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Frizzled receptors transduce signals from the extracellular Wnt ligands through multiple signaling pathways that affect cytoskeletal organization and regulate gene expression. Direct intracellular mediators of Frizzled signaling are largely unknown. We identified FRIED (Frizzled interaction and ectoderm defects) by its association with the C-terminal PDZ-binding motif of Xenopus Frizzled 8. FRIED contains an N-terminal KIND domain, a FERM domain, six PDZ domains, and a tyrosine phosphatase domain, being similar in structure to the protein tyrosine phosphatase PTP-BAS/PTP-BL. We report that FRIED proteins with the FERM domain localize to the apical cortex and can inhibit Wnt8-mediated, but not beta-catenin-mediated, secondary axis induction in Xenopus embryos, suggesting a specific interaction with Wnt signaling. A FRIED construct containing the FERM domain induced reorganization of pigment granules and cortical actin in Xenopus ectoderm. Wnt5a suppressed the depigmentation of ectoderm triggered by FRIED, demonstrating that Wnt5a and FRIED functionally interact to regulate the cytoskeletal organization. Our data are consistent with the possibility that FRIED functions by modulating Rac1 activity. We propose that FRIED is an adaptor protein that serves as a molecular link between Wnt signaling and actin cytoskeleton.
ptpn13 ( protein tyrosine phosphatase non-receptor type 13) gene expression in Xenopus laevis embryo,assayed via in situ hybridization, NF stage 26, lateral view, anteriorleft, dorsal up.
Figure 3. Spatial distribution of FRIED transcripts in Xenopus embryos. Wholemount in situ hybridization analysis has been carried out with digoxygenin-labeled FRIED anti-sense RNA probes with embryos at different developmental stages. A: Stage 10, animal pole is at the top, dorsal is to the right. FRIED RNA is detectable in animal pole ectoderm of a bisected embryo. B,C: At stage 10+, FRIED transcripts are visible in the dorsal blastopore lip. A,B: Saggital view. C: Vegetal pole view. D: Stage 14, dorsal view, anterior is at the top. FRIED is expressed in anteriorectoderm area and around closed blastopore at the beginning of neurulation. E: Tailbud stage, anterior is to the left. FRIED transcripts are clearly detectable in the brain and spinal cord, eyes and otic vesicles, and in lateral plate mesoderm. A control RNA probe did not produce similar staining patterns (data not shown).
Fig. 4. FRIED-2 inhibits secondary axis induc- tion by Xwnt8, but not -catenin. A–D: One ventral vegetal blastomere of 8-cell embryos was injected with Xwnt8 RNA (6 pg) or -catenin RNA (0.2 ng) alone or together with 1 ng of FRIED-2 or FRIED- C RNA as indicated. E,F: Combined data for three experiments are shown. Secondary axis induction was scored when sibling embryos reached stage 40. Complete secondary axes include head structures with eyes and cement glands, while partial secondary axes are induced up to the hindbrain level and lack eyes. FRIED-2, but not FRIED C, RNA inhibits secondary axis induction by Xwnt8, but neither RNA affects -catenin-in- duced secondary axes.
Fig. 5. The effect of FRIED constructs on embryonic ectoderm. Depigmentation of blastula animal pole cells induced by FRIED- C (A), FRIED-FERM (B), but not FRIED-6PDZ (C) RNAs. D: Uninjected embryo. E: Stage 8.5, ectodermal lesion in an embryo injected with FRIED-FERM RNA. F: Stage 10, healing of the lesion shown in E. Bar in A (applies to B–D) 80 m. G: In vivo expression levels of FRIED mutant constructs in embryos shown in A–C. Loading is shown by a major yolk protein stained with Coomassie blue. RNAs were injected into each blastomere of four-cell embryos as indicated. H: The FERM domain of FRIED defines its cortical localization. Two animal blastomeres of 8-cell embryos were injected with the following RNAs: Myc-FRIED C (1 ng), FRIED-6PDZ (1 ng), or FRIED-FERM (0.3 ng). When sibling embryos reached stage 10, ectoderm cells of the injected embryos were stained with anti-Myc antibodies. FRIED C and FRIED-FERM are localized preferentially in the apical cortex (*), whereas FRIED-6PDZ is found mainly in the cytoplasm.
Fig. 6. Xwnt5a suppresses the effect of FRIED-FERM on ectoderm. Four animal blastomeres of 8-cell embryos were injected with 0.3 ng of FRIED-FERM, 0.8 ng of Xwnt5 RNA, separately (A,C) or in combination (B). Animal view of midblastula stage embryos is shown. The depigmentation of ectoderm cells overexpressing FRIED-FERM (A) is rescued by Xwnt5a RNA (B), restoring the normal pigmentation pattern.
Fig. 8. Rac1L61 rescues the effects of FRIED on embryonic ectoderm. A–D: Different degrees of depigmentation induced by FRIED RNA were scored as (A), (C), and – (D, uninjected embryo). A,B: Cell autonomy of the FRIED effect. Embryo coinjected with 1 ng of FRIED-2 RNA and 0.1 ng of -gal RNA reveals -galactosidase activity (identified by RedGal staining) in the cells that undergo depigmentation (compare A and B). The same embryo before and after Red-Gal staining is shown (A, B). E,F: Rac1L61 RNA suppresses ectoderm lesions induced by higher doses of FRIED-FERM RNA.
ptpn13 ( protein tyrosine phosphatase non-receptor type 13) gene expression in a Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10, blastoporal view, dorsal up.
