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Heparan sulfate proteoglycans (HSPGs) are abundant cell surface molecules, consisting of glycosaminoglycan (GAG) chains bound to a protein core. There is high diversity in the sulfation pattern within each GAG chain, creating specific binding sites for many proteins including cell signalling factors, whose activities and distribution are modified by their association with HSPGs (Danesin et al., 2006; Freeman et al., 2008). Here, we describe the distinct expression of three enzymes which modify the 6-O-sulfation state of HSPGs: two 6-O-endosulfatases (Sulf1 and Sulf2), and a 6-O-sulfotransferase (6OST-1). We use in situ hybridisation to determine the spatial distribution of transcripts during tailbud stages of Xenopus laevis development, with a particular focus on neural regions where the 6-O-sulfatases are expressed ventrally and the 6-O-sulfotransferase is restricted dorsally. The complementary expression of these enzymes in the hindbrain and neural tube suggest a role for specific HSPG structure in dorsoventral patterning, possibly through modifying the activity or distribution of signalling molecules such as BMP, Sonic hedgehog, Wnt and/or FGF.
Fig. 1. Expression of Sulf1, Sulf2 and 6OST during tailbud stages in Xenopus laevis. (A) Lateral view of a stage 28 embryo hybridised to a probe for Sulf1A (BG408276). (B) Lateral view of a stage 34 embryo hybridised to a probe for Sulf1A. (C) Lateral view of a stage 28 embryo hybridised to a probe for Sulf2 (BG814048). (D) Lateral view of a stage 34 embryo hybridised to a probe for Sulf2. (E) Lateral view of a stage 28 embryo hybridised to a probe for 6OST (XL037105). (F) Lateral view of a stage 34 embryo hybridised to a probe for 6OST. Abbreviations: BA, branchial arches; CG, cement gland; CMZ, ciliary marginal zone; FP, floorplate; L, liver; LLP, lateral line placodes; MHJ, midbrain–hindbrain junction; NP, nasal placode; PN, pronephros; arrowheads denote some of the somites repeated along the embryonic axis.
Fig. 2. Cross-sections of tailbud (stage 34) Xenopus laevis embryos hybridised to probes for Sulf1 (A and D), Sulf2 (B and E), and 6OST (C and F). Panels A–C are sections taken at the level of the otic vesicle. Panels D–F are sections taken at the level of the pronephros. Diagram shows level of sections taken. ov, otic vesicle; pn, pronephros.
Fig. 3. Complementary expression of Sulf2 and 6OST in the brain of Xenopus laevis embryos at stage 28. (A) Sulf2 is expressed in the ventralmidbrain and hindbrain, the cement gland, the nasal placodes (red arrowheads), the retina, the midbrain–hindbrain junction, and the pineal gland (red asterisk). (B) 6OST is expressed in the dorsal part of the midbrain and hindbrain, but a reduced level of 6OST expression is found at the midbrain–hindbrain junction (MHJ). 6OST expression is also detected in the olfactory placodes, and in the three lateral line placodes posterior to the eye (red arrowheads). (C) A parasagital-section through the brain shows the ventral expression of Sulf2 in the midbrain and hindbrain. (D) A parasagital-section through the brain shows the tight restriction of 6OST expression to the dorsal part of the midbrain and hindbrain. (E) Double in situ hybridisation to En2 (blue) and Sulf2 (purple). Expression in the midbrain–hindbrain junction is indicated with an arrowhead. (F) Double in situ hybridisation to En2 (blue) and 6OST (purple). The midbrain–hindbrain junction is indicated with an arrowhead, and the gap in 6OST expression is indicated with an asterisk. (G) A section through the head of an embryo showing the expression of Sulf2 (purple) and En2 (blue) in the midbrain–hindbrain junction as indicated with an arrowhead. (H) A section through the head of an embryo showing the expression of En2 (purple) and 6OST (blue). The midbrain–hindbrain junction is indicated with an arrowhead, and the gap in 6OST expression is indicated with an asterisk.
Fig. 4. Sulf1 is expressed in the ventricular zone of the forebrain and midbrain and the floorplate of the neural tube. (A) A cross-section taken at an oblique angle through a stage 28 Xenopus laevis embryo hybridised to Sulf1 showing Sulf1 expression in the ventricular zone in the forebrain. (B) Cross-section through the midbrain showing Sulf1 expression in the ventricular zone. Arrowhead indicates expression in CMZ. (C) Cross-section through the hindbrain showing ventral expression of Sulf1. (D) Sagital-section through the brain showing the ventral expression of Sulf1. (E) Sagital-section through the neural tube showing the expression of Sulf1 in the floorplate and ventralhindbrain. Arrow indicates midbrainâ hindbrain junction.
Supplementry Fig. 2. (A) Vegetal view of a stage 11.5 embryo. The zygotic expression of Xenopus laevis Sulf1 is first detected in the mesoderm during mid-gastrula stage, but is excluded from the most dorsal mesoderm (arrowhead). (B) Dorsal view of a stage 14 embryo. Sulf1 is expressed in the paraxial mesoderm, but is excluded from the dorsal midline. (C) Lateral view of a stage 23 embryo. Sulf1 is expressed in the pronephros (arrowhead), somites and posteriormesoderm as well as anterior neural tissue. (D) Vegetal view of a stage 11.5 embryo hybridised to Xenopus laevis Sulf2. Zygotic Sulf2 expression is not detected. (E) Dorsal view of a stage 14 embryo. Zygotic Sulf2 expression is not detected. (F) Lateral view of a stage 23 embryo. Sulf2 expression is detected in a ventral region that could include haemangioblasts (arrowhead), in the mesendoderm underlying the cement gland (arrow), and in anterior neural tissue. (G) Vegetal view of a stage 11.5 embryo. The zygotic expression of Xenopus laevis6-O-sulfotransferase is only detected in endoderm as the punctuate staining indicated by the arrowhead. (H) Dorsal view of a stage 14 embryo. 6OST is expressed in the two bars in the presumptive hindbrain (arrowheads) and in the paraxial mesoderm. (I) Lateral view of a stage 23 embryo. 6OST is expressed in the endoderm (arrowhead) and in anterior neural tissues (arrow).
Supplementry Fig. 3. Expression of the two alleles coding for Xenopus laevis Sulf1. (A) Shows the expression of Sulf1A (BG408276) in the head of a Stage 28 embryo. (B) Shows the expression of Sulf1A in the head of a Stage 34 embryo. (C) Shows the expression of Sulf1 B (BQ733206) in the head of a Stage 28 embryo. (D) Shows the expression of Sulf1B in the head of a Stage 34 embryo. Abbreviations: BA, branchial arches; CMZ, ciliary marginal zone; FP, floorplate; PN, pronephros.
Supplementry Fig. 4. Sections taken through the eyes of stage 28 embryos subsequent to in situ hybridisation for Sulf2 and 6OST reveal the expression of Sulf2 and 6OST in the retina as indicated by arrows.
