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The interplay of fibroblast growth factor (FGF) and nodal signaling in the Xenopus gastrula marginal zone specifies distinct populations of presumptive mesodermal cells. Cells in the vegetal marginal zone, making up the presumptive leading edge mesoderm, are exposed to nodal signaling, as evidenced by SMAD2 activation, but do not appear to be exposed to FGF signaling, as evidenced by the lack of MAP kinase (MAPK) activation. However, in the animal marginal zone, activation of both SMAD2 and MAPK occurs. The differential activation of these two signaling pathways in the marginal zone results in the vegetal and animal marginal zones expressing different genes at gastrulation, and subsequently having different fates, with the vegetal marginal zone contributing to ventralmesoderm (e.g. ventralblood island) and the animal marginal zone giving rise to dorsal fates (e.g. notochord and somite). We report here the cloning of a cDNA encoding a novel nuclear protein, Xmenf, that is expressed in the vegetal marginal zone. The expression of Xmenf is induced by nodal signaling and negatively regulated by FGF signaling. Results from animal cap studies indicate that Xmenf plays a role in the pathway of ventralmesoderm induction in the vegetal marginal zone.
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Fig. 4. Expression of Xmenf. (A) Expression of Xmenf in staged Xenopus embryos. Expression was first seen at the mid-blastula transition (stage 9). The expression persisted and peaked during gastrulation, and was no longer detectable after gastrulation, with the exception of late tailbud stage (stage 36). Ethidium staining for 28S and 18S RNAs served as a loading control. (B–D, F–H). Double in situ hybridization in gastrula embryos for Xmenf (black, arrows) and Xbra (red, arrowheads). Embryos in (B–D) are in vegetal views, oriented with the Spemann organizer at the bottom. (F–H) Midline sections with the Spemann organizer are to the right. At stage 10, staining for Xbra expression is very faint (B, F), while Xmenf is expressed in cells in the marginal zone just above the blastopore lip in the Spemann organizer (the right arrow in F), and in cells in the superficial layer of the side opposite from the organizer (the left arrow in F). Xmenf is also expressed superficially in vegetal cells (B). At stage 10.25, the expression domains of Xmenf and Xbra are not only mutually exclusive, most obviously near the Spemann organizer (the bottom part of the embryo in C and the right part of the embryo in G), but also opposite from the Spemann organizer (the left part of the embryo in G). Although the expression domains of the genes look as if they overlap in surface view (the top part of the embryo in C), the section shows that Xmenf is expressed in cells in the superficial layer of the animal marginal zone, while Xbra is expressed in cells in the deep layer (the left arrow and arrowhead in G). By stage 10.5, Xmenf is no longer expressed near the Spemann organizer, nor on the opposite side of the embryo in the superficial layer. At this stage, the expression domains of Xmenf and Xbra remain mutually exclusive in the marginal zone opposite from the Spemann organizer (the top part of the embryo in D and the left arrow and arrowhead in H). (E, I) Double in situ hybridization at stage 10.25–10.5 for Xmenf (black, arrows) and Xbra (red, arrowheads) in embryos that had been injected with XFD RNA in the marginal zone (B4 and C4 blastomeres at the 32-cell stage). The marginal zone on the injected side no longer expresses Xbra and shows very intense expression of Xmenf (arrow in E). The section in (I) reveals that Xmenf expression is expanded toward the region (arrow in I), where Xbra is normally expressed (arrowhead in H).
Fig. 5. Regulation of Xmenf expression. (A) Northern blot of RNA from stage 10.5 animal caps injected with 1 ng of Xnr2 RNA with or without 400 pg of XFD RNA. Shown are results of hybridization for Xmenf and EF1α (loading control). Also shown is RNA from a whole embryo at stage 32 (W, lane 5). Xnr2 RNA alone induces a low level of Xmenf expression in animal caps (lane 3), while coinjection of XFD RNA causes a much higher level of Xmenf expression (lane 4). (B) Northern blot of RNA from stage 10 animal caps that had been injected with 400 pg of XFD RNA at the two-cell stage and cultured after dissection at stage 8 in activin-containing COS cell media (Hansen et al., 1997), with or without 5 μg/ml of cycloheximide (CHX). Results of hybridization for Xmenf, eomes and EF1α (loading control) are shown. Induction of Xmenf expression by activin was significantly reduced in the presence of CHX (lane 5 compared to lane 4), while eomes expression was not affected by CHX as described previously (Ryan et al., 1996). ‘W’ represents RNA from a whole embryo at stage 10 (lane 1). (C) In situ hybridization for Xmenf in embryos that had been injected with 500 pg of CmXnr2 RNA in one blastomere at the four-cell stage. Vegetal view with the Spemann organizer at the top. β-Gal staining in blue (arrowhead) is indicative of descendants of the injected cell. Xmenf expression is lost in descendants of the CmXnr2-injected blastomere (arrowhead).
