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The blastulaChordin- and Noggin-expressing (BCNE) center located in the dorsal animal region of the Xenopus blastulaembryo contains both prospective anteriorneuroectoderm and Spemann organizer precursor cells. Here we show that, contrary to previous reports, the canonical Wnt target homeobox genes, Double knockdown of these genes using antisense morpholinos in Xenopus laevis blocked head formation, reduced the expression of the other BCNE center genes, upregulated Bmp4 expression, and nullified hyperdorsalization by lithium chloride. Moreover, gain- and loss-of-function experiments showed that Siamois and Twin expression is repressed by the vegetal transcription factor VegT. We propose that VegT expression causes maternal beta-Catenin signals to restrict Siamois and Twin expression to the BCNE region. A two-step inhibition of BMP signals by Siamois and Twin-- first by transcriptional repression of Bmp4 and then by activation of the expression of the BMP inhibitors Chordin and Noggin--in the BCNE center is required for head formation.
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Fig. 3. Siamois and Twin are required for head formation. (A) Twn-MO injected embryo. Small eyes were detected in 10.3% of injected embryos (3/29). (B) Sia-MO injected embryo. Small eyes were detected in 87.5% of embryos (42/48). (C) Twn-MO and Sia-MO co-injected embryos. 80% of embryos (28/35) lost head structures. (D) Co-injection of Twn-MO and Siamois mRNA rescued the Twn-MO phenotype to normal or mild dorsalization (big eyes in 42.9% (15/35) of the embryos). (E) Sia-MO and Twin mRNA coinjected embryo. Small eyes were detected in 14.8% of embryos (4/27). (F) Control uninjected sibling embryo for (A–E). (G) Sagittal histological section of the embryo shown in C. (H) Sagittal histological section of embryo shown in F. (I) Whole-mount in situ hybridization analysis of the anterior neural marker Otx2 in stage 26 embryo co-injected with Twin-MO and Siamois-MO. (J) Otx2 expression in control embryo at stage 26. fb, forebrain; mb, midbrain; hb, hindbrain; nt, neural tube; no, notochord; he, heart.
Fig. 1. Siamois and Twin are regulated by β-Catenin. (A) The positioning of two dorsal signaling centers at blastula. The BCNE center expresses Chordin (Chd), Noggin (Nog), Siamois (Sia), Twin (Twn), Xenopus nodal-related 3 (Xnr3), FoxA4a/Pintallavis/HNF3β, Anti-dorsalizing morphogenetic protein (ADMP). D, dorsal; V, ventral. (B) The effect of β-Cat-MO, demonstrating that BCNE center genes require β-Catenin. (C–F) Blocking of Xnr inductive events by CerS mRNA injection inhibits Chordin expression at gastrula but not at blastula (when the BCNE is active), while Siamois and Twin are independent of Xnrs. Ornithine decarboxylase (ODC) was used as loading control.
Fig. 2. The function of Xenopus laevis Siamois and Twin is blocked by the antisense morpholino oligomer reagents designed in this study. (A) The MOs for Siamois and Twin target the translation initiation site. (B) In vitro translated Siamois and Twin protein were blocked only by each specific MO. (C) Secondary axes induced by ventral injection Siamois mRNA (5 pg); 77.3% (17/22) of embryos have secondary axes with formation of complete heads. (D) Secondary axis-inducing activity by Siamois mRNA is blocked by Sia-MO (4 ng); 0% (0/25) of the embryos formed secondary axes. (E) Twn-MO (4 ng) did not block the effect of Siamois mRNA; 78.9% (15/19) of embryos had secondary axes with heads.
Fig. 4. Dorsalization by LiCl and planar neural induction require Siamois and Twin. (A) Embryo treated with LiCl at the 32-cell stage. The average of dorso-anterior index (DAI); was 9.58 (Kao et al., 1986). (B) Embryo co-injected with Sia-MO and Twn-MO prior to LiCl treatment. The average of DAI was 5.38. (C) Control embryo for (A) and (B). In all cases 8 ng of MOs and 1 pg of mRNA were injected. (D) Phenotype of dorsal-animal injection of Sia-MO (total 4 ng) and Twn-MO (total 4 ng); 76.0% (19/25) of embryos lost head structures. (E) The phenotype of dorsal-vegetal injection of Sia-MO and Twn-MO. 7.7% (2/26) of embryos lost head structures. (F) Sibling control embryo at stage 38 for (A) and (B). (G) RT-PCR analyses of the effects of Sia-MO and Twn-MO on whole embryos at stage 9 and 11. (H) Neuroectoderm (NE) region of Keller explants used for molecular studies. (I) RT-PCR analyses of the effect of Sia-MO, Twn-MO, Chd-MO, and Chordin mRNA on the NE region shown in (H).
Fig. 5. Siamois and Twin are key regulators of the transcription of BCNE center genes. (A) RT-PCR analyses of the effects of Sia-MO, Twn-MO, or both on whole embryos. (B) Chordin and Noggin expression at blastula in the animal cap and marginal zone (BCNE) of normal embryos. (C) Co-injection of Twn-MO and Sia-MO eliminates Chordin and Noggin expression. Embryos are shown in animal view.
Fig. 6. Two-step inhibition of BMP4 by Siamois and Twin. (A) Experimental procedure. (B) RT-PCR analyses for the effects of Siamois, Twin, and the BMP antagonists Chordin and Noggin on Bmp4 and its target gene Vent1. The nodal-related target gene Mix1 was used to indicate that CerS was effective in blocking mesoderm and endoderm formation in these experiments. (C) Diagram showing a two-step model for the regulation of BMP4 via Siamois and Twin. BMP signaling is blocked in two steps (1) transcriptional repression and (2) BMP antagonists such as Chordin and Noggin which are induced by Siamois and Twin.
Fig. 7. The BCNE center is restricted to the dorsal-animal region by the maternally localized VegT transcription factor. (A) The phenotype of VegT-MO. White arrowhead indicates enlarged cement gland. (B) Control embryo for (A). (C) RT-PCR analyses of the effects of Wnt8 and VegT mRNA for animal caps. 1, 4, or 16 pg of Wnt8 mRNA and 100 pg of VegT mRNA were injected. (D) Chordin expression in control embryo at stage 9. White and black arrowheads indicate the animal and vegetal poles, respectively. (E) Siamois expression in control embryo. (F) Chordin expression of VegT-MO (16 ng) injected embryo. (G) Siamois expression of VegT-MO injected embryo. Note the expansion of Chordin and Siamois expression towards the vegetal side upon inhibition of VegT. (H) Real-time PCR analyses of Chordin mRNA expression levels from stage 8 to 12. (I) Siamois mRNA expression levels in the same samples. (J) Model of the regulation of the two blastula signaling centers. The border between the BCNE and Nieuwkoop centers is generated by repression by two maternally provided transcription factors: B1-type Sox (animal; Zhang et al., 2004) and VegT (vegetal).
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