PMC

The glia of the outer and inner chiasm. A.1: Schematic of the optic lobes and their chiasms. The outer chiasm connects lamina and distal medulla, the inner chiasm connects proximal medulla, lobula and lobula plate. A.2: Labeling of chiasm glia (XG) in outer (XGO) and inner (XGI) chiasm; 1 µm section. B.1,2: XG and EGN are distinct populations in outer chiasm; B.1: 15 µm projection; B.2: 4 µm projection. C.1–5: Individual XG cell morphologies in outer chiasm; C.1: 30 µm projection; C: 2,3: single sections. Neuronal tracts are enveloped by XGO (arrows in C.2,3). C.4,5: 3D reconstruction of C.1, view from medial medulla into chiasm (C.4), view onto lamina portion of chiasm (C.5). Different fibers crossing the chiasm at perpendicular angles can be ensheathed by a single XGO cell. D.1–4 Individual XG cell morphologies in inner chiasm; D.1: 93 µm projection; D.2: 60 µm projection. D.1: A single XGI cell may extend its sheath‐like processes along the entire coronal plane of the inner chiasm. D.2: XGI send many long protrusions (open arrows) into all three neighboring neuropiles. Most XGI cell bodies lie outside the chiasm (filled arrow). D.3,4: 3D reconstruction of D.1, view of the dorso‐ventral axis showing the multilayered structure of the chiasm (D.3); D.4: cross section through D.3, XGI envelop neuronal tracts. Scale bar = 10 µm in all images.

A. Schematic demonstrating the stereotypical extension of ipsilateral (dark green) and contralateral axons (light green) across the optic chiasm in wild-type mice.
B. Schematic demonstrating different misrouting defects of ipsilateral axons (orange) and contralateral axons (red) in animals lacking HS modifications or CS. Specific HS modifications guide contralateral retinal axons towards the optic tract. HS 2-O-sulfation is required to maintain axons organized in bundles and at the chiasm. HS 6-O-sulfation is required to prevent axons from extending away from the chiasm and the optic tract. Asterisks denote defects observed in a double mutant with Slit1. CS inhibits the crossing of ipsilateral axons at the midline in the optic chiasm and repels contralateral axons towards the optic tract.
C. Schematic showing the expression of HS/CS glycans with important morphogens at the optic chiasm. The expression pattern of particular HS epitopes in and around the chiasm is highlighted: HS 2-O-sulfated moieties (indicated as green bubbles) are abundant throughout the optic chiasm, and high levels of this modification concentrate at regions where the optic nerve reaches the brain; HS 6-O-sulfated moieties (indicated as red bubbles) show a unique expression pattern at the chiasm, concentrating at the borders of this structure. Slit1 and Slit2 (colored hexagons) expression correlates with the HS 2-O-sulfated and HS 6-O-sulfated structures at the chiasm, respectively. The interaction between this network of genes guides retinal axons in a context-dependent manner. Similar to the expression pattern observed at the dorsal and ventral borders in HS 6-O-sulfated glycans, Shh (blue circles) is found off the chiasm. CS glycans (blue chains) are found at the midline of the chiasm, sharing a similar expression pattern to the EphB2 ligand (triangles). Both of these molecules are crucial for the turning of ipsilateral axons, hinting at a possible interaction between them.

(A) Sema6D and Nr-CAM are expressed in radial glia at the chiasm midline at E15.5, but Sema6D extends further dorsally in the VZ. (B) Sema6D mRNA is expressed in Nr-CAM⁺ (green)/RC2⁺ (red) radial glia at E14.5. Retinal axons stained with neurofilament antibody (NF, blue) extend through radial glial processes. (C) Sema6D mRNA is expressed in radial glial cell bodies at the floor of the third ventricle in the optic chiasm and along the ventricular zone at E13.0, E15.5 and E17.5. (D) Plexin-A1 expression at the E15.5 optic chiasm in cells in the ventral diencephalon. In frontal sections, Plexin-A1 protein (green) is colocalized with SSEA-1 (red) in chiasmatic neurons at the midline raphe and also on neurofilament⁺ retinal axons (blue). (E) Schema of Sema6D and Plexin-A1 expression at the optic chiasm, depicted in frontal planes. Note that Sema6D is expressed by Nr-CAM⁺/ephrin-B2⁺ radial glial cells, and Plexin-A1 is expressed by SSEA-1⁺ chiasm neurons. Scale bars: 100µm.

Quantitation of optic chiasm width in Hs2st;Hs6st1 compound mutants. Labelling of optic nerve following unilateral DiI injection (denoted by *) into E15.5 eyes. Labelled axons appear white in these grey-scale images and reveal the anatomy of the optic chiasm in (A) wild-type, (B) Hs2st^−/− and (C) Hs6st1^−/− embryos. In Hs2st^−/− mutants, the optic chiasm appeared wider along the rostro-caudal midline due to the formation of an ectopic tract rostral to the chiasm (white arrow in B). In mutants lacking Hs6st1 sulphation the optic chiasm appeared wider along the rostro-caudal midline due to ectopic axon navigation along the caudal midline of the ventral diencephalon (white arrow in C). The width of the optic chiasm, taken to consist of any DiI RGC axon that could be detected using confocal microscopy, was measured along the midline (red dotted line). (F) Graph showing the width (mean ± SEM) of the optic chiasm in wild-type and compound mutant embryos. The optic chiasm was significantly wider in both the Hs6st1^−/− mutant (435 ± 25 μm, n = 5) and Hs2st^−/− mutant (448 ± 30 μm, n = 7) than in wild-types [345 ± 51 μm, n = 5; one way anova, multiple comparisons vs. a control group (Holm–Sidak method), P < 0.05]. The optic chiasm in Hs2st^−/−;Hs6st1^+/− mutants (n = 4) was 438 ± 45 μm wide, and in Hs2st^+/−;Hs6st1^−/− mutants (n = 3) it was 447 ± 44 μm wide. These values are not significantly different to those obtained in Hs2st^−/− and Hs6st1^−/− single mutants, respectively. OC, optic chiasm; ON, optic nerve; OT, optic tract. Scale bars: 200 μm.