Enzymatic removal of chondroitin sulphates abolishes the age‐related axon order in the optic tract of mouse embryos

Abstract

Retinal axons undergo an age-related reorganization at the junction of the chiasm and the optic tract. We have investigated the effects of removal of chondroitin sulphate on this order change in mouse embryos aged embryonic day 14, when most axons are growing in the optic tract. Enzymatic removal of chondroitin sulphate but not keratan sulphate in brain slice preparations of the retinofugal pathway abolished the accumulation of phalloidin-positive growth cones in the subpial region of the optic tract. The loss of chronotopicity was further demonstrated by anterograde filling of single retinal axons, which showed a dispersion of growth cones from subpial to the whole depth of the tract. The enzyme treatment neither produced detectable changes in growth cone morphology and growth dynamic of retinal neurites nor affected the radial glial processes in the tract, indicating a specific effect of removal of chondroitin sulphate from the pathway to the axon order in the tract. Although chondroitin sulphate was also found at the midline of the chiasm, growth cone distribution across the depth of fibre layer at the midline was not affected by the enzyme treatment. These results suggest a mechanism in which retinal axons undergo changes in response to chondroitin sulphate at the chiasm-tract junction, but not at the midline, that produce a chronotopic fibre rearrangement in the mouse retinofugal pathway.

Heparan Sulphation Patterns Generated by Specific Heparan Sulfotransferase Enzymes Direct Distinct Aspects of Retinal Axon Guidance at the Optic Chiasm

“…The most obvious interpretation of this is that high levels of Hs2st and/or Hs6st1 are required at these locations to supply navigation cues to RGC axons. The chronotopic and retinotopic organization of the optic nerve means that younger axons navigate along the outside of the optic nerve as it approaches and enters the chiasm (Colello and Guillery, 1998;Leung et al, 2003), so cells surrounding the optic nerve and chiasm, in which we have detected high levels of Hs2st and/or Hs6st1 expression, are well placed to direct their growth. This is a plausible mechanism because HSPGs are known to bind to axon guidance molecules including Slit and netrin and so might be involved in tethering them at the chiasm in the correct configuration to direct RGC axons.…”

“…At the chiasm, RGC axons are sorted into the optic tracts and grow on toward their targets in the thalamus and superior colliculus. The topographical relationships between RGC axons and the tissues they traverse is tightly regulated throughout the developing visual pathway with axons executing a particularly intricate set of maneuvers as they approach, grow through, and exit the chiasm Colello and Guillery, 1998;Leung et al, 2003). Here we examine the contribution made to this process by the sulfation status of heparan sulfate proteoglycans (HSPGs).…”

Disruption of Sonic Hedgehog Signaling Affects Axon Routing in the Mouse Optic Chiasm

“…Brain slices containing the primary visual pathway were prepared using the protocol described in our previous reports [13,14] . Embryos aged from E13 to E15 were removed by cesarean section, decapitated and maintained in Dulbecco's modified Eagle's medium/F12 medium.…”

Selective inhibition of ventral temporal but not dorsal nasal neurites from mouse retinal explants during contact with chondroitin sulphate

“…The explants were then allowed to grow in DMEM/F12 supplemented with N1, 1% bovine serum albumin and 0.4% methylcellulose (all from Sigma) for 18 h at 37°C. The specificity of CS to neurite growth was examined in cultures treated with chondroitinase ABC (0.5 U/ml; Seikagaku, Japan), which has been demonstrated to be able to remove effectively the CS epitopes from the brain slice preparation of the mouse optic pathway (Chung et al 2000b) and produce no obvious alteration to the morphology and growth dynamics of retinal growth cones in vitro (Leung et al 2003). The enzyme was added at the start of the culture.…”