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We have used differential display to identify genes inducible by activin and isolated a novel member of the T-box gene family that includes the Xenopus genes Xbrachyury and Eomesodermin. Here we show that this novel gene is unique within the T-box family because it is maternally expressed at a high level. Furthermore, it belongs to a rare class of maternal mRNAs in Xenopus that are localised to the vegetal hemisphere of the egg and we have therefore named it Antipodean. We show here that low amounts of Antipodean injected into ectoderm (animal cap cells) strongly induce pan mesodermal genes such as Xbrachyury and ventral mesodermal genes such as Xwnt-8. Overexpression of Antipodean generates mesoderm of ventral character, and induces muscle only weakly. This property is consistent with the observed late zygotic Antipodean mRNA expression in the posterior paraxial mesoderm and ventral blastopore, and its exclusion from the most dorsal mesodermal structure, the notochord. Antipodean is induced by several molecules of the TGF-beta class, but in contrast to Xbrachyury, not by bFGF. This result suggests that the expression of these T-box genes may be under the control of different regulatory pathways. Finally, we demonstrate that Antipodean and Eomesodermin induce each other and both are able to induce Xbrachyury. The early zygotic expression of Antipodean is not induced by Xbrachyury, though later it is to some extent. Considering its maternal content, Antipodean could initiate a cascade of T-box gene activations. The expression of these genes may, in turn, sustain each other's expression to define and maintain the mesoderm identity in Xenopus.
Fig. 3. Localisation of maternal Apod mRNA throughout oogenesis and early cleavage stages in Xenopus. Apod mRNA was visualised by in situ hybridisation to previously sectioned material using DIG-labelled antisense riboprobes (A-D) or sense probes as negative controls (E). Cells are orientated such that the animal pole is uppermost. (A) Stage 3 oocyte showing presence of Apod mRNA in both the vegetal cortex and yolk mass. (B) Stage 4 oocyte. (C) Mature stage 6 oocyte showing the particulate localisation of Apod mRNA in the vegetal yolk mass. The mRNA is now localised in a more subequatorial position. (D) Egg showing more diffuse localisation of Apod mRNA, still distributed in the vegetal yolk mass. (E) Egg hybridised with a sense probe showing very faint non-specific staining throughout the animal hemisphere only. A and B are shown at a greater magnification than C-E for convenient comparison. (gv, germinal vesicle)
Fig. 4. Apod mRNA shows a more widespread distribution than Xbra mRNA in the gastrula. In situ hybridisation was performed on whole-mount (A,B,E,F) or sectioned (C,D) embryos. The dorsal side is to the right in A-D, and the position of the dorsal lip is indicated with a solid arrow. Late stage gastrulae were also hybridised with probes for Apod mRNA as whole mounts (E,F) and show its presence in the posterior paraxial mesoderm and ventralblastopore. Apod mRNA is notably excluded from the notochord (indicated with an open arrow). (A,C) Apod probe, stage 10.5. (B,D) Xbra probe, stage 10.5. (E) Posterior view, dorsal side at top; Apod probe stage 13. (F) Dorsal view, anterior end at top; Apod probe, stage 13.
Fig. 5. Apod mRNA can induce mesoderm. Apod mRNA (500 pg) was ectopically expressed in the whole embryo by injection of synthetic mRNA into the animal pole region of two-cell embryos. These were reared until uninjected siblings had reached stage 26. Embryos ectopically expressing Apod failed to gastrulate and developed as exogastrulae (A). Apod-injected and uninjected embryos were sectioned and then analysed for the expression of Xbra by in situ hybridisation at stage 10.25 (B,C, respectively). The ectopic expression of mesodermal markers in the animal cap region of the embryo suggests that Apod is able to induce mesoderm. To further characterise Apod mesoderm- inducing properties, the indicated amounts of Apod mRNA were injected into the animal pole region of embryos at the 2-cell stage. At stage 9, animal caps were isolated and cultured as explants until sibling embryos had reached stage 10.25. Total RNA was extracted from the equivalent of two embryos and analysed for the expression of mesodermal markers by RNase protection (D). Apod mRNA is able to strongly induce Xwnt-8, Xbrachyury (Xbra), Eomesodermin (Eomes) and Mix.1. At high Apod mRNA concentrations there is a weak activation of goosecoid (gsc). Siamois (Sia) is not activated. Histological examination shows that whereas uninjected controls form atypical epidermis (E), Apod-injected animal caps form mesoderm of ventral character as judged by the presence of mesenchyme and vesicles (F). Immunostaining of explants in E and F with the muscle- specific 12/101 antibody showed no muscle (see also Table 1).
