Ku M and Melton DA (1993), Xwnt-11: a maternally expressed Xenopus wnt gene.

XB-ART-21903

Development 1993 Dec 01;1194:1161-73.

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Xwnt-11: a maternally expressed Xenopus wnt gene.

Ku M , Melton DA .

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We have isolated and characterized a novel Xenopus wnt gene, Xwnt-11, whose expression pattern and overexpression phenotype suggest that it may be important for dorsal-ventral axis formation. Xwnt-11 mRNA is present during oogenesis and embryonic development through swimming tadpole stages. Xwnt-11 mRNA is ubiquitous in early oocytes and is localized during mid-oogenesis. By late oocyte stages, Xwnt-11 mRNA is localized to the vegetal cortex, with some mRNA in the vegetal cytoplasm. After egg maturation, Xwnt-11 mRNA is released from the vegetal cortex and is found in the vegetal cytoplasm. This early pattern of Xwnt-11 mRNA localization is similar to another vegetally localized maternal mRNA, Vg1 (D. A. Melton (1987) Nature 328, 80-82). In the late blastula, Xwnt-11 mRNA is found at high levels in the dorsal marginal zone. As gastrulation proceeds, Xwnt-11 mRNA appears in the lateral and ventral marginal zone and, during tadpole stages, it is found in the somites and first branchial arch. Injection of Xwnt-11 mRNA into UV-ventralized embryos can substantially rescue the UV defect by inducing the formation of dorsal tissues. The rescued embryos develop somitic muscle and neural tube; however, they lack notochord and anterior head structures.

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Species referenced: Xenopus
Genes referenced: fn1 gdf1 mtor wnt1 wnt11b wnt4 wnt8a
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	Fig. 1. Sequence of Xwnt-11 and comparison with other Xwnt genes. (A) The nucleotide sequence of Xwnt-11 is shown, with the amino acid sequence underneath. Numbers in bold refer to the amino acid sequence. (B) Amino acid sequence comparisons are shown for Xwnt-1, Xwnt-4, Xwnt-8 and Xwnt-11, for which complete coding region sequence is known. Cysteines that are conserved between all other known mouse wnt genes are printed in bold. Potential N-linked glycosylation sites are underlined in the Xwnt-11 sequence. The regions to which degenerate PCR primers were made are indicated by double underlining. The asterisks mark the end of the coding region. (C) Amino acid sequence similarities are compared between Xwnt-1, Xwnt-4, Xwnt-8 and Xwnt-11. The numbers above the diagonal are absolute amino acid similiarities, and the numbers below the diagonal include conserved amino acid substitutions (serine-threonine, isoleucineleucine- methionine-valine, glutamic acidaspartic acid, glutamine-asparagine and lysine-arginine).
	Fig. 2. Maternal expression and vegetal localization of Xwnt-11 RNA. Northern blot probed with Xwnt-11 and with fibronectin as a control for RNA loading. The numbers refer to developmental stages (Nieuwkoop and Faber, 1967), V is the vegetal hemisphere, A is the animal hemisphere and T is total embryo. The vegetal and animal dissections were done at the 8-cell stage.
	Fig. 3. In situ hybridization to sectioned tissue from oocytes and embryos. Middle and late stage oocytes were oriented during embedding such that sections cut through the animal-vegetal axis. Early stage oocytes were randomly oriented during embedding. (A) Early stage I-II oocytes (small arrows) have Xwnt-11 mRNA distributed uniformly in the cytoplasm. Middle stage III-IV oocytes (large arrows) are beginning to localize Xwnt-11 mRNA at one pole of the oocyte. The germinal vesicle is visible as a dark circle in the middle of each oocyte. The sections shown in B, C and D are all oriented with the animal pole facing up and the vegetal pole facing down. (B) Late stage V-VI oocyte shows that Xwnt-11 mRNA is localized to the vegetal cortex and to patches in the vegetal cytoplasm. The germinal vesicle is in the animal hemisphere. (C) Unfertilized egg shows that Xwnt-11 mRNA is diffusely localized to the vegetal hemisphere. (D) 8-cellstage embryo shows that Xwnt-11 mRNA remains diffusely localized to the vegetal hemisphere. The horizontal cleavage plane of the third cell division (arrowheads) confines the mRNA to vegetal blastomeres.
	Fig. 4. Whole-mount in situ hybridization with a Xwnt-11 probe on embryos of different stages. (A) Late blastula (stage 9), vegetal view, dorsal is up. Staining is present in a broad arc in the marginal zone of the embryo on the dorsal side. This arc is much broader than the organizer. (B) Early gastrula (stage 10.5), vegetal view, dorsal is up. The marginal zone staining has extended laterally and ventrally. Highest levels of expression are on the dorsal side in an area roughly corresponding to the organizer (60°around the dorsal mid-line). The lateral and ventral marginal zone staining does not extend down to the blastopore lip. (C) Late gastrula (stage 12.5), vegetal view, dorsal is up. A ring of staining around the closing blastopore is present. The few dorsal-most staining cells extending out along the dorsal midline probably include presumptive posterior neural plate and archenteron roof cells. (D) Late neurula (neural folds have closed), view from posterior-ventral side, dorsal is up. The blastopore is almost completely closed and is visible only as a small slit. Xwnt-11 expressing cells are present both dorsal and ventral to the blastopore. (E) Early tailbud (stage 22-23), anterior to the right, dorsal is up. The predominant staining is around somite nuclei, which are aligned vertically in each somite, and in the first branchial arch. (F) Tadpole (stage 28), anterior to the right, dorsal is up. Xwnt-11 staining is present around the somite nuclei, which are aligned vertically in each somite, and in the first branchial arch. Scale bars all represent 500 μm.
	Fig. 5. Sections of whole-mount embryos hybridized with Xwnt-11 antisense probe. All sections were oriented during embedding such that the sections cut through the dorsal and ventral midline. (A). This section is late stage 9 or early stage 10, and Xwnt-11 mRNA (between arrowheads) is beginning to appear in a region that will later be above the dorsal blastopore lip. No involution has occurred yet at this stage, and the mRNA is present in surface and deep layer cells. The embryo is oriented with the animal pole up and the vegetal pole (vg) down. The blastocoel floor is marked (bf). Fissures are an artifact of sectioning. (B) Mid gastrula (stage 12) shows Xwnt-11 mRNA (between arrowheads) in the dorsal blastopore lip (area below dotted line). Cell boundaries are difficult to discern, but the positively staining cells probably include both surface epithelial cells and deeper cells but not cells that have already involuted. The archenteron is marked (a).
	Fig. 6. Histological sections of UVventralized eggs injected with Xwnt-11 mRNA. UV-ventralized embryos, UV embryos injected with Xwnt-11 mRNA and normal embryos were fixed at stage 26-28, sectioned and stained to determine what structures had formed. (A) UVventralized embryo, cross section. (B) UV embryos injected with Xwnt-11 mRNA. Mid-trunk cross section, dorsal is up. (C) Higher magnification of a UV embryo injected with Xwnt-11 mRNA. The neural tube (nt) consists of a dark- and a palestained region. There is no lumen in this section, but other sections sometimes do show a lumen. The pale-stained region is the ‘marginal zone (mz)’, where axon tracts are located. There is one large marginal zone in the ventral midline (compare with Fig. 6E). There is also one large mass of muscle (m) that extends across the midline. (D) Normal embryo. Mid-trunk cross-section, dorsal is up. (E) Higher magnification view of a normal embryo. Neural tube (nt) and notochord (n) are visible. One of the two files of somitic muscle on each side of the notochord is marked (m). Note the two lateral pale-stained ‘marginal zones’ (mz) separated by ventral floor plate cells. The scale bars in A, B and D) represent 100 μm and in D and E represent 50 μm.
	Fig. 7. Whole-mount immunohistochemistry of Xwnt-11-injected embryos. UV-ventralized embryos (A,D), UV embryos injected with Xwnt-11 mRNA (B,E) and normal embryos (C,F) were fixed at stage 26-28, and whole-mount immunohistochemistry was done with tissue-specific antibodies. UV-ventralized embryos have neither notochord nor muscle. UV embryos injected with Xwnt-11 mRNA have somitic muscle but no notochord. Normal control embryos have both muscle and notochord. (A-C) Incubated with the notochord-specific monoclonal antibody (mAb) Tor 70 (Kushner, 1984; Bolce et al., 1992). (D-F) Incubated with the muscle-specific monoclonal (mAb) antibody 12101 (Kintner and Brockes, 1984). (A) UV-ventralized embryo plus Tor 70 mAb. There are no Tor 70-positive cells. (B) UV embryo injected with Xwnt-11 mRNA plus Tor 70 mAb. There are a few scattered Tor 70-positive cells but no organized notochord is present. (C) Normal embryo plus Tor 70 mAb. An extensive notochord is present along the dorsal axis. (D) UV-ventralized embryo plus 12101 mAb. There are no 12101-positive cells. (E) UV embryo injected with Xwnt-11 mRNA plus 12101 mAb. Extensive somitic muscle is present along the dorsal axis. (F) Normal embryo plus 12101 mAb. Somitic muscle is present. Scale bars all represent 100 μm.