Landesman Y and Sokol SY (1997), Xwnt-2b is a novel axis-inducing Xenopus Wnt, w...

XB-ART-16529

Mech Dev 1997 May 01;632:199-209.

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Xwnt-2b is a novel axis-inducing Xenopus Wnt, which is expressed in embryonic brain.

Landesman Y , Sokol SY .

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Xwnt-2b is a novel member of the Wnt gene family and is 73-74% similar to human and mouse Wnt-2 proteins. Starting from stage 15, Xwnt-2b transcripts are localized to a non-contiguous stripe in the anterior neural plate of the Xenopus embryo. In the tailbud, Xwnt-2b is expressed along the dorsoanterior side of the prosencephalon-mesencephalon boundary. At the tadpole stages, the brain-specific expression fades, but the total amount of Xwnt-2b mRNA does not decline due to activation of its expression in non-brain areas. Microinjection of Xwnt-2b mRNA into a ventral blastomere of 4-8-cell embryos results in the formation of complete secondary body axes. These results suggest that Xwnt-2b is a member of the axis-inducing Wnts and that it is involved in brain development and in later organogenesis.

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Species referenced: Xenopus
Genes referenced: en2 fn1 gdf1 tbx2 wnt1 wnt2 wnt2b wnt3a wnt4 wnt8b

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	Fig. 1. Sequence comparison between Xwnt-2b and other Wnt-2 sub-family members including human Wnt-13. (A) Amino-acid sequence alignment of Xwnt-2b, human Wnt-13 (Katoh et al., 1996), mouse Wnt-2 (McMahon and McMahon, 1989), human Wnt-2 (Wainwright et al., 1988), rat Wnt-2 (Levay- Young and Navre, 1992) and zebrafish Wnt-2 (Blader et al., 1997). Conserved amino acid residues are shown on dark background. (B) Partial nucleotide sequence alignment of Xwnt-2b, human Wnt-13 (Katoh et al., 1996), Xenopus Wnt-2 (Wolda and Moon, 1992) and human Wnt-2 (Wainwright et al., 1988). The partial Xenopus Wnt-2 cDNA is aligned with the numbered (in parentheses) sequences of the other Wnts. Conserved bases are shown on dark background.
	Fig. 2. Developmental expression of Xwnt-2b transcripts. Total RNA was analyzed by Northern hybridization using Xwnt-2b antisense probe. Fibronectin antisense probes were used as loading control. A major transcript of 3.5 kb and a minor band of 7 kb (*) are visible. Lanes 1–10 contain total RNA extracted from embryos at the following stages: 8.5, 10, 10.5, 12.5, 17, 19, 23, 30, 33 and 35, respectively.
	Fig. 3. Spatial localization of Xwnt-2b transcripts in neurula and in early tailbud embryos using whole-mount in situ hybridization with Xwnt-2b and En-2 antisense probes. (A,B) Stage 15 neurulae. Expression of both Xwnt-2b and En-2 is restricted to two stripes in the anterior neural plate. (C,D) Stage 22, frontal view. Expression of Xwnt-2b and En-2 is visible as a continuous band across the anterior part of the brain. (E,F) Stage 22, lateral view. Xwnt-2b staining is anterior to En-2 staining. (G,H) Stage 22, dorsal view. Xwnt-2b expression is restricted to the dorsal part of the brain, whereas the En-2 staining has a ringlike shape on both the dorsal and ventral sides. Embryos in (E,F,G,H) were cleared in benzyl benzoate/benzyl alcohol solution.
	Fig. 4. Spatial localization of Xwnt-2b transcripts in stage 30 and 35 embryos using whole-mount in situ hybridization with Xwnt-2b (A,C,E–G,I) and En-2 (B,D,H) antisense probes. (A,B) Stage 30, lateral view. (E) Stage 30, dorsal view. (F) Stage 35, dorsal view. (G,H) Stage 35, lateral view. Xwnt-2b is expressed in the prosencephalon-mesencephalon boundary. Additional staining is seen above the heart primordium (arrowheads in A,G). The cephalic expression of En-2 is seen at the mesencephalon-rhombencephalon boundary. Embryos in (G,H) were cleared in benzyl benzoate/benzyl alcohol. Wholemount samples from stage 30 (C,D) or stage 35 (I) embryos were sectioned either transversally (C,D) or sagittally (I) and revealed Xwnt-2b expression in the dorsal mesencephalon (C). En-2 expression is detected posterior to the Xwnt-2 expression where it expands across the mesencephalon (D). Arrowheads in (I) point to non-brain staining of Xwnt-2b in stage 35 embryo between the heart and the pronephros. Abbreviations: c, cement gland; e, eye; h, heart; m, mesencephalon; o, otic vesicle; p, prosencephalon; ph, pharynx; pr, pronephros; r, rhombencephalon. Bar, 50 mm.
	Fig. 5. Ectopic expression of Xwnt-2b mRNA leads to induction of a secondary body axis. Four to eight-cell embryos were injected into one ventrovegetal blastomere with either 60 pg of Vg-1 mRNA (A), or 12 pg of Xwnt-2b mRNA (B).
	Fig. 6. A schematic localization of different Xwnts in the brain of late tailbud embryos. The expression patterns of specific Wnt transcripts are indicated by dark regions. Distribution of Xwnt-2b transcripts is based on our observations. Data on Xwnt-1, Xwnt-3A, Xwnt-4 and Xwnt-8b are based on whole-mount in situ analysis and sectioning reported by others. The cephalic expression of En-2 (Hemmati-Brivanlou et al., 1991) is shown at the bottom. Domains of expression: Xwnt-1 (stage 31 embryos; Wolda et al., 1993), mesencephalon-rhombencephalon boundary and the dorsal midline of the brain and spinal cord with a gap in a part of rhombencephalon; Xwnt-3A (stage 31; Wolda et al., 1993), the brain around the prosencephalon-mesencephalon boundary and the dorsal midline posterior to the prosencephalon-mesencephalon boundary, descending ventrally along the inner surface of the mesencephalon; Xwnt-4 (stages 27–38; McGrew et al., 1992), the prosencephalon-mesencephalon boundary and two areas in the rhombencephalon. The floor plate of the rhombencephalon and spinal cord; Xwnt-8b (stage 33; Cui et al., 1995), the dorsal diencephalon, prosencephalon-mesencephalon boundary and the anterior mesencephalon.