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Abstract Frodo is a novel conserved regulator of Wnt signaling that has been identified by its association with Dishevelled, an intracellular component of Wnt signal transduction. To understand further how Frodo functions, we have analyzed its role in neural development using specific morpholino antisense oligonucleotides. We show that Frodo and the closely related Dapper synergistically regulate head development and morphogenesis. Both genes were cell-autonomously required for neural tissue formation, as defined by the pan-neural markers sox2 and nrp1. By contrast, beta-catenin was not required for pan-neural marker expression, but was involved in the control of the anteroposterior patterning. In the mesoderm, Frodo and Dapper were essential for the expression of the organizer genes chordin, cerberus and Xnr3, but they were not necessary for the expression of siamois and goosecoid, established targets of beta-catenin signaling. Embryos depleted of either gene showed a decreased transcriptional response to TCF3-VP16, a beta-catenin-independent transcriptional activator. Whereas the C terminus of Frodo binds Dishevelled, we demonstrate that the conserved N-terminal domain associates with TCF3. Based on these observations, we propose that Frodo and Dapper link Dsh and TCF to regulate Wnt target genes in a pathway parallel to that of beta-catenin.
Fig. 3. Frodo and Dapper are required for neural development. (A) Localization of Frodo RNA visualized by whole-mount in situ hybridization on a half-embryo at stage 10, sagittal view. (B-M) Morpholinos and RNAs were injected as indicated in Table 3 into a single right animal-dorsal blastomere of 8- to 16-cell stage embryos with (B-H) or without (I-M) nβgal mRNA. Whole-mount in situ hybridization has been carried out with antisense probes for sox2 (B-F), myoD (G,H) and nrp1 (I-M). Suppression of sox2 was observed in cells injected with FrdMO (or DprMO) and nβgal RNA at stage 10.5 (C) or 13 (E,F). (C) The inset is shown on the right at higher magnification. CoMO-injected embryos at stage 10.5 (B), 13 (D) and 20 (I). (G,H) Lack of effect of FrdMO on myoD expression at stage 14. (I-K) Nrp1 expression on the injected side is severely reduced in both anterior and posterior neural tube in FrdMO- and DprMO-injected embryos at stage 20. (L) The nrp1 expression domain becomes narrow posteriorly, but expands anteriorly in the embryos injected with β-catenin morpholino (βcatMO). Morphology of an embryo injected with βcatMO is shown on the right. (M) The effect of FrdMO on nrp1 is restored by Frodo RNA (see also Table 3). (B-H,M) Dorsal view. (I-L) Dorsal view (left), anterior view (right).
Fig. 4. The effect of FrdMO and DprMO on organizer markers. (A) Four-cell stage embryos were injected in the dorsal equatorial region of two blastomeres with morpholinos and mRNAs as indicated in Table 4, were cultured until stage 10.5 and were subjected to whole-mount in situ hybridization with organizer markers, chordin (left panels), Xnr3 (middle panels) and gsc (right panels) as probes. Co-injection of FrdMO and DprMO can reduce chordin and Xnr3 but not gsc, whereas βcatMO strongly inhibited expression of all three genes, implying that Frodo and Dapper function in a gene-specific manner. The reduction of chordin and Xnr3 by co-injection of FrdMO and DprMO was reversed not only by Frodo RNA, but alsoβ -catenin RNA. (B) Four-cell stage embryos were injected in the equatorial region of each blastomere with morpholinos and mRNAs as indicated and were analyzed at stage 10 and 11 by RT-PCR, using primers specific for chordin, cerberus, Xnr3, siamois, gsc and vent1. EF1α primers were used to control loading.
nodal3.1 (nodal homolog 3, gene 1) gene expression in Xenopus laevis embryos, NF stage 10.5, as assayed by in situ hybridization, vegetal view, dorsal up.