Wolda SL et al. (1993), Overlapping expression of Xwnt-3A and Xwnt-1 in...

XB-ART-22970

Dev Biol 1993 Jan 01;1551:46-57. doi: 10.1006/dbio.1993.1005.

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Overlapping expression of Xwnt-3A and Xwnt-1 in neural tissue of Xenopus laevis embryos.

Wolda SL , Moody CJ , Moon RT .

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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 mesoderm tissue. 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.

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Species referenced: Xenopus laevis
Genes referenced: ces2.8 wnt1 wnt3 wnt3a

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	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.