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Heparan sulfate (HS), a structurally diverse molecule comprising distinct sequences of sulfated disaccharide units, is abundant in the developing brain and binds to axon guidance molecules. Addition of HS to the developing Xenopus optic pathway causes severe targeting errors yet it is not known how the structural diversity of this molecule relates to its role in axon guidance. We have used an in vivo brain assay to identify the structural characteristics of HS that induce aberrant axon targeting. Inhibiting sulfation of endogenous HS with chlorate causes axons to bypass their target, the tectum, and treatment with chemically modified heparins reveals that 2-O- and 6-O-sulfate groups have potent bypass-inducing activity. Experiments with purified heparin saccharides show that bypass-inducing activity correlates with distinct structures, particularly those containing a combination of 2-O- and 6-O-sulfate groups. Taken together the results indicate that specific sequences, rather than gross structural composition, are critical for activity. In situ hybridisation revealed that HS 6-O-sulfotransferase is regionally expressed along the border of the dorsal optic tract whereas 2-O-sulfotransferase is expressed broadly. Our results demonstrate that specific HS sequences are essential for regulating retinotectal axon targeting and suggest that regionalised biosynthesis of specific HS structures is important for guiding axons into the tectum.
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.
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 anteriortectum (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.
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.
