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First published online August 4, 2003
doi: 10.1242/10.1242/dev.00674

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

Nobue Itasaki^1,*, C. Michael Jones^2,, Sara Mercurio^1,2, Alison Rowe¹, Pedro M. Domingos^1,, James C. Smith^2, and Robb Krumlauf^1,¶,*

¹ Division of Developmental Neurobiology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK
² Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, Mill Hill, London NW7 1AA, UK

View larger version (57K): [in a new window]   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).

View larger version (56K): [in a new window]   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.

View larger version (47K): [in a new window]   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.

View larger version (69K): [in a new window]   Fig. 5. Wise requires components of the Wnt pathway for en2 induction and activates the canonical Wnt pathway. (A,B) RT-PCR of noggin-injected animal caps assayed for en2 induction. Induction of en2 is attenuated or blocked by dominant-negative (dn)-Wnt8 (Wnt8), dn-LRP6 (LRP6), dn-Dishevelled [Dsh (dd1)], GSK3-ß (GSK3), dn-Lef1 (LEFN) (A) and dn-Frizzled8 (Fz8) (B). (C) Wise does not interfere with the ability of Wint8 to induce Krox20 in noggin-injected animal caps. Wnt8-induced Krox20 expression is blocked by Wnt8, but not by Wise. (D) Long-exposure of RT-PCR result showing that Wise-injected animal caps express siamois and Xnr3, although very weakly in comparison with the induction by Wnt8. (E-G) Staining for subcellular localisation of endogenous ß-catenin detected immunocytochemically in Xenopus animal caps after RNA injection of (E) Tcf3, (F) Wnt8+Tcf3 and (G) Wise+Tcf3. Both Wnt8 and Wise promote nuclear accumulation of ß-catenin.

View larger version (58K): [in a new window]   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, Dsh (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 ß-catenin (ß-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).

View larger version (83K): [in a new window]   Fig. 7. Wise blocks cell movements in activin-treated animal caps. (A) Control animal caps at stage 15. (B) Control animal caps treated with activin (8 units/ml). They undergo gastrulation-like movements in response to activin and become elongated. (C) Animal caps from Wise RNA (1 ng) injected embryos, treated with activin. Elongation is blocked. (D) RT-PCR of animal caps assayed for muscle-actin (m.a.) and ef1. All caps were assayed at stage 15. Induction of muscle actin by activin is not interfered by preceded injection of 500 pg (+) or 1 ng (++) of Wise RNA, showing that failure in elongation in response to activin in C is not due to failure in muscle induction.

View larger version (58K): [in a new window]   Fig. 2. Molecular structure of Wise. (A) Alignment of the predicted amino acid sequence of Zebrafish, Xenopus, chick, mouse and human Wise proteins. Shaded boxes represent identical amino acids between species; thick line underneath shows conserved amino acids in SOST, asterisks indicate residues conserved in Drosophila Slit and dots identify residues conserved in Cef10 (chick homologue of Cyr61 in CCN family). Circles mark conserved cysteine residues. The arrowhead delineates the site of signal peptide cleavage predicted in the chick protein. (B) Diagram showing alignment of conserved amino acids between Wise, Cef10 and Drosophila Slit. Red filled ovals and lines indicate cysteine residues conserved between Wise, Cef10 and Slit. Open ovals indicate additional cysteines in Cef10 conserved in the CT domain of CCN family members but not in Wise or Slit. Black dotted lines show other conserved amino acids.

View larger version (42K): [in a new window]   Fig. 8. Wise interacts with the extracellular domain of LRP6. (A-C) All components were provided as concentrated conditioned medium for the immunoprecipitation study. Asterisks show samples of input loaded directly on to the gel. Extracellular domains of LRP6 and Frizzled8 are fused to human IgG Fc domain. E1-2 and E3-4 are deletion constructs of LRP6 in which the first two or last two EGF repeats respectively are missing (Mao et al., 2001). (A) Wise binds to LRP6 and a deletion construct of LRP6 (E3-4), but not to Frizzled8 or LRP6 E1-2. Dkk1 binds to LRP6 and E1-2 LRP6, but not to E3-4 LRP6 or Frizzled8. Wnt8 binds to LRP6 and Frizzled8. (B) Wise does not bind to Wnt8. Dkk1 and extracellular domain of Frizzled1 (Fz1 ECD) are used as a negative and a positive control for binding to Wnt8, respectively. (C) Binding of Wnt8 and LRP6 is attenuated in the presence of Wise, but binding of Wnt8 and Frizzled8 is not. (D) A possible model of Wise action on Wnt signalling. Open arrows in each panel show weak (1,3) and strong (2) activation of the downstream pathway. (1,2) Wise and Wnt share the same binding domain of LRP6, while Dkk1 interacts with a different domain on LRP6. Wise induction of downstream targets of the canonical Wnt pathway is weaker compared with Wnt. (3) Wise competes with Wnt for binding to LRP6, resulting in attenuation of the Wnt action (compare 2 with 3).