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Figure 1. Organizer-inducing factors activate Xhairy2b expression. A-F: Spatial expression patterns of Xhairy2b by whole-mount in situ hybridization at stage 10.5. Cleared embryos are shown in lateral view and dorsal is to the left. A: Uninjected control embryo. B-F: Ectopic induction of Xhairy2b expression in embryos ventrally microinjected with mRNAs encoding organizer-inducing factors. mRNAs of activin (B), BVg1 (C), Xnr1 (D), Xnr2 (E), and follistatin (F) were injected at the eight-cell stage. Arrowheads show the ectopic expression of Xhairy2b.
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Figure 2. Ventral expression of Xhairy2b induces a secondary axis and inhibits ventralizing factors. A,B: Uninjected control embryo and Xhairy2b mRNA ventrally injected embryo, respectively, at stage 35. Xhairy2b mRNA ventral injection results in the induction of secondary axes lacking head structures (arrowhead). C: Transverse section of the embryo shown in B. Secondary axis (arrowhead) has neural tube and somitic mesoderm but lacks notochord. no, notochord, nt, neural tube, so, somite. D,D′′: Uninjected control embryos (D) and Xhairy2b mRNA (800 pg) ventrally injected embryos (D′′) are shown in vegetal view, dorsal side up at stage 10.5. D′′: Expression of organizer genes, follistatin (D′), frzb1 (E′), and admp (F′) is ectopically induced by Xhairy2b (arrowheads). G′: The expression of Xvent1 and Xvent2 in the ventral region (G,H) is reduced by Xhairy2b expression (G′,H′, arrowheads).
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Figure 3. Xhairy2b expression inhibits head formation and represses expression of some trunk and head organizer genes. A′: Uninjected control embryos (A) and 800 pg of Xhairy2b mRNA dorsally injected embryos (A′′) at stage 30. The overexpression of Xhairy2b results in a deficit of the expression of nkx2.4 (A,A′), otx2 (B,B′), and en2 (C,C′) but not that of krox20 (D,D′). Arrowheads indicate the remainder of the expression. E,E′: β-catenin ventrally injected embryo (E) and β-catenin and Xhairy2b coinjected embryo (E′). β-catenin expression induces a secondary axis containing a complete head (arrowhead in E), whereas the coinjection of Xhairy2b and β-catenin induces a secondary axis lacking head structures (arrowhead in E′). F′: Uninjected control embryo (F), 800 pg of Xhairy2b mRNA dorsally injected embryos (F′′), 400 pg of β-catenin mRNA ventrally injected embryos (L), and 400 pg of β-catenin and 800 pg of Xhairy2b mRNA ventrally coinjected embryos (L′′) are shown in vegetal view, dorsal side up at stage 10.5. The expression of Xhex (F,F′), Xdkk1 (G,G′), chordin (I, I′), and Xnot (J,J′) is repressed by Xhairy2b (green arrowheads), whereas that of cerberus (H,H′) and noggin (K,K′) is not affected by Xhairy2b. Light blue staining is coinjected β-gal. These genes are ventrally induced by β-catenin expression (red arrowheads in L). The ectopic expression of Xhex (L′), Xdkk1 (M′), chordin (O′), and Xnot (P′) is repressed by Xhairy2b coexpression with β-catenin (green arrowheads). Meanwhile, the expression of cerberus (N′) and noggin (Q′) is not affected (red arrowheads).
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Figure 4. Comparison of the expression domain of Xhairy2b with that of anterior endodermal genes and chordamesodermal genes at the early gastrula stage. A: Neighboring sections in the mid-sagittal plane of stage 10.5 embryos. Dorsal is to the right. Arrowheads indicate the blastopore lip. B,D,F:Xhairy2b is expressed in the ectoderm and the deep layer of the dorsal blastopore lip at the early gastrula stage. A,C: The expression of Xhex (A) and Xdkk1 (C) in the anterior endoderm is hardly overlapped with the expression of Xhairy2b. E: The expression of chordin in the deep layer of the organizer is overlapped with that of Xhairy2b.
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Figure 5. Xhairy2b morpholino oligonucleotide (mo) specificity and change of axial mesoderm in Xhairy2b-depleted embryos. A: Alignment of the injected Xhairy2b mRNA constructs and morpholino oligos (1: five-mismatch control mo; 2: Xhairy2b mo). Xhairy2byc Tag (MT) constructs (3) contain the 5′-untranslated region binding site for the Xhairy2b morpholino, whereas the -mo Xhairy2bT constructs (4) do not. B: Western blot analysis of Xhairy2bT. The efficacy of the morpholinos is tested by blocking the in vivo translation of Xhairy2bT mRNA and morpholino-injected embryos. Uninjected control (lane 1) and embryos injected with Xhairy2bT mRNA (lane 2), Xhairy2bT mRNA+five-mismatch control mo (lane 3), Xhairy2bT mRNA+Xhairy2b mo (lane 4), -mo Xhairy2bT mRNA (lane 5), or -mo Xhairy2bT mRNA+Xhairy2b mo (lane 6). The translation of Xhairy2bT is repressed by coinjecting Xhairy2b mo but not the five-mismatch control mo. On the other hand, the translation of -mo Xhairy2b mo, which does not contain the morpholino binding site, is not suppressed by Xhairy2b mo. Coomassie staining of the cell pellets served as a loading control. C,C′: In situ hybridization of chordin and pax2 in control mo-injected embryo (C) and Xhairy2b morpholino antisense oligo-injected embryo (C′) at stage 13. The expression of chordin is expanded in the mo-injected embryo. Dorsal view and anterior to the top. D′: Neighboring section in the mid-sagittal plane of control mo-injected embryo (D,E) and Xhairy2b mo-injected embryo (D′,E′). At the early neurula stage, the mesodermal expression of Xhairy2b is restricted to the anterior prechordal mesoderm and is distinct from the expression of chordin. In the mo-injected embryo, the expression of chordin (D′) and Xhairy2b (E′) is overlapped in the anterior prechordal mesoderm. Arrowheads indicate the posterior limits of Xhairy2b expression in the axial mesoderm. Anterior is to the left. F,F′:goosecoid expression in control mo-injected (F) and Xhairy2b mo-injected (F′) embryos. The goosecoid expression is significantly decreased in Xhairy2b mo-injected embryo. Light blue staining is in situ hybridization for coinjected YFP mRNA. G′: Mid-sagittal section of stage 14 control mo-injected embryo (G) and Xhairy2b mo-injected embryo (G′′). Anterior is to the left. Prechordal mesoderm-specific goosecoid expression is decreased in the posterior region (G,G′) and chordamesoderm-specific Xbra and Xnot expression is anteriorly increased (H′). Arrowheads indicate the limit of the gene expression domain. The dotted line is the border between the anterior neuroectoderm and the prechordal mesoderm. J: Schematic diagram of the axial mesodermal tissue in Xhairy2b mo-injected embryo. Anterior prechordal mesoderm (apm) is decreased, and the region of the posterior prechordal mesoderm (ppm) and the notochord (nc) is anteriorly expanded.
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