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Recent advances in analyzing wnt signaling have provided evidence that frizzled proteins can function as wnt receptors. We have identified Xfz3, a Xenopus frizzled family member. The amino acid sequence is 89% identical to the product of the murine gene Mfz3, and is predicted to be a serpentine receptor with seven transmembrane domains. Xfz3 is a maternal mRNA with low levels of expression until the end of gastrulation. The expression level increases significantly from neurulation onward. Whole-mount in situ hybridization analysis shows that expression of Xfz3 is highly restricted to the central nervous system. High levels of expression are detected in the anterior neural folds. Low levels of expression are also detected in the optic and otic vesicles, as well as in the pronephros anlage. In addition, Xfz3 mRNA is concentrated in a large band in the midbrain. Overexpression of Xfz3 blocks neural tube closure, resulting in embryos with either bent and strongly reduced anteroposterior axis in a dose-dependent manner. However, it does not affect gastrulation, the expression and localization of organizer-specific genes such as goosecoid, chordin and noggin. Therefore, Xfz3 is not involved in early mesodermal patterning. Injection of RNA encoding GFP-tagged Xfz3 shows that overexpressed proteins can be detected on the cell surface until at least late neurula stage, suggesting that they can exert an effect after gastrulation. Our expression data and functional analyses suggest that the Xfz3 gene product has an antagonizing activity in the morphogenesis during Xenopus development.
Fig. 2. Expression of Xfz3 during early development. RNAs were prepared from indicated stages and the temporal expression was analyzed by RNase
protection. The level of expression is low at cleavage and gastrula stages and increases significantly at neurulation. FGFR-1 probe was used as a loading
control.
Fig. 3. Whole-mount in situ hybridization analysis of Xfz3 mRNA expression. (A) Stage 14, dorsal view. Xfz3 mRNA is detected in the anterior neural fold.
(B) Stage 17, dorsal view. Expression of Xfz3 mRNA extends to the trunk neural fold. (C) Stage 20, dorsal view. Xfz3 mRNA is detected in the entire neural
fold along a rostral and caudal direction with high levels of expression in the anterior region. A strong expression site can be found as a large band in the
midbrain (arrowheads). (D) Stage 20. Lateral view shows Xfz3 mRNA expression in the neural fold but also in the region corresponding to the optic lobe. (E)
Stage 26, dorsal view. Expression of Xfz3 mRNA decorates the anterior central nervous system. In the trunk region, expression of Xfz3 mRNA is located in
the dorsal region of the neural tube. Arrowheads indicate the strong expression site of Xfz3 in the midbrain. (F) Lateral view of an embryo at stage 26,
showing strong expression of Xfz3 mRNA in the neural tube. Lower levels of expression can be observed in the optic lobes (op), in the otic vesicles (ov) and
in the pronephros anlage (arrowhead). (G) Stage 35, lateral view. Xfz3 mRNA is strongly expressed in the anterior nervous system. Lower levels of
expression are found in the spinal cord. (H, I) Cross-sections corresponding to the embryo in (G) at the levels of midbrain (H) and rhombencephalon (I).
Xfz3 mRNA is localized to the ventricular zone of the midbrain (H) and in the dorso-lateral region in the rhombencephalon (I).
Fig. 4. Xfz3 overexpression phenotypes. (A) Normal embryos at stage 18.
(B) Embryos injected with 0.25 ng Xfz3 RNA show open and curved
neural plates. (C) Normal embryo at stage 45. (D) Embryos injected
with 0.1 ng Xfz3 RNA show either dorsally bent head or tail. (E) Embryos
that had been injected with 0.25 ng Xfz3 RNA. These embryos show
absence of the trunk region.
Fig. 5. Whole-mount immunocytochemistry for somites (A,B) and notochord (C,D). All embryos were fixed at stage 30. (A,C) Normal embryos. (B,D)
Embryos were injected with 0.25 ng Xfz3 at the two-cell stage near the animal pole region. Somites (B) and notochord (D) are well-differentiated in these
embryos although they were slightly reduced compared with normal embryos.
Fig. 6. Whole-mount in situ hybridization of the expression of anteroposterior
neural markers in affected embryos. Embryos were injected with
0.25 ng Xfz3 RNA near the animal pole and allowed to develop until
stage 278. (A) Hybridization with Otx-2. (B) Hybridization with En-2.
(C) Hybridization with XlHbox-6. Normal embryos are shown on the top.
Fig. 7. Lineage of injected cells and localization of overexpressed proteins on the cell surface. (A) Transverse section of an embryo injected with 1 ng GFP
RNA plus 0.25 ng Xfz3 RNA near the animal pole region. Notice the contribution of injected cells to presumptive neuroectoderm (N) and epidermis. The
neural tube did not form due to expression of Xfz3. (B) Section made from (A) showing the nuclear and cytoplasmic localization of GFP. (C) Expression of
GFP-tagged Xfz3 (with six transmembrane segments) on the cell surface. Two-cell stage embryos were injected with 1 ng RNA encoding GFP-tagged Xfz3
and sections were made at neurula stages. Notice the presence of GFP-tagged Xfz3 on the cell surface (arrowheads). Scale bars: (A) 350 mm, (B) 60 mm, (C)
35 mm.
Fig. 8. Overexpression of Xfz3 had no effect on the expression and localization of organizer-specific genes. Whole-mount in situ hybridization. (A,C,E)
Normal embryos. (B,D,F) Embryos injected with 0.25 ng Xfz3 RNA near the animal pole region. (A, B) Expression of goosecoid in normal (A) and injected
(B) embryos was examined at stage 111.5. Notice that their expression patterns are identical. (C,D) Stage 10.5, expression and localization of chordin
mRNA are indistinguishable between normal (C) and injected (D) embryos. (E,F) Stage 10.5, both normal (E) and injected (F) embryos show an identical
expression pattern of noggin.
Fig. 1. (A) Protein sequence alignment between Xfz3 and Mfz3. Identical amino acid residues are indicated by vertical lines. The ten invariant cysteines are
indicated by bold face. Dashes indicate the absence of amino acid residues at the corresponding position. (B) Hydropathicity profile of Xfz3 protein with the
signal peptide (sp) and the seven transmembrane segments (TM 1â7) indicated. The nucleotide sequence has been deposited to the EMBL database (access
number AJ001754).
fzd3 (frizzled class receptor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, lateral view, anteriorleft, dorsal up.
fzd3 (frizzled class receptor 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 35, lateral view, anteriorleft, dorsal up.
