Ephrin-B regulates the Ipsilateral routing of retinal axons at the optic chiasm

Abstract

In Xenopus tadpoles, all retinal ganglion cells (RGCs) send axons contralaterally across the optic chiasm. At metamorphosis, a subpopulation of EphB-expressing RGCs in the ventrotemporal retina begin to project ipsilaterally. However, when these metamorphic RGCs are grafted into embryos, they project contralaterally, suggesting that the embryonic chiasm lacks signals that guide axons ipsilaterally. Ephrin-B is expressed discretely at the chiasm of metamorphic but not premetamorphic Xenopus. When expressed prematurely in the embryonic chiasm, ephrin-B causes precocious ipsilateral projections from the EphB-expressing RGCs. Ephrin-B is also found in the chiasm of mammals, which have ipsilateral projections, but not in the chiasm of fish and birds, which do not. These results suggest that ephrin-B/EphB interactions play a key role in the sorting of axons at the vertebrate chiasm.

Figure 1

“Old” Retinal Neurons from Ipsilaterally Projecting Region of Frog Retina Send Axons Contralaterally in Young Tadpoles (A and B) “Fillet” whole-mounted brains 2 days after the transplantation. Temporoventral retina of stage 64 frog was labeled with PKH26 and transplanted into temporoventral retina of stage 33/34 tadpole. Anterior is at the bottom; lateral is to the sides. More than 99% of axons navigate contralaterally as shown in (A), while less than 1% of axons go ipsilaterally as in (B). The dashed lines indicate the outline of the brains. (C–F) Conservation of the topographical mapping after the transplantation. Ventral retina of stage 58 frog was labeled and transplanted into dorsal (C and D) or ventral (E and F) retina of stage 33/34 tadpoles. Axons from the transplants (C and E) projected to the dorsal part of the tectum in both cases at stage 43. Whole retinal projections at stage 43 are labeled with GFP (D and F) following grafts of eye primordia from GFP-RNA-injected embryos into unlabeled hosts. Abbreviations: ite, ipsilateral tectum; cte, contralateral tectum; ch, chiasm; dt, dorsal tectum; vt, ventral tectum. Scale bars, 40 μm.

Figure 2

Thyroid Hormone Is Not Sufficient to Generate Ipsilateral Projection in the Early Tadpole Stage 52 (A–G) or stage 54 (H) tadpoles were reared in the absence (A, C, and D) or presence (B and E–H) of thyroxine (10⁻⁹ M) for 4 days. (A and B) Lateral view of whole-mount dissected brains. (C–F) Cryo-section of the marginal region of retina showing BrdU incorporation in the dorsal (C and E) or ventral (D and F) marginal retinas. (G and H) Transverse vibratome section at chiasm level. DiI was placed on a cut stump of optic nerve to label retinal projection. Arrowheads in (G) indicate the nonspecific ipsilaterally projecting axons in the deep optic pathway, which project to optic tectum on adjacent sections. Arrows in (H) indicate hormone-induced ipsilateral projection. Note that they are found close to the pial surface. Dashed lines in (G) and (H) indicate the midline. Abbreviaitons: fb, forebrain; ch, chiasm; te, tectum. Scale bars: 200 μm (A and B), 20 μm (C–F), 100 μm (G and H).

Figure 3

Ephrin-B Expression at the Chiasm Begins at Metamorphosis Expression pattern of ephrin-B was revealed by AP tag staining on transverse vibratome sections except for the inset in (B), which is ventral view. Arrowheads indicate the position of the chiasm midline. (A and B) Ephrin-B in stage 54 (A) and stage 60 (B) tadpoles. Inset in (A) shows the ephrin-B staining in the olfactory bulb of the same tadpole. These samples were processed under identical conditions at the same time. (C) Distribution of retinal axons at the chiasm. Sections were taken from the same stage and the same axial level as (B). The DiI-labeled retinal axons cross the ephrin-B-expressing region. Arrows indicate the ipsilaterally projecting axons. (D and E) Expression of ephrin-B in hormone-treated tadpoles. Stage 52 (D) or stage 54 (E) tadpoles were reared in the presence of thyroxine for 4 days. Abbreviation: on, optic nerve. Scale bars, 100 μm.

Figure 4

Receptors for Ephrin-B Are Expressed in the Ipsilaterally Projecting Region of the Retina Expression patterns of EphBs (A and B) or EphAs (C) in intact whole-mount retinas of stage 35/36 (A) and stage 62 (B and C) tadpoles revealed by AP tag staining. Arrows indicate the position of the ventral fissure. (D–G) Distribution of DAPI-positive ipsilaterally projecting cells (D and F) and immunolabeled EphB2-expressing cells (E and G) of stage 57 (D and E) or stage 66 (F and G) flat-mounted retinas. Asterisks in (D) and (F) indicate the position of the optic nerve head. The inset in (E) shows a higher magnification view of the optic nerve head (dashed box in [E]). (H–K) Double staining of EphB2 (red) and retrogradely transported DAPI (green). (H and I) Ipsilateral eyes. (J and K) Contralateral eyes. (H and J) Transverse cryo-sections of ventral retina. (I, K) Transverse cryo-sections of dorsal retina. Small arrows indicate EphB2 expression on RGC axons. Note that ipsilaterally projecting RGCs are located only in ventral retina (H) but not in dorsal retina (I), while contralaterally projecting cells are found both in ventral (J) and dorsal (K) retina. Dorsal is up in (A) through (G). Abbreviations: inl, inner nuclear layer; ipl, inner plexiform layer; gcl, RGC layer. Scale bars: 100 μm (A), 200 μm (B–G), 20 μm (H–K).

Figure 5

Ectopic Gene Expression in the Chiasm Region by Targeted Lipofection (A and B) DNA–lipid complex was injected into either the future chiasm region (A) or the retinal primordium (B). Arrows indicate the position of the tip of the injection needle. (C and D) Spatial distribution of the ectopic expression. GFP expression vector/lipid complex was microinjected into the chiasm (C) or retinal (D) region of stage 19 embryos. Transverse cryostat sections were stained with anti-GFP antibody. Insets show the retina at the level of the optic nerve head. (E–H) Time course of the ectopic expression in the chiasm region. Dashed lines indicate the midline of the ventral neural tube. HRP was injected into the retina to visualize the growing retinal axons (E and F). The tadpoles were fixed at stage 33/34 (E and G) or stage 41 (F and H) and double stained for HRP (E and F) and GFP (G and H). Abbreviations: te, tectum; ch, chiasm. Scale bars: 200 μm (A–D), 20 μm (G–H).

Figure 6

Ephrin-B Overexpression at the Chiasm Induces Precocious Ipsilateral Projection in the Early Tadpole (A–D) Double staining for the HRP-labeled retinal axons (red) and lipofected cells (green). GFP-myc (A and C) or ephrin-B2-myc (B and D) was lipofected at stage 19, and HRP was injected in the retina at stage 41. Arrows indicate the lipofected chiasm cells and arrowheads indicate the ipsilaterally projecting axons. Since this region of the chiasm is largely composed of crossing axons, only a few lipofected cells are observed on these sections. Insets in (A) and (B) show the tectal region of the ipsilateral brain. (E and F) DiI was placed on dorsal (E) or ventral (F) regions of the retina in the ephrin-B-lipofected tadpole. Arrowheads indicate ipsilaterally projecting axons to the tectum (top) and to the diencephalic nuclei (bottom), respectively. The dashed lines indicate the outlines of the brains. The orientation of the brains is the same as in Figures 1A and 1B. (G) Statistical analysis (Student’s t test) of the number of ipsilaterally projecting axons in the lipofected embryos. The ipsilateral optic tract was divided in three regions—dorsal (DOT), middle (MOT), and ventral (VOT)—and the number of axons was counted. Scale bars: 20 μm (C and D) (insets in [A] and [B]), 100 μm (A, B, E, and F).

Figure 7

Expression of Ephrin-B at the Chiasm of Other Species from Ventral View Expression pattern of ephrin-B was revealed by AP tag staining on horizontal vibratome sections (A, C, and D) or dissected whole-mount ventral diencephalon (B). In all cases, anterior is top and posterior is bottom. (A) 48 hr zebrafish. (B) E8 chicken. (C) E13.5 mouse. (D) E16.5 mouse. The arrowheads indicate the position of the chiasm. Abbreviation: hy; hypothalamus. Scale bars: 20 μm (A), 200 μm (B–D).