Fellgett SW et al. (2015), Sulf1 has ligand-dependent effects on canonical...

XB-ART-50160

J Cell Sci April 1, 2015; 128 (7): 1408-21.

Sulf1 has ligand-dependent effects on canonical and non-canonical Wnt signalling.

Fellgett SW , Maguire RJ , Pownall ME .

Abstract
Wnt signalling plays essential roles during embryonic development and is known to be mis-regulated in human disease. There are many molecular mechanisms that ensure tight regulation of Wnt activity. One such regulator is the heparan-sulfate-specific 6-O-endosulfatase Sulf1. Sulf1 acts extracellularly to modify the structure of heparan sulfate chains to affect the bio-availability of Wnt ligands. Sulf1 could, therefore, influence the formation of Wnt signalling complexes to modulate the activation of both canonical and non-canonical pathways. In this study, we use well-established assays in Xenopus to investigate the ability of Sulf1 to modify canonical and non-canonical Wnt signalling. In addition, we model the ability of Sulf1 to influence morphogen gradients using fluorescently tagged Wnt ligands in ectodermal explants. We show that Sulf1 overexpression has ligand-specific effects on Wnt signalling: it affects membrane accumulation and extracellular levels of tagged Wnt8a and Wnt11b ligands differently, and inhibits the activity of canonical Wnt8a but enhances the activity of non-canonical Wnt11b.

PubMed ID: 25681501
PMC ID: PMC4379729
Article link: J Cell Sci
Grant support: [+]

Species referenced: Xenopus laevis
Genes referenced: atf2 chrd.1 ctnnb1 dvl2 fzd8 gnl3 lrp6 mmut myc nodal3.1 nodal3.2 odc1 ror2 sia1 sulf1 wnt11 wnt11b wnt3a wnt8a
Antibodies: HA Ab3 Myc Ab7

Article Images: [+] show captions

	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.
	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.

References [+] :

Ai, QSulf1 remodels the 6-O sulfation states of cell surface heparan sulfate proteoglycans to promote Wnt signaling. 2003, Pubmed