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Fig. 1. Sulf1 inhibits the ability of Wnt8a to activate canonical Wnt signalling. (A–M) Xenopus laevis embryos were microinjected with mRNA encoding Wnt8a
(5 pg) and/or Sulf1 (1 ng) into a single ventral blastomere at the four-cell stage. (A–F) Whole embryo phenotypes of (A) uninjected control embryo, and embryos
injected with (B) Sulf1, (C) Wnt8a or (D–F) Sulf1 and Wnt8a. White arrowheads in C mark a fully duplicated axis; red arrowheads in F mark duplicated trunk
and cement gland. (G) The data shown in A–F is quantified in G. **P,0.01 (Chi squared test). (H–M) In situ hybridisation shows the expression of chordin at NF
stage 10.5. (H) Uninjected control embryos; (I–K) embryos injected with (I) Sulf1, (J) Wnt8a and (K) Sulf1 and Wnt8a. (L) Wnt8a induces an ectopic domain of
chordin expression. (M) Sulf1 inhibits the ability of Wnt8a to induce ectopic chordin resulting in the formation of a partial secondary organiser domain. The red
arrowheads in L and M mark the edges of the ectopic chordin domains. (N) The data shown in H–M is quantified in N, **P,0.01 (Chi squared test). (O–U) In situ
hybridisation shows the expression of Xnr3 at NF stage 10. (O) Uninjected control embryos; (P–T) embryos injected with (P) Sulf1, (Q) Wnt8a and (R) Sulf1 and
Wnt8a. Enlarged images of embryos shown in Q and R are shown in S and T. The data shown in O–T is quantified in (U). N, number of embryos.
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Fig. 2. Sulf1 inhibits the ability of Wnt8a to activate canonical Wnt signalling. (A) A graph showing the response of the Topflash reporter in whole embryos
and (B) in animal caps injected with the mRNAs indicated. Results are mean6s.e.m. (C) An RNase protection analysis shows the expression of Chordin and
Siamois in gastrula stage 10 embryos and in animal caps injected with mRNAs indicated. The expression of ODC serves as a loading control, and hybridisation
to tRNA controls for specificity. (D) Western blotting for b-catenin shows protein levels in animal caps injected with the mRNAs indicated. Antibodies
against MCM and GAPDH serve as loading controls. (E) Xenopus laevis embryos were microinjected with mRNA coding for b-catenin delta-N (labelled as bcatenin,
150 pg) or chordin (150 pg), alone or together with Sulf1 (1 ng) into a single ventral blastomere at the four-cell stage. The number of embryos with
duplicated axes was counted at NF stage 20. (F) Immunoprecipitation of epitope-tagged proteins expressed in animal caps injected with the mRNAs indicated.
The top panel shows protein immunoprecipitated with an antibody against HA (Wnt8a is tagged with HA) and immunoblotted with an antibodies against Myc
(LRP6 is tagged with Myc). The bottom two panels are the protein lysates prior to immunoprecipitation.
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Fig. 3. Sulf1 does not inhibit the ability of Wnt3a to induce axis duplication. (A–F) Xenopus laevis embryos were microinjected with mRNA encoding Wnt3a
(5 pg) and/or Sulf1 (1 ng) into a single ventral blastomere at the four-cell stage. In situ hybridisation for the gene chordin was performed at NF stage 10.5.
(A) Uninjected control embryos; (B–D) embryos injected with (B) Sulf1, (C) Wnt3a or (D) Sulf1 and Wnt3a. The areas indicated in the white boxes in C and D are
enlarged in E and F, respectively. (G) The data shown in A–F is quantified in G. NS, not significant (Chi squared test), N, number of embryos.
(H) Immunoprecipitation (IP) of epitope-tagged proteins expressed in animal caps injected with the mRNAs indicated. The top panel shows protein
immunoprecipitated with an antibody against HA (Wnt3a is tagged with HA) and immunoblotted with an antibodies against Myc (LRP6 is tagged with Myc). The
bottom two panels are protein lysates prior to immunoprecipitation.
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Fig. 4. Sulf1 enhances the ability of Wnt11b to activate both canonical and non-canonical Wnt signalling. (A–F) Xenopus laevis embryos were
microinjected with mRNA encoding Wnt11b (600 pg) and/or Sulf1 (1 ng) into a single ventral blastomere at the four-cell stage. In situ hybridisation for the gene
chordin was performed at NF stage 10.5. (A) Uninjected control embryos; (B–D) embryos injected with (B) Sulf1, (C) Wnt3a or (D) Sulf1 and Wnt11b. The areas
indicated in the white boxes in in C and D are enlarged in E and F, respectively. (G) The data in A–F is quantified in G. **P,0.01 (Chi squared test). (H–L)
Embryos were microinjected bilaterally in the animal hemisphere with mRNA encoding Wnt11b (50 pg) and Sulf1 (500 pg). Embryos were cultured until NF
stage 8, animal explants were taken and cultured until NF stage 10.5 in either the presence or absence of activin. (H,I) Control animal explants culture in either
the absence (H) or presence (I) of activin. (J–L) Animal explants injected with (J) Wnt11b, (K) Sulf1 or (L) Wnt11b and Sulf1. (M) The classification system used
to score animal cap explants shown in N. (N) The data shown in H–L is quantified in N. N, number of embryos.
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Fig. 5. Sulf1 enhances Wnt11b induced
Dvl–GFP translocation to the cell
membrane. (A–P) Xenopus laevis embryos
were microinjected bilaterally with mRNA
encoding mRFP (500 pg) and Dvl–GFP
(500 pg) into the animal hemisphere at the
two-cell stage. In addition embryos were
injected with mRNA encoding Sulf1 (4 ng),
LacZ (4 ng), Wnt11b (400 pg) or a mixture of
the three. (A–D) Control animal explants
overexpressing mRFP and Dvl–GFP. Animal
explants injected with (E–H) Sulf1,
(I–L) Wnt11b and LacZ or (M–P) Sulf1 and
Wnt11b. The white boxes in C, G, K and O
mark the areas that are enlarged in D, H, L and
P, respectively. mRFP is shown in magenta,
Dvl–GFP is shown in green. Scale bars:
20 mm. (Q) The data shown in A–P is
quantified in Q. Quantification was done using
a program written in MatLab, results are
mean6s.e.m. **P,0.01; NS, not significant
(Mann–Whitney U test). N, number of
embryos. (R) Immunoprecipitation (IP) of
epitope-tagged proteins expressed in animal
caps injected with the mRNAs indicated. The
top panel shows protein immunoprecipitated
with an antibody against HA (Wnt11b is tagged
with HA) and immunoblotted with an antibodies
against Myc (Ror2 is tagged with Myc). The
bottom two panels are protein lysates prior
to immunoprecipitation.
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Fig. 6. Sulf1 enhances the accumulation
of Wnt11b–GFP on the cell membrane.
(A–P) Xenopus laevis embryos were
microinjected bilaterally with mRNA
encoding mRFP (500 pg) into the animal
hemisphere at the two-cell stage. In
addition, embryos were injected with mRNA
encoding LacZ (4 ng), Sulf1 (4 ng), Wnt8a–
GFP (500 pg), Wnt11b–GFP (1 ng) or a
mixture of the four. (A–D) Control animal
explants overexpressing LacZ and Wnt8a–
GFP. (E–H) Animal explants
overexpressing Sulf1 and Wnt8a–GFP.
(I–L) Control animal explants
overexpressing LacZ and Wnt11b–GFP.
(M–P) Animal explants overexpressing
Sulf1 and Wnt11b–GFP. The white boxes in
C, G, K and O mark the areas that are
enlarged in D, H, L and P, respectively.
mRFP is shown in magenta, Wnt8a– or
Wnt11b–GFP is shown in green. Scale
bars: 20 mm. (Q,R) Graphs quantifying the
relative levels of (Q) Wnt8a–GFP and
(R) Wnt11b–GFP on the cell membrane.
Data was quantified using a programme
written in Matlab, results are mean6s.e.m.
**P,0.01; NS, not significant (Mann–
Whitney U test). N, number of embryos.
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Fig. 7. Sulf1 enhances the secretion and range of diffusion of Wnt8a–GFP in animal explants. (A) Diagram depicting the assay used to measure Wnt8a–
GFP secretion and diffusion through a control background, see Materials and Methods for details. (B,C) mRNA encoding either (B) mCerulean (600 pg), LacZ
(4 ng) and Wnt8a–GFP (2 ng) or (C) mRFP (600 pg), Sulf1 (4 ng) and Wnt8a–GFP (2 ng) was injected into the animal hemisphere of one blastomere
at the four-cell stage. (D) The range of diffusion of Wnt8a–GFP through a control background was quantified using Fiji Image J. (E) Diagram depicting the assay
used to measure Wnt8a–GFP diffusion through a background expressing Sulf1. (F–I) mRNA encoding mCerulean (600 pg) and Wnt8a–GFP (2 ng) was injected
into the animal hemisphere of one blastomere at the four-cell stage. An adjacent blastomere was injected with mRNA encoding (F,G) mRFP (600 pg) and LacZ
(4 ng) or (H,I) mRFP (600 pg) and Sulf1 (4 ng). (J) The range of Wnt8a–GFP through a background expressing either LacZ or Sulf1 was quantified using Fiji
Image J. Scale bars: 20 mm.
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Fig. 8. Sulf1 enhances both the secretion and the amount of Wnt11b–GFP diffusing in animal explants. (A) Diagram depicting the assay used to measure
Wnt11b–GFP secretion and diffusion through a control background. (B,C) mRNA encoding either (B) mCerulean (600 pg), LacZ (4 ng) and Wnt11b-GFP (2 ng)
or (C) mRFP (600 pg), Sulf1 (4 ng) and Wnt11b–GFP (2 ng) was injected into the animal hemisphere of one blastomere at the four-cell stage. (D) The range of
diffusion of Wnt11b–GFP through a control background was quantified using Fiji Image J. (E) Diagram depicting the assay used to measure Wnt11b–GFP
diffusion through a background expressing Sulf1. (F–I) mRNA encoding mCerulean (600 pg) and Wnt11b–GFP (2 ng) was injected into the animal hemisphere
of one blastomere at the four-cell stage. An adjacent blastomere was injected with mRNA encoding (F,G) mRFP (600 pg) and LacZ (4 ng) or (H,I) mRFP
(600 pg) and Sulf1 (4 ng). (J) The range of Wnt11b–GFP through a background expressing either LacZ or Sulf1 was quantified using Fiji Image J. Scale bars:
20 mm.
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Figure 1
[A-F] Xenopus laevis embryos were microinjected with mRNA encoding Wnt3a (0.1pg) and Sulf1 (1ng) into
a single ventral blastomere at the four cell stage. In situ hybridisation for the gene chordin was performed at
NF stage 10.5. [A] Uninjected control embryos. [B-D] Embryos injected with [B] Sulf1, [C] Wnt3a or [D]
Sulf1 and Wnt3a. The white boxes in [C] and [D] were used to create panels [E] and [F] respectively. [G]
The data shown in [A-F] is quantified in [G], Chi squared test (NS=not significant), N=number of embryos.
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Figure 2
[A-L] Xenopus laevis embryos were microinjected bilaterally with mRNA encoding mRFP (500pg) and Dvl-GFP (500pg)
into the animal hemisphere at the two cell stage. In addition embryos were injected with increasing amounts of Wnt11b
mRNA. [A-D] Control animal explants over expressing mRFP and Dvl-GFP. Animal explants injected with [E-H] Wnt11b
(400pg) or [I-L] Wnt11b (800pg) mRNA. The white boxes in [C], [G] and [K] mark the areas used to create panels [D],
[H] and [L] respectively. [M] The data shown in [A-L] is quantified in [M]. Quantification was done using a program
written in MatLab, Mann-Whitney U (**P<0.01), error bars represent s.e.m, N=number of embryos. mRFP (magenta),
Dvl-GFP (green), scale bars represent 20um. [N] A diagram of the ATF2 reporter used for this assay is shown above a
graph depicting the results obtained when 100pg of ATF reporter plasmid DNA and 1pg of Renilla was injected into the
marginal zone of all four cells of four cell stage Xenopus laevis embryos. In addition embryos were injected with mRNA
encoding Sulf1 (4ng) or Wnt11b (200pg) with a control mRNA or with Wnt11b (200pg) together with Sulf1. Embryos
were lysed and analysed in a luminometer at stage 10 to assay luciferase activity. The graph illustrates the effects of
Sulf1 and Wnt11b on the activation of the ATF reporter.
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Figure 3
Wnt8a-GFP and Wnt11b-GFP are biologically active.
mRNA encoding Wnt8a (10pg) or Wnt8a-HA-GFP (10pg) was injected into one ventral blastomere of
an embryo at the four cell stage. Embryos were cultured until NF stage 38 and then examined for
phenotype. [A] Lateral view of an uninjected embryo. [B]. An example of an embryo injected with
Wnt8a. [C]. An example of an embryo injected with Wnt8a-HA-GFP.[ D]. Graph depicting the
quantification of the frequency of axis duplication in embryos over-expressing Wnt8a and Wnt8-HAGFP.
Wnt8a-HA-GFP showed a similar level of activity to Wnt8a in axis duplication assays.
mRNA encoding Wnt11-HA or Wnt11-HA-GFP was injected bilaterally into the animal hemisphere of
embryos at the two cell stage. Embryos were cultured until NF stage 8 and then animal cap explants
were explanted and cultured in either the presence or absence of activin until NF stage 19. [A-B]
Uninjected animal caps cultured in either the absence [E] or presence [F] of activin. [G-I] Animal caps
treated with activin and injected with increasing amounts of Wnt11b-HA. [H-L] Animal caps treated
with activin and injected with increasing amounts of Wnt11b-HA-GFP. Over-expression of increasing
amounts of Wnt11-HA or Wnt11-HA-GFP inhibited activin induced convergent extension of animal
caps to a similar extent.
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Figure 4
Expression of Wnt8a-GFP and Wnt11b-GFP in Xenopus animal caps.
Western blot analysis of Xenopus embryos injected with 400ng of mRNA coding for Wnt fusion
proteins (Wnt8a-HA-GFP or Wnt11b-HA-GFP) together with 4ng of mRNA coding for Sulf1 or the
control LacZ. Wnt proteins are detected at similar levels in the presence and absence of Sulf1.
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