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Fig. 1. Zygotic expression of XtSulf1 analysed by whole mount in situ hybridisation. (A) Stage 13 embryo shows XtSulf1 expression in the posterior paraxial mesoderm (dorsal view; anterior to the left). (B) Stage 20 embryo shows XtSulf1 expression in the pre-segmented posterior mesoderm, the somites, and the ventral part of the hind brain and the floorplate of the neural tube (lateral view; anterior to the left). (C) Stage 26 embryo shows expression in the posterior mesoderm, the somites, the hindbrain, the floorplate of the neural tube, the retina of the eye, some neural crest (arrowhead), and the pronephros (arrow). (D) At later tailbud stages, XtSulf1 is expressed in the mesoderm of the tailbud, the somites, the hindbrain, the neural crest (arrowhead), the floorplate of the neural tube, the fin, the pericardium, the lens, the ciliary marginal zone (CMZ) of the retina, and in the first branchial arch. (E) Vibratome section through D showing expression of XtSulf1 in the ventricular part of the midbrain, the lens (arrowhead) and CMZ of the eye (arrow). (F) Vibratome section through C showing the expression in the pronephros (arrowhead) and the floorplate (arrow). (G) Vibratome section of D showing the expression of XtSulf1 in the somites and floorplate.
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Fig. 3. XtSulf1 enhances the axis-duplicating activity of Wnt11. (A–F) Whole mount in situ hybridisation shows that maternal expression of XtSulf1 co-localises with maternal Wnt11 in cleavage stage embryos (A, C, E, Wnt11; B, D, F, XtSulf1; all vegetal view). (G) Graph quantifying the frequency of double axes formed in embryos when increasing amounts XtSulf1 mRNA is co-injected with 600 pg of Wnt11 mRNA into a single ventral blastomere at the 4-cell stage (OB = open blastopore, DA = double axis, DH = double head). (H) Uninjected control X. laevis embryo. (I) Embryo injected with 600 pg of mRNA coding for XtWnt-11 into one ventral blastomere at the 4-cell stage. (J) Embryo injected with 600 pg of mRNA coding for XtSulf1 into one ventral blastomere at the 4-cell stage. (K–M) Embryos co-injected with 600 pg of XtWnt-11 and 600 pg XtSulf1 into one ventral blastomere at the 4-cell stage show duplicated axes. (N–P) Embryos co-injected with 600 pg of XtWnt-11 and 4 ng of XtSulf1 into one ventral blastomere at the 4-cell stage show duplicated heads and gastrulation defects. (Q) Section through a control embryo. (R) Section though an embryo shown in K showing duplicated notochord (arrows) and neural tissue. (S–U) In situ hybridisation showing the expression of chordin in a control stage 10 embryo (S), or embryos co-injected with 600 pg of XtWnt-11 and 600 pg XtSulf1 into one ventral blastomere at the 4-cell stage (T, U) showing ectopic expression of chordin in the ventral marginal zone (arrows). (V–X) In situ hybridisation showing the expression of Xnr3 in a control stage 10 embryo (V), or embryos co-injected with 600 pg of XtWnt-11 and 600 pg XtSulf1 into one ventral blastomere at the 4-cell stage (W, X) showing ectopic expression of Xnr3 in the ventral marginal zone (arrows). (Y) Co-immunoprecipitation of Wnt11-HA with LRP6-myc is enhanced when XtSulf1 is also expressed in Xenopus embryos.
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Fig. 4. XtSulf1 inhibits BMP signaling in Xenopus embryos. (A–I) show the effects of over-expression of XtSulf1 on BMP4 activity, while (J–P) show the effects of XtSulf1 knock-down on BMP4 activity. (A) Western blotting to detect phospho-SMAD1/5/8 in stage 10 animal caps injected with mRNA coding for BMP4 or the constitutively active BMP receptor Alk3. Over-expression of XtSulf1 results in the inhibition of BMP4 activation of pSMAD1, while it has no effect on the pSMAD1 activation by Alk3. (B) A GST-BMP receptor fusion protein was used to pull down an HA-tagged BMP4 in whole embryos. In the presence of XtSulf1 less BMP4 is associated with the BMP receptor. (C) XtSulf1 over-expression in stage 18 animal caps results in low levels of NCAM expression as analysed by RNAase protection analysis. In situ hybridisation shows that the BMP target genes sizzled (D–F) and Msx1 (G–I) are down-regulated in embryos over-expressing XtSulf1. Panels D and G are uninjected stage 10 control embryos, panels E and H have been injected unilaterally at the 2-cell stage with 2 ng of XtSulf1 mRNA, and panels F and I have been injected into all blastomeres at the 4-cell stage with a total of 4 ng of XtSulf1 mRNA. (J) Western blotting to detect phospho-SMAD1/5/8 in X. tropicalis embryos at stage 14. Embryos where XtSulf1 has been knocked down show elevated levels of pSMAD1; this effect is rescued by co-injecting mRNA coding for XtSulf1. In situ hybridisation of X. tropicalis embryos at stage 16 shows that embryos unilaterally injected at the 2-cell stage with an AMO against XtSulf1 (L, N, P) have reduced levels of Otx2 (K and L) and Pax6 (M–P) which are known to be negatively regulated by BMP4 signaling.
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Fig. 5. XtSulf1 inhibits FGF signaling in Xenopus embryos and disrupts somitogenesis. (A–G) show the effects of over-expression of XtSulf1 on FGF activity, while (H–V) show the effects of XtSulf1 knock-down on FGF activity. (A) Western blotting shows the induction dpERK in stage 10 animal caps treated with FGF4 protein from stage 8 is inhibited in animal caps over-expressing XtSulf1. When the MAPK pathway is intracellularly activated by a drug inducible FGF receptor, XtSulf1 does not affect the induction of dpERK. In situ hybridisation shows that the FGF target genes MyoD (B–D) and Xbra (E–G) are down-regulated in embryos over-expressing XtSulf1. Panels B and E are uninjected stage 10 control embryos, panels C and F have been injected unilaterally at the 2-cell stage with 2 ng of XtSulf1 mRNA, panels D and G have been injected into all blastomeres at the 4-cell stage with a total of 4–8 ng of XtSulf1 mRNA. Western blotting to detect dpERK in X. tropicalis embryos at stage 14 in embryos where XtSulf1 has been knocked down shows elevated levels of dpERK; this effect is rescued by co-injecting mRNA coding for XtSulf1. In situ hybridisation shows that the FGF regulated gene Cdx4 (I–K) is up-regulated in neurula embryos where XtSulf has been knocked down. (I) uninjected control, (J) unilaterally injected at the 2-cell stage with AMO3, (K) injected into both blastomeres at the 2-cell stage. Panels L and M show the two sides of an embryos unilaterally injected with AMO3 and probed with MyoD. The uninjected side (L) shows normally segmented somites (arrowheads), while the side where XtSulf1 has been knocked down (M) continues to express MyoD, but does not segment to form somites. Whole mount immunostaining for dpERK (N–P) shows staining in the posterior mesoderm in stage 14 embryos unilaterally injected with a control morpholino (N). In embryos unilaterally injected with AMO3 (O and P) dpERK increases and extends anteriorly (shift is indicated by horizontal lines). In situ hybridisation for bowline and thylacine panels Q–V shows an anterior shift of the onset of somitogenesis where XtSulf1 is knocked down. (Q) Bowline expression in a stage 17 embryo unilaterally that was injected with a control morpholino at the 2-cell stage. (R and S) Bowline expression in stage 17 embryos that were unilaterally injected with AMO3 at the 2-cell stage. (T) Thylacine expression in a stage 17 embryo unilaterally that was injected with a control morpholino at the 2-cell stage. (U and V) Thylacine expression in stage 17 embryos that were unilaterally injected with AMO3 at the 2-cell stage. Arrow heads in panels Q–V indicate the most posterior, newly determined somitomere.
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Fig. 1. Zygotic expression of XtSulf1 analysed by whole mount in situ hybridisation. (A) Stage 13 embryo shows XtSulf1 expression in the posterior paraxial mesoderm (dorsal view;
anterior to the left). (B) Stage 20 embryo shows XtSulf1 expression in the pre-segmented posteriormesoderm, the somites, and the ventral part of the hind brain and the floorplate of
the neural tube (lateral view; anterior to the left). (C) Stage 26 embryo shows expression in the posteriormesoderm, the somites, the hindbrain, the floorplate of the neural tube, the
retina of the eye, some neural crest (arrowhead), and the pronephros (arrow). (D) At later tailbud stages, XtSulf1 is expressed in the mesoderm of the tailbud, the somites, the
hindbrain, the neural crest (arrowhead), the floorplate of the neural tube, the ï¬n, the pericardium, the lens, the ciliary marginal zone (CMZ) of the retina, and in the ï¬rst branchial
arch. (E) Vibratome section through D showing expression of XtSulf1 in the ventricular part of the midbrain, the lens (arrowhead) and CMZ of the eye (arrow). (F) Vibratome section
through C showing the expression in the pronephros (arrowhead) and the floorplate (arrow). (G) Vibratome section of D showing the expression of XtSulf1 in the somites and
floorplate.
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Fig. 1. Sulf1 expression at stg 20
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