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Xwnt-3A is a member of the Xenopus-Wnt gene family, a class of secreted, cysteine-rich proteins implicated in intercellular signaling during early development. Here we describe the full-length coding sequence of Xwnt-3A, as well as the spatial expression pattern of this Xwnt gene as determined by whole-mount in situ hybridization analysis. While Xwnt-3A shares considerable amino acid identity with both Wnt-3 (87%) and Wnt-3A (85%), its spatial expression pattern is most like that of Wnt-3A. Xwnt-3A, which is first detected at the neurula stage of development, is expressed exclusively along the dorsal midline of the developing brain and neural tube and along the dorsal surface of the otic vesicle. While the expression of Xwnt-1 extensively overlaps that of Xwnt-3A, Xwnt-1 is uniquely expressed along the midbrain/hindbrain boundary and is absent from the otic vesicle. The expression of Xwnt-3A in neural ectoderm is dependent upon neural induction as determined by experiments with recombined ectoderm and mesodermtissue. These results suggest that Xwnt-3A may participate in patterning the central nervous system during early Xenopus development. Last, the ectopic expression of Xwnt-3A induces the formation of a secondary axis at the anterior end of the embryo.
FIG. 1. Comparison of the predicted amino acid sequences of Xumt-
3A, Wnt-3, and Wnt-3A. Dashes represent identical amino acids compared
to the Xumt-3A sequence. The periods within the contiguous
sequence indicate gaps introduced to align the sequences. Conserved
cysteine residues are marked with an asterisk and two putative glycosylation
sites are marked with solid bars. The cysteine at amino acid
position 3 of Xwnt-3A is not conserved.
FIG. 2. Spatial expression pattern of Xwnt-3A during early development using whole-mount in situ hybridization. Embryos in A through E
were hybridized with an Xwnt-3A antisense probe while the embryo in F was hybridized with an Xwnt-SA sense probe. (A) Neurula stage
embryo (stage 16). Arrow indicates patch of signal at the anterior end of the embryo. (B) In a stage 22 embryo, the hybridization signal extends
dorsally along the anterior/posterior axis and appears most abundant in the head (arrow 1) and tail (arrow 2) regions. (C) In a stage 24 embryo,
the hybridization signal continues to extend along the anterior/posterior axis (arrow). (D) By stage 27 (hatching), the hybridization signal is
seen along the dorsal midline of the mesencephalon (arrow 1), the anterior portion of the neural tube (arrow 2), the dorsal surface of the otic
vesicle (arrow 3), and the tip of the tail (arrow 4). (E) At stage 31, theXwnt-3A hybidization signal extends continuously along the dorsal midline
from the forebrain/ midbrain junction (arrow 1) to the anterior portion of the neural tube (arrow 2) and is still detected in the otic vesicle (arrow
3) and at the tip of the tail (arrow 4). (F) A stage 27 embryo has no hybridization signal when probed with an Xwnt-3A sense probe. Stage 27 or 31
embryos shown in G and H, respectively, were hybridized with either an Xwnt-3A antisense probe (top embryo) or an Xwnt-1 antisense (bottom
embryo). Xwnt-1 is uniquely expressed at the midbrain/ hindbrain boundary (arrow 1) and is completely absent from the otic vesicle (arrow 2).
FIG. 3. Comparison of Xwnt-3A and Xwnt-1 expression in the developing brain and neural tube of tailbud stage ~mbryos. Tailbud stage
embryos (stage 31) hybridized with either an antisense Xwnt-3A or an Xwnt-1 probe were embedded in paraplast, sectioned transversely (10
.Jtm), and stained with Eosin as described under the Materials and Methods. (A) Transverse sections through the prosencephalon/mesencephalon
boundary. (B) Transverse sections through the eye vesicle at the mesencephalon/rhombencephalon boundary. (C) Transverse sections
through the otic vesicle. (D) Transverse sections through the anterior neural tube. Arrows indicate specific expressi~n of either Xwnt-3A or
Xwnt-1 as indicated. Abbreviations: E, eye; N, notochord; NT, neural tube; OV, otic vesicle; P, prosencephalon; M, mesencephalon; R, rhombencephalon.
Bar = 50 J-Lm.
FIG. 4. Analysis of Xwnt-SA expression in the developing auditory system. Embryos at several stages of development were hybridized with an
Xwnt-3A antisense probe and processed as described in Fig. 3. (A) Transverse section through a stage 19 embryo. Hybridization signal is
localized to the sensorial neural ectoderm (arrows; SN). (B) Transverse section through the otic placode of a stage 22 embryo. Xwnt-3A
expression is localized to the dorsal most portion of the placode in close proximity to the developing rhombencephalon (arrow; R). (C and D)
Transverse sections through stage 31 and 46 embryos, respectively. Xwnt-3A expression is localized to the dorsal most portion of the otic vesicle
(arrows). Abbreviations: N, notochord; NT, neural tube; OP, otic placode; OV, otic vesicle; R, rhombencephalon; S, somite; SM, somitic mesoderm;
SN, sensorial neurectoderm. Bar = 50 ~m.
FIG. 5. Neural induction is necessary for the expression of Xumt-3A. Ectoderm was isolated from stage 10 embryos and cultured alone or in the
presence of mesoderm . The recombinants were grown until control embryos reached stage 27 of development. The recombinants were then
proces!ged for whole-mount in situ hybridization with an antisense Xwnt-3A probe and sectioned. (A) Mesoderm/ectoderm recombinant; open
arrow, dorsal midline; closed arrow, otic vesicle. {B) Ectoderm grown in the absence of mesoderm. (C-E) Transverse sections through mesoderm/
ectoderm recombinants. (C) Neural tube signal, open arrow; bar= 50 #J.-m. (D) Apparent otic vesicle signal, closed arrow; bar = 25~tm. (E)
Transverse section through an ectoderm tissue mass; bar = 100 IJ.ffi.
FIG. 6. Ectopic expression of Xwnt-SA leads to formation of a secondary
body axis. Embryos were microinjected at the 4-ceH stage with
synthetic Xwnt-SA RNA generated from an Xwnt-3AISP64T construct.
Injections were made into either each of the two presumptive
dorsal cells (A) or the two presumptive ventral cells (B) and allowed to
develop to the late neurula stage of development.
