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First published online 25 August 2004
doi: 10.1242/dev.01369

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The involvement of Frodo in TCF-dependent signaling and neural tissue development

Hiroki Hikasa and Sergei Y. Sokol^*,

Department of Microbiology and Molecular Genetics, Harvard Medical School, and Department of Medicine, Beth Israel Deaconess Medical Center, 330 Brookline Avenue, Boston, MA 02215, USA

View larger version (99K): [in a new window]   Fig. 1. Frodo and Dapper are required for head development. Two blastomeres of four-cell stage embryos were injected in the dorsal equatorial region with morpholinos and mRNAs as indicated in Table 1. (A) Control morpholino (CoMO)-injected embryos. (B,C) Embryos injected with FrdMO or DprMO show shortened axes with head defects. (D) Co-injection of FrdMO and DprMO leads to severe head defects. (E) The effects of FrdMO and DprMO are reversed by Frodo RNA lacking the target sequences. (F) Head development is rescued in FrdMO and DprMO-injected embryos by stabilized ß-catenin (ßcat) RNA. Shortened axes are rescued by Frodo RNA, but not by ßcat RNA.

View larger version (81K): [in a new window]   Fig. 2. The effect of FrdMO and DprMO on neural plate closure. (A-C) CoMO (A), FrdMO (B) or DprMO (C) were injected into the right animal dorsal blastomere of 8- to 16-cell stage embryos without (left panels) or with (right panels) nuclear ß-galactosidase (nßgal) RNA, a lineage tracer. Doses of MOs and mRNAs are as indicated in Table 2. (B,C) Neural plate closure and neural fold formation is severely disturbed on the injected side as evidenced by RedGal staining. Arrowheads indicate the neural plate border. Scale bars: 150 µm.

View larger version (75K): [in a new window]   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) nßgal 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 nßgal 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 ß-catenin morpholino (ßcatMO). Morphology of an embryo injected with ßcatMO 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).

View larger version (45K): [in a new window]   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 ßcatMO 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 also ß-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. EF1 primers were used to control loading.

View larger version (39K): [in a new window]   Fig. 5. Physical and functional interactions of Frodo and TCF3. (A) Dose-dependent effects of Frodo and Dapper RNA on the activation of pSiaLuc reporter by Dsh. (B) Levels of non-phosphorylated ß-catenin are not changed in stage 10 embryos injected with FrdMO and DprMO in all blastomeres at the eight-cell stage. (C) Frodo constructs used for transfections and microinjections. (D) The association of GST-TCF3 with HA-Frodo and HA-Frd337, but not HA-Frd186, in COS7 cells, revealed in a GST pull-down assay. (E) TCF3 associates with Frodo in Xenopus gastrulae. Four- to eight-cell stage embryos were injected with 200 pg of HA-TCF3 RNA and 1 ng of Flag-Frd337 RNA. Protein complexes were immunoprecipitated with anti-Flag antibodies from gastrula stage lysates. (F) The activation of the pSiaLuc reporter by TCF3-VP16 is suppressed by HA-Frd337, but not by HA-Frd186.

View larger version (30K): [in a new window]   Fig. 6. The involvement of Frodo and Dapper in different steps of Wnt signal transduction. (A) Frodo and Dapper synergize with TCF3-VP16 to activate the pSiaLuc reporter in Xenopus embryos. (B) The amount of ß-catenin associated with GST-TCF3 is not altered by Frodo. (C) Frodo/Dapper are not required for ß-catenin-dependent reporter activation. (D) An inhibitory activity of Frodo and Dapper revealed in Wnt8-dependent stimulation of pSiaLuc.

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