Moon RT et al. (1993), Xwnt-5A: a maternal Wnt that affects morphogene...

XB-ART-22248

Development 1993 Sep 01;1191:97-111.

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Xwnt-5A: a maternal Wnt that affects morphogenetic movements after overexpression in embryos of Xenopus laevis.

Moon RT , Campbell RM , Christian JL , McGrew LL , Shih J , Fraser S .

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To contribute to an understanding of the roles and mechanisms of action of Wnts in early vertebrate development, we have characterized the normal expression of Xenopus laevis Wnt-5A, and investigated the consequences of misexpression of this putative signalling factor. Xwnt-5A transcripts are expressed throughout development, and are enriched in both the anterior and posterior regions of embryos at late stages of development, where they are found primarily in ectoderm, with lower levels of expression in mesoderm. Overexpression of Xwnt-5A in Xenopus embryos leads to complex malformations distinct from those achieved by ectopic expression of Xwnts -1, -3A, or -8. This phenotype is unlikely to result from Xwnt-5A acting as an inducing agent, as overexpression of Xwnt-5A does not rescue dorsal structures in UV-irradiated embryos, does not induce mesoderm in blastula caps, and Xwnt-5A does not alter the endogenous patterns of expression of goosecoid, Xbra, or Xwnt-8. To pursue whether Xwnt-5A has the capacity to affect morphogenetic movements, we investigated whether overexpression of Xwnt-5A alters the normal elongation of blastula cap explants induced by activin. Intriguingly, Xwnt-5A blocks the elongation of blastula caps in response to activin, without blocking the differentiation of either dorsal or ventral mesoderm within these explants. The data are consistent with Xwnt-5A having the potential activity of modifying the morphogenetic movements of tissues.

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Species referenced: Xenopus laevis
Genes referenced: eef1a2 gsc inhba lsamp tbx2 tbxt wnt1 wnt5a wnt8a
???displayArticle.antibodies??? Lsamp Ab1 Somite Ab1

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	Fig. 1. Comparison of the predicted amino acid sequences of Xwnt-5A (Genbank accession number L19716), murine Wnt-5A (Gavin et al., 1990), and Xwnt-1 (Noordermeer et al., 1989). Identical residues are marked by asterisks, and gaps indicated by dashes were introduced to yield the highest degree of identity.
	Fig. 2. Localization of Xwnt-5A transcripts in tailbud embryos by RNase protection. (A) Tailbud stage embryos were dissected into head (H), mid-dorsal (MD), mid-ventral (MV) and tail (T) regions as indicated by the solid lines. (B) RNA was isolated from the dissected regions, and subjected to an Xwnt-5A-specific RNase protection assay. The P lane contains undigested probe, and the remaining lanes, labeled as in A display protected fragments of the predicted size. (C) RNA employed in the RNase protection assay in B was employed in an RNA blot analysis with an EF1a probe to provide a reference for the relative levels of expression of Xwnt-5A.
	Fig. 3. Localization of Xwnt-5A transcripts by whole-mount in situ hybridization. Developing embryos at (A) the late gastrula (approximately stage 11 1/2); (B, lateral view, C, dorsal view) late neurula (stage 20); and (E) tadpole (stage 34) were hybridized with an antisense Xwnt-5A probe. A dorsal view of control neurula stage embryo hybridized with a sense Xwnt-5A probe is shown in D. Random tadpole embryos were then embedded and sectioned to analyze further the expression of Xwnt-5A in the head (F) and tail (G). White arrows in B and C denote anterior Xwnt-5A hybridization signals. Black arrows (E,F,G) denote specific Xwnt-5A hybridization signals. For reference, the eye is denoted by a white arrowhead in E and F, the cement gland is indicated by a black arrowhead in F, and the blastopore (bp), notochord (nc), tail ectoderm (e), and skeletal muscle (m) are noted.
	Fig. 4. Phenotypes of embryos injected with Xwnt-5A RNA. Control embryos (A,C,E), and embryos injected with Xwnt-5A RNA into the upper marginal zone of dorsal blastomeres (B,D,F) or ventral blastomeres (G) at the four cell stage were cultured to neurula stage (A,B), tailbud stage (C,D), or tadpole stages (E,F,G). Black arrowheads denote the location of the cement gland, and white arrowheads the position of the eye. The black arrows in G indicate supernumerary tails.
	Fig. 5. Whole-mount immunocytochemistry of tailbud and tadpole embryos injected with Xwnt-5A RNA. Control embryos (A,C) and embryos injected with Xwnt-5A RNA in the upper marginal zone of both dorsal blastomeres at the 4-cell stage (B,D) were stained with the pan-neural mAb 2G9 (A,B), or the skeletal muscle-specific mAb 12/101 (C,D). Arrows in A and B denote the anterior limit of neural staining in dorsally-injected embryos. Arrows in D denote the anterior widening between somitic mesoderm in embryos dorsally injected with Xwnt-5A relative to controls (C).
	Fig. 6. Histological analysis of embryos injected with Xwnt-5A RNA in the upper marginal zone of both dorsal blastomeres at the 4-cell stage. (A) Control embryo (stage 18-19) sectioned transversely through the trunk region. (B) Xwnt-5A-injected (high dose) embryo (stage 18- 19) sectioned transversely through the trunk region showing the morphology and positional relationship between the notochord, somites, and neural plate. A pointer marks the position of the dorsal midline with respect to the neural axis. (C) Horizontal section of a control embryo (stage 34-36). (D) Horizontal section through a Xwnt-5Ainjected (high dose) embryo (stage 34-36). This embryo shows a mild phenotype of fused nasal placode, malformed forebrain, and malformed anterior notochord. A subtle bifurcation can also be seen in the notochord, indicated by the dark septum down the middle of the notochord. (E) In some Xwnt-5A-injected embryos the notochord clearly bifurcates, as evident in this cross section through the tail of an embryo at stage 34-36. (F) In most Xwnt- 5A-injected embryos the notochord does not duplicate as in E, rather, it appears as an enlarged bulb anteriorly. (G) This embryo demonstrates that the phenotype observed in B persists at stage 34-36, in horizontal section. (H) This Xwnt-5A-injected embryo demonstrates that occasionally there is a position specific disruption of axial development in the tail. In this stage 34-36 embryo, sectioned horizontally, three somite segments along with the notochord and neural tube in these segments are seriously disrupted (downward arrow). Axial tissue rostral (upward arrow) and caudal (downward arrowhead) to this disrupted area appear normal. (I) Horizontal section through control embryo at stage 34-36 to denote interocular distance. (J) Horizontal section through Xwnt-5A-injected embryo at stage 34-36 to denote the reduction in interocular distance relative to the sibling control in I. Abbreviations: e, eye; en, endodermal cavity; cg, cement gland; di, diencephalon; np, neural plate; nc, notochord; na, nasal placode; nt, neural tube; ot, otic vesicle; s, somite; tel, telencephalon. The bar in A serves as a reference to the following scales: A and B, 28 mm; C-H, 88 mm; G, 105 mm; and I and J, 65 mm.
	Fig. 7. Determination of blastomere sensitivity to overexpression of Xwnt-5A. Mixed Xwnt-5A and beta-galactosidase RNAs were injected into both dorsal blastomeres at the 4-cell stage (A, B), or into two dorsal midline tier one (C), tier two (D), or tier three (E) blastomeres at the 32-cell stage. Hatched embryos were processed for whole-mount immunocytochemical localization of betagalactosidase and photographed (C,D,E), or embedded and sectioned through anterior (A) or trunk (B) regions. Arrows denote examples of specific beta-galactosidase staining, and the black arrowheads (C,D,E) denote the position of the cement gland. Abbreviations: b, brain; e, eye; ec, ectoderm; fb, forebrain; nc, notochord; nt, neural tube; pe, pharyngeal endoderm.
	Fig. 8. Effects of overexpression of Xwnt-5A on the endogenous patterns of expression of goosecoid, Xbra, and Xwnt-8. Goosecoid is expressed exclusively in the gastrula organizer of control embryos (A), as well as in embryos injected with Xwnt-5A RNA in the marginal zone of both dorsal (B) or ventral (C) blastomeres at the 4-cell stage. Arrow in B denotes slight bulging of the dorsal side. The pattern of expression of endogenous Xbra (D) is similarly unaffected by dorsal (E) or ventral (F) injection of Xwnt-5A RNA. Xwnt-8 is expressed in future ventral and lateral mesoderm of control embryos, and is excluded (between arrows) from the gastrula organizer field (G). UV irradiation of eggs ventralizes the embryo, leading to expression of Xwnt-8 throughout the marginal zone (H), a pattern that is unaffected by injection of Xwnt-5A RNA into the marginal zone (I).
	Fig. 9. Effects of overexpression of Xwnt-5A on the differentiation of isolated blastula caps. Histological examination of blastula caps from control embryos (A) and embryos injected with Xwnt-5A RNA in the animal poles of both blastomeres at the 2-cell stage (B) reveals formation of atypical epidermis. Treatment of control explants with recombinant activin A induces extensive elongation of the explants (C), whereas parallel treatment of explants from Xwnt-5A-injected embryos does not induce elongation (D). Histological examination of dorsal halves of control blastula caps (E) reveals skeletal muscle (m) and notochord (nc) in the explants treated with activin A, similar to the mesodermal types in the activin A-treated dorsal halves of caps from Xwnt-5A-injected embryos (F). Ventral halves of blastula caps from control embryos (G) and from Xwnt-5Ainjected embryos (H) treated with activin A display similar ventral mesodermal types (shown here), as well as skeletal muscle (in explants not shown).