Roles of the telencephalic cells and their chondroitin sulfate proteoglycans in delimiting an anterior border of the retinal pathway

Abstract

The axons of the retinal ganglion cells run on the diencephalotelencephalic boundary on their way to the tectum; however, they do not invade the telencephalon anteriorly. To investigate the mechanisms that prevent the retinal axons from entering the telencephalic territory, the effects of the telencephalic cells were examined on the outgrowth of the retinal axons in vitro; the retinal outgrowth was selectively inhibited by the cellular substrate derived from the telencephalon. The responsible factor for the selective inhibition was, furthermore, found in the telencephalic membranes and the fraction of peripheral membrane molecules from the telencephalon. Because the inhibitory effect was destroyed by chondroitinase ABC but not by heat, this inhibition was attributable to the carbohydrate chains of chondroitin sulfate proteoglycans (CSPGs) adhering to the membranes of the telencephalic cells. To understand the function of the telencephalic CSPGs on the retinal pathfinding in vivo, their carbohydrate chains [chondroitin sulfate glycosaminoglycan (CS-GAG)] were removed from the embryonic brains by intraventricular injection of chondroitinase ABC; the removal of CS-GAG resulted in an anterior enlargement of the optic tract. The results indicate that the telencephalic cells delimit the anterior border of the optic tract with their CSPGs and prevent the retinal axons from aberrantly entering the anterior territory.

Fig. 1.

Axonal trajectories of the retinal ganglion cells of E7 chickens. A, The retinal axons run on the diencephalon dorsocaudally to the tectum. Chiasm, Optic chiasm; Dien, diencephalon; Tectum, optic tectum; Tel, telencephalon. B, In the anterior diencephalon, they run on the diencephalotelencephalic boundary (arrowhead) but do not enter the telencephalic territory. Positions of the following axonal scaffolds are indicated:DVDT, the dorsoventral diencephalic tract;TPOC, the tract of the postoptic commissure;SOT, the supraoptic tract. Scale bars, 50 μm.

Fig. 2.

Schematic drawings of the experiments in theryomen chamber and the telencephalic substrates.A, A pair of stainless steel rings holds the Nuclepore filter. B, An axon of a retinal ganglion cell grows on the processes of telencephalic cells that have penetrated the filter.C, A scanning electron micrograph shows the telencephalic substrate on the retinal side of the filter. The telencephalic substrates consist of cellular processes that are positive for MAP2 (D), vimentin (E), and G4/NgCAM (F). Scale bars, 25 μm.

Fig. 3.

Axonal outgrowth on the substrates in theryomen chambers. The axons are observed from the retinal side of the filter after 3 d. The black areas in the middle of the pictures are the nitrocellulose filters that mechanically support the retinal tissues (A, B, D,E). Few retinal axons grow on the E6 (A) and E8 (B) telencephalic substrates, although they grow well on the E6 (D) and E8 (E) anterior tectal substrates. The DRG axons grow well on either the E8 telencephalic substrate (C) or E8 anterior tectal substrate (F). Scale bars, 300 μm.

Fig. 4.

Axonal outgrowth in the conditioned media. The retinal (A–D) and DRG (E–H) explants are cultured in the F-12 culture medium (A, E), in the medium conditioned by the E8 anterior tectal cells (B,F), in the medium conditioned by the E8 telencephalic cells (C, G), and in the heat-treated medium conditioned by the E8 telencephalic cells (D, H). Scale bars, 300 μm.

Fig. 5.

Axonal outgrowth in the media with the membrane fractions or peripheral membrane molecules. The retinal (A–C, G–I) and DRG (D–F, J–L) explants are cultured in the F-12 minimum medium (A, D), in the medium with the anterior tectal membranes (B,E), in the medium with E8 telencephalic membranes (C, F), in the medium with peripheral membrane molecules extracted from E8 anterior tectal membranes (G, J), in the medium with peripheral membrane molecules extracted from E8 telencephalic membranes (H, K), and in the medium with E8 telencephalic membranes treated with chondroitinase ABC (I, L). Scale bar, 300 μm.

Fig. 6.

Outgrowth of the retinal axons in the experimentsin vitro. A, The diameters of axonal halos are shown on the substrates in the ryomenchambers. Matrigel, The Nuclepore filter coated with the Matrigel solution; AT-E6, the E6 anterior tectal substrate; Tel-E6, the E6 telencephalic substrate;AT-E8, the E8 anterior tectal substrate;D-E8, the E8 diencephalic substrate;Tel-E8, the E8 telencephalic substrate.B, The total areas covered with the axonal bundles are shown in the conditioned media. F12CM, The F-12 culture medium; AT, the medium conditioned by the E8 anterior tectal cells; Tel, the medium conditioned by the E8 telencephalic cells; Tel-H, the heat-treated medium conditioned by the E8 telencephalic cells. C, The total areas covered with the axonal bundles are shown in the media with the membrane fractions or peripheral membrane molecules.F12MM, The F-12 minimum medium; AT, the medium with the E8 anterior tectal membranes; E8-Tel, the medium with the E8 telencephalic membranes; E13-Tel, the medium with the E13 telencephalic membranes; heat-Tel, the medium with the E8 telencephalic membranes treated with heat; chondroitinase ABC-Tel, the medium with the E8 telencephalic membranes treated with chondroitinase ABC; PM-Tel, the medium with the peripheral membrane molecules from the E8 telencephalon.Asterisks indicate the significant differences from theATs in each graph (p < 0.01).

Fig. 7.

The enzymatic removal of CS-GAG and its effect on the trajectories of the retinal axons. A, Distribution of CS-GAG is immunofluorescently shown on a horizontal section around the diencephalotelencephalic boundary in the control E7 chicken treated with the inactivated chondroitinase ABC. C, The cytoarchitecture of the junctional region is shown by double-staining with DAPI. B, Removal of CS-GAG and its residual distribution are shown in the E7 chicken treated with chondroitinase ABC. D, The cytoarchitecture is shown by double-staining with DAPI. The distribution of CS-GAG is not influenced in the neural retina in the E7 chicken treated with chondroitinase ABC; the immunofluorescent signal is intense in the peripheral retina (E), although it is weak in the central retina (F). G, The retinal axons labeled with HRP are shown on a horizontal section around the diencephalotelencephalic boundary in the E7 embryo treated with the inactivated chondroitinase ABC. I, The cytoarchitecture is shown with DAPI; the retinal axons are situated posterior to the groove (arrows) above the thin layer of cells on the SOT. H, The retinal axons labeled with HRP are shown in the E7 chicken treated with chondroitinase ABC. J, The cytoarchitecture is shown with DAPI; the retinal axons are situated over the anterior groove (arrows) above the thin layer of cells on the SOT. Dien, Diencephalon;Tel, telencephalon; SOT, the supraoptic tract. The anterior sides are left (A–D,G–J). The exposures in the photographs are the same between A and B, Cand D, E and F,G and H, and I andJ. Scale bars: A–D, 25 μm;E–J, 50 μm.

Fig. 8.

Anterior enlargement of the retinal trajectories at the diencephalotelencephalic boundary by the enzymatic removal of CS-GAG. Relative positions of the most anteriorly situated retinal axons to the groove above the thin layer of cells on the SOT, the diencephalotelencephalic boundary, are plotted as filled diamonds in the embryos treated with the inactivated enzyme (−) or the chondroitinase ABC (+). The mean values are also indicated (open diamonds). DTB, The diencephalotelencephalic boundary.