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The forkhead type Brain Factor 2 from mouse and chicken help pattern the forebrain, optic vesicle and kidney. We have isolated a Xenopus homolog (Xbf2) and found that during gastrulation it is expressed in the dorsolateral mesoderm, where it helps specify this territory by downregulating BMP-4 and its downstream genes. Indeed, Xbf2 overexpression caused partial axis duplication. Interference with BMP-4 signaling also occurs in isolated animal caps, since Xbf2 induces neural tissue. Within the neurulaforebrain, Xbf2 and the related Xbf1 gene are expressed in the contiguous diencephalic and telencephalic territories, respectively, and each gene represses the other. Finally, Xbf2 seems to participate in the control of neural crest migration. Our data suggest that XBF2 interferes with BMP-4 signaling, both in mesoderm and ectoderm.
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Fig. 2. Expression pattern of Xbf2 in Xenopus embryos at different developmental stages. (A) Dorsal views, anterior is up; (F) lateral view, anterior is up. (A) At stage 10 Xbf2 is initially detected in the dorso-lateral prospective mesoderm. (B) Stage 11 embryo showing Xbf2 expression in the uninvoluted dorsolateral prospective mesoderm. (C) Stage 12.5; at this stage expression appears in the anterior neural plate (arrow) and in the anterior neural folds (arrowhead). (D,E) At stages 14 and 17, respectively, Xbf2 expression becomes stronger in the anterior neural plate (arrows) and in the anterior neural folds (arrowheads). At stage 17, Xbf2 is clearly expressed in the segmented premigratory cephalic neural crest (arrowhead). (F) Stage 20 embryo showing Xbf2 expression in the eye (arrow) and in the paraxial mesoderm (arrowhead). Note that at this stage the expression in the migratory neural crest has disappeared.
Fig. 3. Effect of Xbf2 overexpression on embryonic development. Embryos were injected at the 1-cell stage (A) or 2-cell stage (B) with different concentrations of Xbf2 mRNA and 0.3 ng of lacZ mRNA as a lineage marker (blue dots). Embryos were cultured until stage 16 or 25, when the expression of different genes was analyzed by in situ hybridization. Views are from the dorsal side, anterior is up. (A) Reduced head and absence of anterior structures, such as eyes or cement gland (arrow) and partial secondary axis (arrowhead) in an embryo injected with 4 ng of Xbf2 mRNA (95%, n = 22). (B) Low level of Xtwist (B) or Xslug (C) expression in the secondary axis of an embryo injected with 0.25 ng of Xbf2 mRNA. This duplicate axis is clearly detected by Xsox2 (D) expression (arrowheads point to the double axis that were found in about 5% of the injected embryos, n = 32, 43 and 54, respectively). (E) Ectopic expression of Xsox2 close to the ventral region of an embryo injected with 4 ng of Xbf2 mRNA (arrow; 93%, n = 54). (F) Embryos injected with 4 ng of Xbf2 mRNA also showed expansion of the XMyoD domain in the injected side (arrow; 75%, n = 40).
Fig. 4. Xbf2 affects the neural inducer capacity of the mesoderm. Different tissues were dissected from control or Xbf2 injected stage 10 embryos, conjugated and cultured until the equivalent of stage 17, when Xsox2 expression was analyzed. (A) Control animal caps showed no Xsox2 expression (n = 15). (B) All the conjugates of animal cap and dorsal marginal zone strongly expressed Xsox2 (n = 14). (C) Animal caps conjugated with ventral marginal zone did not express Xsox2 (n = 17). (D) Most of the conjugates of animal cap and ventral marginal zone taken from embryos injected with 4 ng of Xbf2 mRNA expressed Xsox2 (70%, n = 13), although at lower levels than conjugates of animal cap and dorsal marginal zone. In contrast, the ventral marginal zone alone taken from injected embryos did not express Xsox2 (inset; n = 10).
Fig. 5. Effect of Xbf2 mRNA on mesodermal development. Embryos were injected in one cell at the 2-cell stage in the equatorial region with 4 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA and cultured until stage 10.5-11.5, when the expression of different mesodermal markers were analyzed. Arrowheads point to the injected sites. Neither gsc (A) nor Xlim1 (B) expression were affected by Xfb2 overexpression (100%, n = 18 and 13, respectively). (C) XMyoD expression was strengthened in the lateralmesoderm in the injected side (60%, n = 34). (D) Ventral or lateralXbf2 injections did not affect Xvent2 expression (94%, n = 48). In contrast, overexpression of Xbf2 strongly inhibited the expression ofXwnt8 (E; 95%, n = 45) and Xvent1 (F; 92% n = 23) and weakly that of BMP-4 (G; 80% n = 15) and Xmsx1 (H; 58%, n = 12) at the injection site.
Fig. 6. Direct neuralization of the ectoderm by Xbf2 expression. Animal caps were dissected from stage 9 embryos and cultured until the equivalent of stage 17, when Xsox2 expression was analyzed. (A) Animal caps taken from embryos injected with 4 ng of Xbf2 mRNA at the 1 cell stage. Xsox2 is expressed in these caps (58%, n = 12). Note also that these caps are not elongated as could be expected if dorsal mesoderm was induced. Inset: control animal caps showing no Xsox2 expression (100%, n = 13). (B) Embryos were injected in one blastomere at the 2-cell stage with 4 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA. Xmsx1 expression was analyzed at stage 16 embryos. Notice the absence of Xmsx1 expression at the injected side (arrowhead; 83%, n = 18).
Fig. 7. Xbf2 participates in patterning of the forebrain. All the embryos are shown from anterior side. The weaker expression of Xbf2 in neural crest cells is not visible in A and B due to low staining development. (A) Double in situ hybridization of an stage 17 embryo. Blue (arrow) corresponds to Xbf2 expression and purple (arrowhead) denotes Xbf1 expression. (B) Xbf2 expression in an stage 17 embryo injected in one blastomere of a 2-cell stage embryo with 0.5 ng of Xbf1 mRNA and 0.3 ng of lacZ mRNA. Notice the complete inhibition of Xbf2 expression at the injected side (58%, n = 12). (C) Embryo injected in one blastomere of a 2-cell stage embryo with 0.25 ng of Xbf2 mRNA and 0.3 ng lacZ mRNA and cultured until stage 17. Xbf1 expression is suppressed in the injected side (72%, n = 22). (D) Similar embryo as in C, but expression of Xcpl1 was analyzed. Notice the inhibition of Xcpl1 in the injected side (53%,n = 15).
Fig. 8. Overexpression of Xbf2 blocks neural crest migration. (A,B) One blastomere of a 2-cell stage embryo was injected with 0.25 ng of Xbf2 mRNA and 0.3 ng of lacZ mRNA, was cultured until stage 25 and the expression of Xtwist was analyzed. (A) Right side view of the embryo shown in (B). (A) View of uninjected right side. Notice the normal expression of Xtwist and the migration of the neural crest cells (arrowhead). (B) View of injected left side. Notice that the cells still express Xtwist but they do not migrate normally, most of them remaining in the dorsal side of the embryo (arrow; 58%, n = 17). (C,D) Grafts of ectodermal tissue, taken from stage 10 embryos previously injected with 0.3 ng of lacZ mRNA without (C) or with (D) 0.25 ng of Xbf2 mRNA, were placed in the prospective neural crest region of a stage 11 embryo. The embryos were cultured until stage 28 and b-galactosidase was analyzed. (C) Control embryo that received a graft containing lacZ mRNA. Note in C the normal migration of the grafted cells (arrowhead; 87%, n = 15) and in D the permanence of the labeled cells in the original site of the graft (arrow; 87%, n = 15). Inset: a similar, but more lateral (to better distinguish b-galactosidase staining from Xsox2 expression) graft, does not show Xsox2 expression (100%, n = 12).
