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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. |
