Itasaki N et al. (2003), Wise, a context-dependent activator and inhibit...

XB-ART-4894

Development 2003 Sep 01;13018:4295-305. doi: 10.1242/dev.00674.

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Wise, a context-dependent activator and inhibitor of Wnt signalling.

Itasaki N , Jones CM , Mercurio S , Rowe A , Domingos PM , Smith JC , Krumlauf R .

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We have isolated a novel secreted molecule, Wise, by a functional screen for activities that alter the anteroposterior character of neuralised Xenopus animal caps. Wise encodes a secreted protein capable of inducing posterior neural markers at a distance. Phenotypes arising from ectopic expression or depletion of Wise resemble those obtained when Wnt signalling is altered. In animal cap assays, posterior neural markers can be induced by Wnt family members, and induction of these markers by Wise requires components of the canonical Wnt pathway. This indicates that in this context Wise activates the Wnt signalling cascade by mimicking some of the effects of Wnt ligands. Activation of the pathway was further confirmed by nuclear accumulation of beta-catenin driven by Wise. By contrast, in an assay for secondary axis induction, extracellularly Wise antagonises the axis-inducing ability of Wnt8. Thus, Wise can activate or inhibit Wnt signalling in a context-dependent manner. The Wise protein physically interacts with the Wnt co-receptor, lipoprotein receptor-related protein 6 (LRP6), and is able to compete with Wnt8 for binding to LRP6. These activities of Wise provide a new mechanism for integrating inputs through the Wnt coreceptor complex to modulate the balance of Wnt signalling.

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Species referenced: Xenopus laevis
Genes referenced: dvl2 egr2 emx2 en2 hoxb9 hsp70 hspa1l lrp6 muc2 muc2l nodal3.2 nog odc1 rax sia1 snai2 sostdc1 sox3 wnt8a
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	Fig. 1. Isolation, characterisation and expression of Wise. (A) Outline of the screen. (B) RT-PCR analysis using Wise RNA. Wise alone (600 pg) does not induce pan-neural (NCAM) or mesodermal (myosin) markers. In the presence of noggin, increasing amounts of Wise RNA (150, 300, 600 and 1200 pg) induce progressively more posterior neural markers (en2, krox20, Hoxb9). (C) Western blot detecting HA-tagged Wise protein secreted into the medium after RNA injection into oocytes. C, control uninjected oocytes. (D,E) Recombination of noggin-expressing and Wise-expressing animal caps assayed for induction of Krox20 (D) or en2 (E). In D, the noggin RNA injected cap was marked with FIDx (to the left of the broken line) and in E the Wise cap with lacZ (to the right of the broken line). Wise induces patches of Krox20 or a ring of en2 expression (arrowheads) in a non-cell-autonomous manner in the noggin-injected cap. (F,G) In situ hybridisation of chick embryo at stage 10. Wise is expressed in the surface ectoderm (se in G) from the level of presomitic mesoderm to the posterior. Expression is also seen faintly in the head surface ectoderm. (H) RNase protection of Xenopus embryos with stages noted above each lane. Wise is first detected at an early gastrula stage (st. 10) and the expression persists into tadpole stages. ODC is a loading control. (I-N) In situ hybridisation of Xenopus embryos at indicated stages. At the neurula stages (I-L), Wise is expressed in the surface ectoderm broadly at anteroposterior levels, strongest at the edge of the neural tube and the posterior edge of the eye (arrowheads in I,J). The expression is also seen in the stomodeal-hypophyseal anlage (arrow in J, front view; arrow in L, transverse section). At the tailbud stage (M,N) the expression appears to be localised in cranial placodes, lateral line placodes and the ventral neural tube at the diencephalon level (arrowhead in N).
	Fig. 3. Phenotypes after blastomere injection at four to eight cell stage of Wise RNA (B,E,H,K,N,Q) or antisense morpholino oligonucleotides (C,F,I,L,O,R) in comparison with control embryos (A,D,G,J,M,P) assayed by whole-mount in situ hybridisation at neurula stages. Marker is noted on the left of the panel. In most embryos, lacZ RNA was co-injected as a lineage tracer (blue staining). Injected side is to the left of each panel.
	sostdc1 expression (sclerostin domain containing 1) assayed through in situ hybridization Xenopus laevis embryo, NF stage 20, dorsal view, anterior to the left.
	sostdc1 expression (sclerostin domain containing 1) assayed through in situ hybridization Xenopus laevis embryo, NF stage 22, anterior view, dorsal up.
	sostdc1 expression (sclerostin domain containing 1) assayed through in situ hybridization Xenopus laevis embryo, NF stage 24, anterior view, dorsal up.
	sostdc1 expression (sclerostin domain containing 1) assayed through in situ hybridization Xenopus laevis embryo, NF stage 35, lateral view, anterior to the left.
	Fig. 4. Analysis with antisense morpholino oligonucleotides against Wise. (A) Antisense morpholino oligonucleotides against Xenopus Wise specifically blocks translation of Xenopus Wise RNA. Embryos were injected as indicated, animal caps were cut at stage 8, and the caps were collected at stage 9. Cell extracts were analysed by western blotting using anti-Flag and anti-HSP70 antibodies. (B-E) Phenotypes arising from whole-embryo injection of control (B,C) or Wise antisense (D,E) morpholino oligonucleotides at stage 43. (B,D) Whole-mount dorsal view; (C,E) transverse sections at the eye level. (F-I) In situ hybridisation analysis of Emx2 at stage 35 after injection of control (F) or Wise (G-I) morpholino oligonucleotides into a dorsal-animal blastomere at four- to eight-cell stages. (F,G) Dorsal view, anterior towards the top. (H,I) Lateral view. Wise morpholino oligonucleotides injection causes loss of olfactory placode. Arrowheads in G and H indicate unaffected placode on the uninjected side. (J,K) Injection of Wise antisense morpholino oligonucleotides causes thick surface ectoderm (* in K) in comparison with the un-injected side or control embryo (J). (L) The eye defect caused by antisense morpholino oligos can be partially rescued by co-injection of chick Wise RNA.
	Fig. 6. Wise interferes with Wnt signalling. (A-C) Wise blocks secondary axes induced by Wnt8. Injection of Wnt8 RNA into a ventral vegetal blastomere of four- to eight-cell stage embryos induces complete secondary axis formation (A). Co-injection of Wise blocks formation of Wnt8-induced secondary axes (B), as does co-injection of a dominant-negative dishevelled, δDsh (DIX) (C). (D) Wise functions extracellularly to block induction of siamois and Xnr3 by the Wnt pathway in ventral marginal zone (VMZ) explants. Wise blocks the ability of Wnt8 to induce siamois and Xnr3, but does not interfere with the ability of dishevelled (dsh) or β-catenin (β-cat) to induce these markers. DMZ, dorsal marginal zone explant. (E,F) Wise acts as a Wnt inhibitor and complements a truncated BMP receptor (tBR) to induce head structures. When BMP signalling is blocked in the ventral marginal zone by injection of tBR, an incomplete secondary axis is formed (E). Co-injection of tBR and Wise induces a complete secondary axis with eyes (arrows) and cement gland (F).