G, despite the injection of mNanog mRNA [21,31] (Fig. 2H). This result suggested that mNanog does not affect canonical Wnt signaling in the embryos stages we examined.mNanog injection promoted (-)-Calyculin A expression of dorsal mesodermal genes, but inhibited ventral mesodermal genes in both AC and embryosThe phenotypes of mNanog-injected embryos and their corresponding ACs suggested to us that mNanog could induce dorsal mesodermal tissues. We next performed RT-PCR analysis to examine the expression of mesodermal genes in 1676428 earlier stages. When 200 pg of mNanog mRNA was injected into 2-cell embryos, the expression of dorsal mesodermal marker genes chd, gsc, and xlim-1 was increased in stage-11 ACs without Activin A treatment (Fig. 2A 1st?rd column; lane 3, 5), and 400 pg of mNanog mRNA injection further increased these gene expressions (Fig. 2A column 1, 2, 3; 22948146 lane 7). The mNanog injections only slightly enhanced the same expressions in Activin A-treated AC (Fig. 2A 1st?rd column; lane 4, 6, 8). On the other hand, Xbra expression was not effectively induced by mNanog injection (Fig. 2A 4th column, lane 3, 5, 7), and induction of Xbra expression by Activin A treatment was clearly inhibited by mNanog (Fig. 2A 4th column, lane 4, 6, 8). Similar inhibition was observed with 
Xwnt8, mix, mixer, Cerberus (Cer), and Sox17a [23?7] (Fig. 2A 5th?th columns). To assess whether the enhancement of dorsal mesodermal gene expressions was specific for mNanog function, we carried out RT-PCR with a deletion mutant of mNanog that produces a protein lacking the Cterminus domain including the W-repeat motif (Madrasin mNanogDCD;mNanog subsidiary utilizes Activin-nodal signaling for dorsal-mesoderm inductionPreviously, it was shown that both mesoderm and endoderm formation requires activation of Activin/nodal signaling. Thus, we next examined whether the expression of Xnr genes is induced by mNanog. RT-PCR analysis indicated that Xnr1 and Xnr2 expressions were increased in a dose-dependent manner (Fig. 3A). On the other hand, expression of Xnr5/6 was not increased in mNanoginjected AC (Fig. 3B). From these results, we proposed that mesoderm induction by mNanog involves the upregulation of not Xnr5/6, but Xnr1/2. To assess whether mNanog overexpression promotes the nuclear transport of Smad2, we coinjected embryos with mNanog and Smad2GFP [32]. Without mNanog injection, GFP signal was observed in the cytoplasm of AC cells (Fig. 3C), whereas 10 pg of Xnr5 injection promoted a nuclear localization of the GFP signal (Fig. 3E). When 200 pg of mNanog was coinjected, Smad2GFP signal was occasionally observed in nuclei, although the efficiency was low (Fig. 3D). This result suggested that, at least in some cases, mNanog regulates Activin/nodal signaling through Xnr1/2. Next, to clarify whether mesodermal gene induction was dependent on Activin signaling, coinjection experiments were performed with a truncated form of the type I Activin receptor (tALK4) [15], which acts as a dominant-negative mutant. Indeed,Dorsal Mesoderm-Inducing Activity of NanogFigure 1. Mesodermal tissues were induced by mNanog mRNA injection. A) Detection of exogenous mNanog protein in Xenopus embryo by Western blotting with antibodies to mNanog (upper) and alpha-tubulin (lower). Non-injected embryo control (lane 1). mNanog-injected embryo (lane 2). B ) Subcellular localization of mNanog protein in stage-10 embryo injected with mNanog mRNA. Ectoderm from normal embryo (B) or mNanoginjected embryo (C). Dissected tissues w.G, despite the injection of mNanog mRNA [21,31] (Fig. 2H). This result suggested that mNanog does not affect canonical Wnt signaling in the embryos stages we examined.mNanog injection promoted expression of dorsal mesodermal genes, but inhibited ventral mesodermal genes in both AC and embryosThe phenotypes of mNanog-injected embryos and their corresponding ACs suggested to us that mNanog could induce dorsal mesodermal tissues. We next performed RT-PCR analysis to examine the expression of mesodermal genes in 1676428 earlier stages. When 200 pg of mNanog mRNA was injected into 2-cell embryos, the expression of dorsal mesodermal marker genes chd, gsc, and xlim-1 was increased in stage-11 ACs without Activin A treatment (Fig. 2A 1st?rd column; lane 3, 5), and 400 pg of mNanog mRNA injection further increased these gene expressions (Fig. 2A column 1, 2, 3; 22948146 lane 7). The mNanog injections only slightly enhanced the same expressions in Activin A-treated AC (Fig. 2A 1st?rd column; lane 4, 6, 8). On the other hand, Xbra expression was not effectively induced by mNanog injection (Fig. 2A 4th column, lane 3, 5, 7), and induction of Xbra expression by Activin A treatment was clearly inhibited by mNanog (Fig. 2A 4th column, lane 4, 6, 8). Similar inhibition was observed with Xwnt8, mix, mixer, Cerberus (Cer), and Sox17a [23?7] (Fig. 2A 5th?th columns). To assess whether the enhancement of dorsal mesodermal gene expressions was specific for mNanog function, we carried out RT-PCR with a deletion mutant of mNanog that produces a protein lacking the Cterminus domain including the W-repeat motif (mNanogDCD;mNanog subsidiary utilizes Activin-nodal signaling for dorsal-mesoderm inductionPreviously, it was shown that both mesoderm and endoderm formation requires activation of Activin/nodal signaling. Thus, we next examined whether the expression of Xnr genes is induced by mNanog. RT-PCR analysis indicated that Xnr1 and Xnr2 expressions were increased in a dose-dependent manner (Fig. 3A). On the other hand, expression of Xnr5/6 was not increased in mNanoginjected AC (Fig. 3B). From these results, we proposed that mesoderm induction by mNanog involves the upregulation of not Xnr5/6, but Xnr1/2. To assess whether mNanog overexpression promotes the nuclear transport of Smad2, we coinjected embryos with mNanog and Smad2GFP [32]. Without mNanog injection, GFP signal was observed in the cytoplasm of AC cells (Fig. 3C), whereas 10 pg of Xnr5 injection promoted a nuclear localization of the GFP signal (Fig. 3E). When 200 pg of mNanog was coinjected, Smad2GFP signal was occasionally observed in nuclei, although the efficiency was low (Fig. 3D). This result 
suggested that, at least in some cases, mNanog regulates Activin/nodal signaling through Xnr1/2. Next, to clarify whether mesodermal gene induction was dependent on Activin signaling, coinjection experiments were performed with a truncated form of the type I Activin receptor (tALK4) [15], which acts as a dominant-negative mutant. Indeed,Dorsal Mesoderm-Inducing Activity of NanogFigure 1. Mesodermal tissues were induced by mNanog mRNA injection. A) Detection of exogenous mNanog protein in Xenopus embryo by Western blotting with antibodies to mNanog (upper) and alpha-tubulin (lower). Non-injected embryo control (lane 1). mNanog-injected embryo (lane 2). B ) Subcellular localization of mNanog protein in stage-10 embryo injected with mNanog mRNA. Ectoderm from normal embryo (B) or mNanoginjected embryo (C). Dissected tissues w.