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Fig. 5. XTcf-4 regulates the expression of midbrain and isthmus marker genes. Ten picomoles of XTcf-4 morpholino were coinjected with 100 pg EGFP-RNA. At late neurula stages, embryos were sorted by the fluorescence signal of the coinjected EGFP. At stage 30, embryos were fixed and stained by in situ hybridization for the expression of the indicated genes. The midbrain marker genes wnt-2b, otx-2, and pax-6 and the isthmus marker genes en-2 and fgf-8were markedly reduced at the XTcf-4 morpholino (Tmo)-injected side (marked with an asterisk), while the forebrain marker bf-1 and the rhombomere 3 and 5 marker krox-20 were not reduced. The control morpholino (Como) did not reduce the expression of any of the marker genes analyzed. In early neurula (stage 13), the XTcf-4 morpholino did not alter the expression of pax-6. f: forebrain, m: midbrain, h: hindbrain. |
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Fig. 1. Knockdown of Xwnt-2b results in reduced expression of tcf-4 and en-2. (A) Alignment of Xwnt-2b with the Xwnt-2b EGFP construct containing the 5′-UTR binding site for the Xwnt-2b morpholino (Wmo) but not for the control morpholino (Como). (B) Wnt-2b morpholino (Wmo), but not control morpholino (Como), represses the translation of exogenously expressed Xwnt-2b (Xwnt-2bEGFP). NOP lysates of a single embryonic equivalent of stage 13 (according to Nieuwkoop and Faber, 1967) stained with α-EGFP antibody. Counterstaining of the blot with α-Gemin was used as loading control. (C) Wnt-2b morpholino blocks the expression of XTcf-4 protein in the midbrain. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. Transverse sections through the midbrain region of stage 30 embryos were stained with α-Tcf3/4 primary antibody and visualized with cy3-labeled secondary antibody. The EGFP signal indicates the injected side. (D) In situ hybridization of stage 30 embryos reveals that the Wnt-2b morpholino blocks the expression of tcf-4 and en-2, but not otx-2 and pax-6. Ten picomoles of the morpholino were injected together with 100 pg of myc-tagged EGFP mRNA into one blastomere of two-cell stage embryos. At late neurula stages, embryos were sorted by the fluorescence signal of EGFP. At stage 30, embryos were fixed and analyzed by whole-mount in situ hybridization. f: forebrain, m: midbrain, h: hindbrain. |
