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Fig. 4. Developmental expression of Wnt8 and Fgf8. (A) Comparison of Wnt8 and Fgf8 expression at the gastrula stage. At the mid-gastrula stage (11.5), Wnt8 and Fgf8 have a complementary expression pattern in the ventrolateral and dorsolateral mesoderm, respectively. The embryos are oriented dorsal (D) to the top. Hemi-sections (red lines in the left panels) of these embryos along the animal-vegetal axis reveal that both genes are co-expressed in the lateral mesoderm. The arrowheads indicate the position of the lateral lip of the blastopore (bl, blastocoel). At stage 12, the Wnt8 expression domain expands anteriorly into the involuting mesoderm, the future paraxial mesoderm, while Fgf8 remains confined to the posterior mesoderm. (B) Comparative expression of Wnt8 and Sox8 at stage 12. Sox8 expression in the ectoderm (arrow) is adjacent to Wnt8 expression in the mesoderm. The red lines indicate the level of the serial sections shown in C,D. (C) Expression of Sox8 and Wnt8 on adjacent sections of a stage 12 embryo highlights the mesoderm expression of Wnt8 underlying the first Sox8-positive cells in the NC-forming region (bracket). At stage 12.5, Sox8 expression is stronger in the NC domain, and Wnt8 becomes more broadly expressed in both the ectoderm and the mesoderm layers. The red dotted lines in the lower panels demarcate the separation between the ectoderm and the mesoderm layers. Lower panels are higher magnifications of the upper panels. (D) Comparative expression of Sox8, Wnt8 and Fgf8 on adjacent sections of a stage 12/12.5 embryo confirms that Fgf8 is not co-expressed with Wnt8 in the mesoderm underlying the NC-forming region. The red dotted lines indicate the separation between the ectoderm and the mesoderm layers. (E) In the posterior region of the same embryo, Fgf8 is detected in the dorsolateral mesoderm, around the yolk plug (yp), while Wnt8 is confined to the ventrolateral region. Dorsal to the top. |
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Fig. 5. Fgf8a is a strong inducer of Wnt8 in animal explants and in whole embryos. (A) Animal explants derived from embryos injected with Fgf8a (F) or with a combination of Fgf8a and Chordin (C+F) show a strong upregulation of Wnt8 after 4 hours in culture. For comparison, Wnt8 (W) or Wnt8 and Chordin (C+W) co-injection had little effect on the expression levels of Fgf8. U, uninjected animal explant. Values (n=3) are presented as mean±s.e.m.; *P<0.05, versus uninjected animal explant (U). (B) In whole embryos, loss of Fgf function by injection (arrow) of Fgf8aMO (50 ng) or a dominant-negative Fgf receptor (XFD; 2 ng) results in a reduction of Wnt8 expression in the involuting mesoderm at stage 12. Conversely, Fgf8a (5 pg) mis-expression strongly upregulates Wnt8. For all injections, dorsal and lateral views (control and injected sides) of the same embryo are shown. Dorsal views, anterior to the top, injected side to the right (arrows). Lateral views, dorsal to the top; for the control side anterior is to the left, for the injected side anterior is to the right. |
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Fig. 6. Fgf8a induces NC at the anterior neural fold indirectly. (A) The anterior neural plate (blue) is devoid of NC tissue (orange) as a result of the activity of a Wnt inhibitor, Dkk1 (Carmona-Fontaine et al., 2007). Snail2 expression is shown in a control embryo at stage 15. (B) β-catenin misexpression (200 pg) can overcome this inhibition to induce NC markers (Snail2) at the anterior neural fold (arrows). (C) Fgf8a (5 pg) misexpression can also induce NC markers (Snail2 and Sox8) at the anterior neural fold (arrows), an activity that is mediated by upregulation of Wnt8 in this region of the embryo (arrows). (D) Normal pattern of expression of Wnt8 in a control embryo at the same stage. In all panels the embryos are viewed from the dorsal side, anterior to the top. |
