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Development
1997 Jun 01;12412:2421-30. doi: 10.1242/dev.124.12.2421.
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Essential role of heparan sulfates in axon navigation and targeting in the developing visual system.
Walz A
,
McFarlane S
,
Brickman YG
,
Nurcombe V
,
Bartlett PF
,
Holt CE
.
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Heparan sulfate (HS) is abundant in the developing brain and is a required co-factor for many types of fibroblast growth factor (FGF) signaling in vitro. We report that some HSs, when added exogenously to the developing Xenopus optic pathway, severely disrupt target recognition causing axons from the retina to bypass their primary target, the optic tectum. Significantly, HS sidechains from a neuroepithelial perlecan variant that preferentially bind FGF-2, HS(FGF-2), cause aberrant targeting, whereas those that preferentially bind FGF-1 do not. Charge-matched fragments of HS(FGF-2) show that the mistargeting activity associates with the FGF-binding fragments. Heparitinase removal of native HSs at the beginning of optic tract formation retards retinal axon elongation; addition of FGF-2 restores axon extension but axons lose directionality. Late HS removal, after axons have extended through the tract, elicits a tectal bypass phenotype indicating a growth promoting and guidance function for native HSs. Our results demonstrate that different HS sidechains from the same core protein differentially affect axon growth in vivo, possibly due to their distinct FGF-binding preferences, and suggest that growth factors and HSs are important partners in regulating axon growth and guidance in the developing visual system.
Fig. 1. Pattern of native HS staining in the embryonic optic pathway. Confocal micrographs of transverse sections of the embryonic brain (left side) immunostained for HS using HepSS-1 antibody. (A) HS immunostaining is localized to a narrow band close to the pial surface of the diencephalon (Di) at stage 35/36 and is not present in the tectum (Tec). (B) Absence of HS staining following exposure of optic tract to heparitinase from stage 32 to 40. (C,D) Double immunolabeled section showing the earliest population of retinal ganglion cell axons (red) growing in the HS-rich region (green) close to the pial surface of the diencephalon. The rhodamine filter only was used in C. Np, neuropil; on, optic nerve. Dorsal is up, lateral is left. Scale bar, 100 μm (A,B) and 50 μm (C,D).
Fig. 2. Exogenously applied FGF-2-binding HS disrupts target recognition. Lateral views of wholemount brains at stage 40 showing the trajectories of HRP-filled optic projections. Brains were exposed to different types of HSs at 0.1 mg/ml for a duration of 18-24 hours, from stage 33/34 to 40. B,D,F are higher power views of A,C and E respectively. (A,B) Control projection forms a defined optic tract (ot) in the diencephalon (Di) and crosses smoothly into the tectum (Tec). Dotted line and arrow demarcate the anterior tectal border. (C,D) Projection exposed to heparin extends correctly through the diencephalon but fails to enter the tectum. The projection bifurcates at the entrance to the tectum and axons course dorsally and posteriorly around the target. (E,F) Retinal axons growing in the presence of the full length HS(FGF-2) veer dorsally at the tectal border and bypass the tectum. (G) FGF-2-binding fragments of the full length HS(FGF-2) also cause axons to grow past the target without entering it, whereas (H) non-FGF-2-binding fragments of HS(FGF-2) have no effect. Brains exposed to HS(FGF-1) (I) and to a non-FGF-2-binding full length HS (J) have normal optic projections that enter the tectum. Tel, telencephalon; dorsal is up, anterior to the left. Scale bar, 200 μm in A,C and E and 100 μm in B,D and F-J.
Fig. 5. Gross organization of neuroepithelium is unchanged by heparin treatment. Embryos were exposed to either control or heparin solutions (0.1 mg/ml) at stage 33/34 and examined at stage 40. (A,B) Confocal images of control (A) and heparin (B) treated brain sections stained with anti-NCAM. NCAM is abundant in the neuropil (Np) as well as in the neuroepithelium and is unaffected by heparin treatment. Tec, tectum, exposed side is to the left, dorsal is up. Scale bar, 50 μm. (C,D) Major axonal tracts are not changed by heparin treatment as seen with anti-acetylated tubulin staining in wholemount control (C) and heparin-treated (D) brains. tAC, tract of the anterior commissure; tPC, tract of the posterior commissure; tPOC, tract of the post-optic commissure. Dorsal is up, anterior to the left. Scale bar, 100 μm.
Fig. 6. Effects of enzymatic removal of native HS on retinal axon growth. (A) Control brain showing a normal optic projection at stage 37/38. (B) arlyheparitinase treatment during the period of axon extension through the optic tract, stage 32 to 37/38, causes abnormally short projections that fail to reach the tectum. (C) When FGF-2 (100 ng/ml) is added concomitant with heparitinase, the growth inhibition is alleviated and axons extend to normal lengths. However, many pathfinding errors occur with axons veering off the optic tract into the telencephalon and diencephalon (arrowheads) and axons bypass the tectum (arrow). (D) ateheparitinase treatment during the period of initial tectal invasion, stage 36/37 to 40, causes the bypass phenotype typical of defective target recognition. Tel, telencephalon; Di, diencephalon; Tec, tectum; ot, optic tract; arrows depict anterior border of the tectum. Scale bar, 200 μm.
