Retinal ganglion cell axon guidance in the mouse optic chiasm: expression and function of robos and slits

Abstract

The ventral midline of the nervous system is an important choice point at which growing axons decide whether to cross and project contralaterally or remain on the same side of the brain. In Drosophila, the decision to cross or avoid the CNS midline is controlled, at least in part, by the Roundabout (Robo) receptor on the axons and its ligand, Slit, an inhibitory extracellular matrix molecule secreted by the midline glia. Vertebrate homologs of these molecules have been cloned and have also been implicated in regulating axon guidance. Using in situ hybridization, we have determined the expression patterns of robo1,2 and slit1,2,3 in the mouse retina and in the region of the developing optic chiasm, a ventral midline structure in which retinal ganglion cell (RGC) axons diverge to either side of the brain. The receptors and ligands are expressed at the appropriate time and place, in both the retina and the ventral diencephalon, to be able to influence RGC axon guidance. In vitro, slit2 is inhibitory to RGC axons, with outgrowth of both ipsilaterally and contralaterally projecting axons being strongly affected. Overall, these results indicate that Robos and Slits alone do not directly control RGC axon divergence at the optic chiasm and may additionally function as a general inhibitory guidance system involved in determining the relative position of the optic chiasm at the ventral midline of the developing hypothalamus.

Fig. 1.

Anatomy of the region of the developing optic chiasm during the major period of RGC divergence.A, Head of an E14.5 mouse embryo indicating the planes of section used in this study. B, Schematic diagrams of horizontal and coronal sections through the optic chiasm of an E14.5 mouse embryo. The path of crossed and uncrossed RGC axons is shown in relation to the specialized cell types (radial glia, dark gray hatching; CD44/SSEA neurons, light gray) present in this region. All axons grow into and contact the radial glia before either crossing the midline, at the tip of the SSEA-1 positive region, or turning back into the ipsilateral optic tract (Marcus et al., 1995). D, Dorsal; V, ventral;A, anterior; P, posterior;N, nasal; T, temporal.

Fig. 2.

Expression patterns of robos andslits in the developing mouse retina. Coronal sections of E12.5 (A–E), E14.5 (F–J), and E17.5 (K–O) retinas stained with an antibody against the LIM homeodomain proteins, Islet 1/2 (an early RGC marker; A, F, K) or by in situ hybridization using digoxigenin-labeled riboprobes for robo1 (B,G, L), robo2(C, H, M)slit1 (D, I,N), or slit2 (E,J, O). Boxed regions inG, H, and O are shown at higher power in P, Q, andR respectively. Dotted line inR marks the central lumen of the retina. Scale bar:A–O, 500 μm; P, R, 80 μm. Dorsal, Top; ventral, bottom.

Fig. 3.

Expression of robos andslits in the ventral diencephalon of E12.5 mouse embryos. A–D, Serial horizontal sections showing both DiI-labeled RGC axons (brown) and SSEA-1 (black). In the most dorsal section (A), no RGC axons are present.Asterisk marks the third ventricle. More ventrally (B), a few axons can be seen at the junction of the optic stalk and the brain (arrow). InC and D, the region of the future optic chiasm, RGC axons have entered the diencephalon and appear to be growing along the border of the SSEA-1-positive cells but have not yet crossed the midline. E–T, Comparable serial sections with those in A–D after in situ hybridization to show patterns of robo1(E–H), robo2(I–L), slit1(M–P), and slit2(Q-T) expression. Orientation is the same as in Figure 1B (anterior, top; posterior, bottom). Scale bar, 500 μm.

Fig. 4.

Expression of robos andslits in the ventral diencephalon of E14.5 mouse embryos. A–D, Serial horizontal sections double labeled with DiI to show the RGC axons (brown) and SSEA-1 (black), which marks the CD44/SSEA neurons posterior to the optic chiasm. In the more dorsal sections (A, B), RGC axons are present in the optic nerve (A) and at the junction of the optic nerve and the brain (B). The axons then grow more ventrally before diverging to form the x-shaped optic chiasm (C, D). The site at which the axons diverge is marked by a thin raphe of the CD44/SSEA neurons.E–T, Comparable serial sections with those inA–D after in situhybridization to show patterns of robo1(E–H), robo2(I–L), slit1(M–P), or slit2(Q–T) expression. U–W, Coronal sections labeled with photoconverted DiI to show the RGC axons (U), the monoclonal antibody RC2 (labels radial glia; V), or after in situhybridization for slit2(W). Asterisks inV and W marks the RGC axons.Arrows point to strong staining of RC2 andslit2 in the radial glial cell bodies. Orientation is the same as in Figure 1B. Scale bar, 500 μm.

Fig. 5.

Expression of robos andslits in the ventral diencephalon of E17.5 mouse embryos. A, Horizontal section, at the level of the optic chiasm, double labeled to show both the RGC axons (brown) and SSEA-1 (black).B–E, Comparable sections after in situhybridization to show the patterns of expression ofrobo1 (B), robo2(C), slit1(D), or slit2(E). Anterior, Up; posterior,down. Scale bar, 250 μm.

Fig. 6.

Effect of hSlit2 on RGC axon outgrowth in vitro. A, Schematic diagram of a flat-mounted E14.5 mouse retina. RGCs that project contralaterally are found throughout the retina, whereas ipsilaterally projecting cells are restricted to the ventrotemporal crescent (black region). To enable a comparison of the behavior of crossed and uncrossed RGC axons, explants were prepared only from the most peripheral part of each retinal quadrant. Consequently, growth from these explants was not radial but originated only from the cut edge (indicated by the dotted lines). D, Dorsal; V, ventral; N, nasal; T, temporal. B–E, Explants from dorsotemporal retina cultured alone (B), with clusters of mock-transfected cells (C), or with clusters of cells transfected with hSlit2 (D,E). Explant in E is oriented such that growth is directed away from the Slit-expressing cells. Scale bar, 500 μm. F, Extent of RGC axon outgrowth from explants cocultured with mock-transfected (open bars) or hSlit2-expressing COS cells (filled bars).Numbersabove bars indicates number of explants. *p < 0.001 compared with growth in the presence of the mock-transfected cells (Student's unpairedt test). Data were pooled from five independent experiments. G, Extent of RGC axon outgrowth in the presence of hSlit2 expressed as a percentage of the outgrowth seen in cultures containing mock-transfected cells.