cytoplasm and nucleus (upper panels), whereas Smad8AAVA together with FoxB1 accumulated in the nucleus (lower panels). Scale bar, 10 μm. (H) Summary of data from independent experiments presented in (G). The intensity of FLAG-Smad8AAVA staining in the nucleus was compared with that in the cytoplasm and scored as follows: no (no significant difference to that in the cytoplasm), weak, moderate, or strong nuclear staining. Numbers of Smad8AAVA-positive cells analyzed in independent fields are indicated below the bars. Image published in: Takebayashi-Suzuki K et al. (2011) Copyright © 2011. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |