As Xhairy2b is expressed dorsally and has axis-inducing activity, we expected that its overexpression in the dorsal marginal zone would either enhance dorsal fate or show no significant effect. However, we found that the dorsal injection of Xhairy2b mRNA caused head defects (82%, n = 215). In Xhairy2b-injected embryos, the expression of anterior neural marker genes, such as nkx2.4 (hypothalamus; Small et al., 2000), otx2 (eye, forebrain, and midbrain; Blitz and Cho, 1995), and en2 (midbrain–hindbrain boundary; Hemmati-Brivanlou et al., 1991), was absent or significantly reduced (nkx2.4 55%, n = 56; otx2 38%, n = 58; en2 36%, n = 59; Fig. 3A–C′). On the other hand, the expression of krox20 (Bradlay et al., 1993) in hindbrain was detected in all injected embryos (n = 54, Fig. 3D,D′). These data indicate that an excess of Xhairy2b can suppress the head structures. This head defect might be caused by a deficit in the anterior endoderm, which is known to be necessary for the head formation (reviewed by Beddington and Robertson, 1998). To confirm this possibility, the expression of anterior endodermal markers, Xhex (Newman et al., 1997), Xdkk1, and cerberus, was analyzed in Xhairy2b dorsally injected embryos. Among them, the endogenous expression of Xhex and Xdkk1 was suppressed by Xhairy2b but that of cerberus was not (Fig. 3F–H′). As shown in Figure 3F–H′, because the expression of Xhex and Xdkk1 was partially intact in the organizer, the capability of Xhairy2b for head suppression might be underestimated. To analyze precisely the head deformation caused by Xhairy2b, we coinjected β-catenin and Xhairy2b mRNA on the ventral side of the embryo and analyzed the induction of the secondary axis and the ectopic expression of the anterior endodermal genes described above. As the ventral expression of β-catenin is known to generate the entire organizer (Guger and Gumbiner, 1995), this coinjection experiment can induce the distribution of Xhairy2b throughout the organizer tissue. In these embryos, the coexpression of Xhairy2b almost completely suppressed the induction of Xhex and Xdkk1 expression but not that of cerberus expression, whose expression is induced by β-catenin alone (Fig. 3L–N′; Table 3). As expected, the formation of head structures in the secondary axis induced by β-catenin overexpression was strongly inhibited in the Xhairy2b and β-catenin coexpressing embryos, although a secondary trunk was induced (78%, n = 226; Fig. 3E,E′; Table 2). Consistent with the results obtained by the Xhairy2b dorsal injection, the expression of krox20 was not affected (100%, n = 17), whereas the expression of en2 and otx2 was significantly affected by Xhairy2b coinjection (en2 50%, n = 16; and otx2 94%, n = 16; data not shown). These results confirm that Xhairy2b has the ability to suppress the formation of forebrain to midbrain. Furthermore, we investigated whether the head defects caused by Xhairy2b mRNA coinjection could be rescued by Xhex, Xdkk1, or cerberus mRNAs. As head development could be rescued only by Xhex and Xdkk1 (data not shown), it was speculated that the depletion of Xhex and Xdkk1 caused the observed head defects. From these results, Xhairy2b is able to suppress the expression of anterior endodermal markers, and this suppression is responsible for the defect of the anterior head structures (forebrain to midbrain).
Figure 3. Xhairy2b expression inhibits head formation and represses expression of some trunk and head organizer genes. A–D′: Uninjected control embryos (A–D) and 800 pg of Xhairy2b mRNA dorsally injected embryos (A′–D′) 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–Q′: Uninjected control embryo (F–K), 800 pg of Xhairy2b mRNA dorsally injected embryos (F′–K′), 400 pg of β-catenin mRNA ventrally injected embryos (L–Q), and 400 pg of β-catenin and 800 pg of Xhairy2b mRNA ventrally coinjected embryos (L′–Q′) 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–Q). 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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