The growth-inhibitory protein Nogo is involved in midline routing of axons in the mouse optic chiasm.

We have investigated the role of Nogo, a protein that inhibits regenerating axons in the adult central nervous system, on axon guidance in the developing optic chiasm of mouse embryos. Nogo protein is expressed by radial glia in the midline within the optic chiasm where uncrossed axons turn, and the Nogo receptor (NgR) is expressed on retinal neurites and growth cones. In vitro neurite outgrowth from both dorsonasal and ventrotemporal retina was inhibited by Nogo protein, and this inhibition was abolished by blocking NgR activity. In slice cultures of the optic pathway, blocking NgR with a peptide antagonist produced significant reduction in the uncrossed projection but had no effect on the crossing axons. This result was confirmed by treating cultures with an anti-Nogo functional blocking antibody. In vitro coculture assays of retina and optic chiasm showed that NgR was selectively reduced on neurites and growth cones from dorsonasal retina when they contacted chiasm cells, but not on those from ventrotemporal retina. These findings provide evidence that Nogo signaling is involved in directing the growth of axons in the mouse optic chiasm and that this process relies on a differential regulation of NgR on axons from the dorsonasal and ventrotemporal retina.

Localization of Nogo and its receptor in the optic pathway of mouse embryos.

We have investigated the localization of Nogo, an inhibitory protein acting on regenerating axons in the adult central nervous system, in the embryonic mouse retinofugal pathway during the major period of axon growth into the optic chiasm. In the retina, Nogo protein was localized on the neuroepithelial cells at E12 and at later stages (E13-E17) on radial glial cells. Colocalization studies showed expression of Nogo on vimentin-positive glia in the retina and at the optic nerve head but not on most of the TuJ1- and islet-1-immunoreactive neurons. Only a few immature neurons in the ventricular and peripheral regions of the E13 retina were immunoreactive to Nogo. In the ventral diencephalon, Nogo was expressed on radial glia, most strongly on the dense radial glial midline raphe within the chiasm where uncrossed axons turn and in the initial segment of the optic tract. In vitro studies showed that the Nogo receptor (NgR) was expressed on the neurites and growth cones from both the ventral temporal and dorsal nasal quadrant of the retina. In the optic pathway, NgR staining was obvious in the vitreal regions of the retina and on axons in the optic stalk and the optic tract, but not in the chiasm. These expression patterns suggest an interaction of Nogo with its receptor in the mouse retinofugal pathway, which may be involved in guiding axons into the optic pathway and in governing the routing of axons in the optic chiasm.

Enzymatic removal of hyaluronan affects routing of axons in the mouse optic chiasm.

Perturbations of interaction of hyaluronan (HA) with its receptor CD44 cause multiple errors in axon routing at the mouse optic chiasm. To investigate this interaction further on the chiasm routing, we studied the axon routing after enzymatic removal of HA from slice preparations of the optic pathway. Hyaluronidase treatment produced an obvious reduction in midline crossing of the first generated axons in E13 chiasms, but had no influence on routing ofthe uncrossed axons in E15 and E16 slices. These findings support a direct role of HA, acting probably through CD44, on axon decussation during early phase of chiasm development, but argue against a direct function of HA on the turning of uncrossed axons in the mouse optic chiasm.

Effects of exogenous hyaluronan on midline crossing and axon divergence in the optic chiasm of mouse embryos.

Perturbation of the transmembrane glycoprotein, CD44, has been shown to cause multiple errors in axon routing in the mouse optic chiasm. In a recent report we have shown that the major CD44 ligand, hyaluronan (HA), is colocalized with CD44 at the midline of the chiasm, suggesting a possible contribution to the control of axon routing in the chiasm. We examined this issue by investigating the effects of exogenous HA on routing of axons in the chiasm in slice preparations of the optic pathway. In preparations of the E13 optic pathway, administration of exogenous HA produced a dose-dependent failure in midline crossing of the first generated optic axons. In E15 slices, when the adult pattern of axon divergence develops in the chiasm, anterograde filling of the optic axons showed an obvious reduction in the uncrossed pathway after HA treatment. This reduction was confirmed by retrograde filling of the ganglion cells in E15 slices, and later in E16 pathways where the uncrossed projection is better developed. Furthermore, we have demonstrated in explant cultures of the retina that HA, when presented in soluble or substrate-bound form, does not affect outgrowth and extension of retinal neurites. These findings together indicate the crucial functions of this matrix molecule in regulating midline crossing and axon divergence, probably through interactions with guidance molecules including CD44, at the midline of the chiasm.

Localization of hyaluronan in the optic pathway of mouse embryos.

CD44 has been shown to be involved in midline crossing and the generation of ipsilateral projections in the mouse optic chiasm. To determine whether these functions involve hyaluronan, the major ligand of CD44, we examined localization of hyaluronan in the mouse optic pathway. Hyaluronan was deposited mainly in vitreal regions of the retina and the optic disk. In ventral diencephalon, it was localized largely on the chiasmatic neurons that project processes to the chiasmatic midline and the optic tract. Colocalization of hyaluronan and CD44 was observed only in the midline but not lateral domains of the chiasmatic neurons, suggesting a hyaluronan/CD44-mediated mechanism that controls axon routing at the midline but not at the optic tract and the retina.