Specific heparan sulfate structures involved in retinal axon targeting
Atsushi Irie1,*,
Edwin A. Yates2,
Jeremy E. Turnbull2,
and
Christine E. Holt1,
1 Department of Anatomy, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK
2 Molecular Cell Biology Research Laboratories, School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
* Present address: The Tokyo Metropolitan Institute of Medical Science, Department of Biochemical Cell Research, Bunkyo-ku, Tokyo 113-8613, Japan
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Fig. 1. Disruption of retinal axon targeting with exogenously applied GAGs. Lateral view of whole-mount brains at stage 40 showing the trajectories of HRP-filled optic projections. One side of the brain was exposed to GAGs from stage 35/36 to 40, the time when axons first grow from the mid-optic tract into the optic tectum (Tec). (A) Control projection showing HRP-filled axons coursing through the optic tract (Ot) in the diencephalon (Di) and crossing the diencephalon/midbrain boundary (dashed line anteriorly) into the tectum. Note the caudalward bend in the projection in the mid-optic tract. (B,C) Projections exposed to GAGs exhibiting the bypass phenotype. Brains were treated with 100 µg/ml of bovine lung heparin (B) and porcine mucosal HS (C), beginning when axons were in the mid-optic tract. The axons extend normally to the mid-optic tract, then take an aberrant route dorsally in the diencephalon, failing to cross the diencephalon/midbrain boundary, and bypass the tectum. Note the absence of a caudalward bend in the mid-optic tract. Tel, telencephalon; dorsal is up, anterior to the left.
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Fig. 2. Dose-response curves for the bypass phenotype induced by GAGs. Various concentrations of heparin (open circles) and HS (filled circles) were exogenously applied to exposed brains and plotted as a percentage of embryos showing the bypass phenotype. Numbers in parentheses indicate total number of embryos tested.
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Fig. 3. Induction of the bypass phenotype by chlorate treatment. Chlorate was applied to exposed brains (stage 35/36 to 40) to inhibit the sulfation of endogenous HS. (A) Control projection crosses the diencephalon/midbrain boundary (dashed line anteriorly) to enter the anterior tectum (Tec). (B) Projection exposed to 30 mM of chlorate veers abnormally around the anterior border of the tectum and extends dorsally within the diencephalon. (C) Dose-response curve for the bypass phenotype induced with chlorate. The number of embryos exhibiting the phenotype is plotted as a percentage of the total number of embryos in each condition. Numbers in parentheses indicate total number of embryos per experimental condition.
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Fig. 4. Chemically modified heparin derivatives show differential bypass-inducing activities. (A) The structure of the disaccharide units in heparin and heparan sulfate. (B) Various concentrations of heparin derivatives were exogenously applied to exposed brains. Results are plotted as a percentage of embryos showing the bypass phenotype. The curves correspond to unmodified bovine lung heparin (filled circles), completely de-N-sulfated/re-N-acetylated heparin (open circles), completely de-6-O-sulfated heparin (filled squares), completely de-2-O-sulfated heparin (open squares), and per-sulfated heparin (filled triangles). (C) Partially de-2-O-sulfated heparin (open squares) and de-6-O-sulfated heparin (filled squares) were applied to exposed brains at 10 µg/ml. The percentage of the bypass phenotype induction is plotted against the degree of desulfation. Numbers in parentheses indicate total number of embryos.
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Fig. 5. Size-fractionated heparin oligosaccharides show different potencies for inducing the bypass phenotype. Exposed brains were treated with size-defined heparin oligosaccharides. (A) Size-dependency of heparin oligosaccharides to induce the bypass phenotype. Brains were exposed with 30 µM of size-fractionated bovine lung heparin fragments. (B) Dose-response curves for the bypass phenotype induction with dp6 (filled circles) and dp12 (open circles). Results are expressed as a percentage of total embryos showing the bypass phenotype. Numbers in parentheses indicate total number of embryos.
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Fig. 6. Ability of purified heparin oligosaccharides to induce the bypass phenotype. (A,B) Purification of heparin oligosaccharides with SAX-HPLC. (A) Oligosaccharides derived by heparitinase treatment of porcine mucosal heparin were separated using SAX-HPLC with a linear NaCl gradient (0-2 M NaCl). Fractions collected between 60 and 90 minutes were combined. (B) The combined sample from A was further purified using SAX-HPLC with a shallower NaCl gradient (0.8-1.3 M NaCl), and peaks 1 to 5 were isolated. The inset shows the complete elution profile. (C) The ability of peaks 1 to 5 at a concentration of 10 µM to induce the bypass phenotype. Results are expressed as a percentage of total embryos showing the bypass phenotype. Numbers in parentheses indicate total number of embryos.
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Fig. 7. Expression pattern of Xenopus HS2ST and HS6ST in the embryonic brain. (A,B) Lateral views of brains (stage 39) after whole-mount in situ hybridisation with antisense HS2ST (A) and HS6ST (B) RNA probes. Retinal axons are stained with HRP in (B). Arrows and arrowheads indicate the borders of the diencephalon/tectum and tectum/hindbrain, respectively. (C,D) Control embryos hybridised with sense probes.
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© The Company of Biologists Ltd 2002
