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In Xenopus, two signaling systems, maternal beta-Catenin and Nodal-related, are required for induction of the Spemann organizer and establishment of the body plan. By screening cDNA macroarrays for genes activated by these two signaling pathways, we identified Xenopus xBtg-x, a novel member of the Btg/Tob gene family of antiproliferative proteins. We show that xBtg-x is expressed in the dorsal mesendoderm (Spemann organizertissue) of gastrula stage embryos and that its expression is regulated by both beta-Catenin and Nodal-related signals. Microinjection of synthetic xBtg-x mRNA into Xenopus embryos induced axis duplication and completely rescued the ventralizing effects of UV irradiation through the activation of the canonical Wnt/beta-Catenin signaling pathway. Interestingly, xBtg-x stimulated beta-Catenin-dependent transcription without affecting the stability of beta-Catenin protein. These data suggest that xBtg-x is a novel component of the Wnt/beta-Catenin signaling pathway regulating early embryonic patterning.
Fig. 1. Cloning and expression analysis of xBtg-x. (A) The �combined organizer graph� of clones regulated by maternal β-Catenin (x axis) and Nodal-related signaling (y axis) in a cDNA macroarray screen (Wessely et al., 2004). Each spot represents one cDNA clone analyzed. The positions of xBtg-x, Xnr-3 (a target of β-Catenin), and chordin (a target of both β-Catenin and Nodal-related signaling at gastrula) are indicated. (B) Schematic diagram of xBtg-x showing the conserved Boxes A and B of the antiproliferative domain characteristic of members of the Btg/Tob gene family. (C) Phylogenetic tree of the Btg/Tob gene family. Alignment was performed using the ClustalW program. (D) RT-PCR analysis of xBtg-x and chordin expression in stage 4 to stage 17 Xenopus embryos. Ornithine Decarboxylase (ODC) serves as loading control. (E�L) Whole-mount in situ hybridization of Xenopus embryos with xBtg-x antisense probe. (E�H) Expression of xBtg-x in gastrula stage embryos viewed from the vegetal side with dorsal to the top: (E) uninjected control; (F) embryo treated with LiCl at the 32�cell stage; (G) embryo injected with 800 pg δN-XTcf-3 mRNA, or (H) 150 pg of the Nodal-related antagonist cer-S mRNA. (I) Expression of xBtg-x at stage 9, lateral view; (J) stage 10 hemi-sectioned along the dorso-ventral axis, dorsal to the right; (K) stage 14 and (L) stage 15 in dorsal view, anterior to the left.
Fig. 2. xBtg-x induces complete secondary axis formation and rescues UV ventralization. (A) Uninjected control embryos at stage 40. (B) Axis duplication in an embryo injected at the 8-cell stage once ventrally with 0.8 ng xBtg-x mRNA (55%, n = 147). (C) Embryo co-injected with xBtg-x mRNA and 200 pg δN-XTcf-3 mRNA. Note the absence of ectopic axes (0%, n = 36). (D, E) Histological analysis of uninjected and xBtg-x-injected embryos; en, endoderm; no, notochord; nt, neural tube; so, somite. (F) Ventralized embryo after UV irradiation. (G) Complete rescue of the ventralized phenotype by a single injection of 0.8 ng xBtg-x mRNA at the 8-cell stage.
Fig. 3. xBtg-x activates the BCNE center gene expression in Xenopus embryos. (A�D) Whole-mount in situ hybridization of blastula stage 9 embryos with Xnr-3 (A, B) and chordin probes (C, D). Embryos were injected four times animally with 0.8 ng xBtg-x mRNA (B, D). Note the xBtg-x-induced expansion of Xnr-3 and chordin expression throughout the animal cap at blastula. All embryos are shown in animal view with dorsal to the top. (E) RT-PCR analysis of uninjected whole embryos (WE) and animal cap (AC) explants at stage 10 of Xenopus embryos left untreated (lane 2), injected into the animal region with 800 pg δN-XTcf-3 (lane 3), 3.2 ng xBtg-x (lane 4), or δN-XTcf-3 and xBtg-x mRNA (lane 5). EF-1α is a control for equal loading. (F) RT-PCR analysis of ectodermal explants at stage 20. Note that xBtg-x mRNA functions as a neural inducer.
Fig. 4. xBtg-x activates the Wnt/β-Catenin signaling pathway. (A) Luciferase assays of gastrula stage animal cap explants from Xenopus embryos injected with a Xnr-3 promoter luciferase construct. (B) Animal caps microinjected with the TOP-flash or FOP-flash Wnt/β-Catenin reporter alone or co-injected with 3.2 ng xBtg-x mRNA. (C) Luciferase reporter assay using the TOP-flash or FOP-flash Wnt/β-Catenin reporter in human 293T kidney cells transfected with xBtg-x, stabilized β-Catenin (δN-β-Catenin), xBtg-x and δN-β-Catenin, dominant-negative Axin (AxinδRGS), or AxinδRGS together with xBtg-x. (D) Western blot analysis of gastrula stage Xenopus embryos injected into the animal pole with 150 pg β-Catenin-myc mRNA at the 4-cell stage. Subsets of the injected embryos were then either left untreated, treated with LiCl at the 32-cell stage, or injected with 3.2 ng xBtg-x mRNA at the 8-cell stage. Note that xBtg-x mRNA does not significantly stabilize β-Catenin protein. (E) Uninjected control Xenopus embryo at stage 40, (F) embryo microinjected once ventrally at the 8-cell stage with 0.8 ng xBtg-x mRNA showing a partial secondary axis (50%, n = 24) and (G) injected with a combination of xBtg-x and 60 pg Nlk mRNA. Note that double axis formation by xBtg-x is blocked by co-injection of Nlk mRNA (0%, n = 18).
Fig. 5. Phenotype of xBtg-x antisense morpholino oligomer injections. (A) In vitro transcription/translation of pCS2-xBtg-x in the presence of control morpholino and xBtg-x-MO1. (B) Histological analysis of uninjected control embryos and (C) embryos injected at the 2-cell stage with xBtg-x-MO1 + 2 at stage 40; en, endoderm; nt, neural tube; no, notochord; so, somites. (D�K) Whole-mount in situ hybridization of uninjected and xBtg-x-MO1 + 2 injected embryos at neurula stage 14. (D, E) Combination of Otx2, Krox20, and HoxB9 probes; (F, G) Sox-2; (H, I) Pax3; (J, K) Slug. Note that xBtg-x-MO1 + 2 inhibits neural plate convergence and extension movements.
btg5.2 (B-cell translocation gene 5, gene 2) gene expression in bisected Xenopus laevis embryos, NF stage 10, as assayed by in situ hybridization. equatorial view: dorsal axis on the right
btg5.2 (B-cell translocation gene 5, gene 2) gene expression in bisected Xenopus laevis embryos, NF stage 14, as assayed by in situ hybridization. Dorsal view: anterior left
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