(A) Gradient-matching model. Graded expression of EphA receptors (blue) in both the retina and primary visual cortex (V1) are used to guide topographic mapping in the superior colliculus (SC), which expresses repulsive ephrin-A ligands (gray) in a gradient in both recipient layers. N, nasal; T, temporal; A, anterior; P, posterior; D, dorsal; V, ventral; L, lateral; M, medial; uSGS, upper stratum griseum superficiale; lSGS, lower stratum griseum superficiale.
(B) Retinal-matching model. Retinocollicular mapping is established first through the use of graded EphAs and ephrin-As. Then, V1 projection neurons terminate in areas with similar activity patterns or with RGCs expressing complementary cell surface molecules.

(A and B) Parasagittal sections of the SC after focal injection of DiI (red) in V1 and whole eye fill with CTB-488 (green) in the contralateral eye, which labels all RGCs. In WT mice lateral V1 injections result in TZs in the anterior SC, while medial V1 injections give rise to TZs in the posterior SC. inserts, schematic of V1 injection site; bar, 500 μm; L, lateral; M, medial; A, anterior; D, dorsal; *, pretectal nucleus.
(C) Corticocollicular TZ location expressed as a percent of SC anterior-posterior axis plotted against the V1 injection site, expressed as percent of the lateral-medial axis of the cortical hemisphere. Line represents best-fit regression, R² = 0.9135. n = 23
(D and E) Parasagittal SC sections after focal injection of DiI (red) in V1 and whole eye fill with CTB-488 (green) in the contralateral eye, which labels all RGCs. In EphA3^ki/ki mice lateral V1 injections result in two termination zones in the anterior and central SC, while medial injections result in two termination zones in the central and posterior SC. inserts, schematic of V1 injection site; bar, 500 μm; *, pretectal nucleus.
(F) Corticocollicular TZ location expressed as a percent of SC A-P axis plotted against the V1 injection site expressed as percent of the lateral-medial axis of the cortical hemisphere. Line represents best-fit regression, R² = 0.7828 and 0.7131 for posterior (blue) and anterior (red) TZs, respectively; n = 18.
(G) Quantification of corticocollicular TZ overlap with the retinal recipient layer in WT and EphA3^ki/ki mice. Data are represented as mean +/− SEM, n > 10.
(H) Quantification of corticocollicular TZ area expressed as a percent of SC area in WT and EphA3^ki/ki mice. Data are represented as mean +/− SEM, n = 18. **, p < 0.01 by ANOVA and Tukey's HSD post-hoc analysis.

(A–D) Parasagittal SC sections after focal injection of DiI (white) in V1. In WT pups corticocollicular axons were observed as early as postnatal day 6 (P6), and a broad TZ was apparent by P8, which refined further at P10 and completed by P12. bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.
(E and F) Whole mount SC images after focal injection of DiI (white) in nasal retina. In WT mice, a broad retinocollicular TZ was apparent at P6 (E), which was fully refined by P8 (F). bar, 500 μm; M, medial; P, posterior.
(G) Quantification of TZ size as a percent of the SC. Data are represented as mean +/− SD, n > 3 for each group.
(H–K) Parasagittal SC sections after focal injection of DiI (white) in V1. In EphA3^ki/ki pups, corticocollicular axons were observed at P6, and broad TZs were apparent by P8, which refined further at P10 and were complete by P12. bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.

(A–D) Intrinsic optical imaging signal obtained from V1 (A and B) and SC (C and D) of WT adult mice presented a drifting bar stimulus along the azimuth (A and C) or elevation (B and D) axis. bar, 500 μm.
(E–H) Intrinsic optical imaging signal obtained from V1 (E and F) and SC (G and H) of EphA3^ki/ki animals presented a drifting bar stimulus along the azimuth (E and G) or elevation (F and H) axis. bar, 500 μm.

(A–C) Parasagittal SC sections after focal injection of DiI (white) in V1. In Math5^−/− mice, injections in lateral, central and medial V1 result in broad TZs in anterior, central and posterior SC, respectively. inserts, schematic of V1 injection site; bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.
(D–F) Parasagittal SC sections after focal injection of DiI (white) in V1. In adult WT mice that were enucleated at P6, injections in lateral, central and medial V1 result in broad TZs in anterior, central and posterior SC, respectively. inserts, schematic of V1 injection site; bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.
(G) Quantification of TZ size as a percent of the SC in WT, Math5^−/−, and enucleated mice. Data are represented as mean +/− SEM, n > 3 for each group; since data from anterior and posterior TZs were not significantly different (p = 0.4, n > 3), these data were pooled. ***, p < 0.001 vs. WT; **, p < 0.01 vs. WT, Kolmogorov-Smirnov test.
(H) Corticocollicular TZ location expressed as a percent of SC anterior-posterior (A-P) axis was plotted against the V1 injection site expressed as percent of the lateral-medial axis of the cortical hemisphere for Math5^−/− (gray squares) and enucleated mice (open triangles). Line represents best-fit regression, R² = 0.9139 for Math5^−/− and R² = 1 for enucleated.

(A) Whole mount SC after focal injection of DiI (white) in nasal retina. In EphA3^ki/ki mice, two distinct retinocollicular TZs were observed (arrowheads) in the appropriate topographic postitions. bar, 500 μm; M, medial; P, posterior
(B) Parasagittal section of the SC in (A) revealing two distinct retinocollicular TZs (arrowheads). insert, schematic of retinal injection site; bar, 500 μm; A, anterior, D, Dorsal. Images in (A) and (B) are from the same SC.
(C) Parasagittal SC section after focal injection of DiI (red) and DiA (green) in V1. In EphA3^ki/ki mice, each single injection results in two corticocollicular TZs, which are interdigitated. insert, schematic of V1 injection sites; bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.
(D) Representative intensity profile plots from two EphA3^ki/ki mice after focal injection of DiI (red) and DiA (green) in V1.
(E) Whole mount SC after focal injection of DiI (white) in nasal retina. In EphA3^ki/ki/β2^−/− mice, showing two broad TZs (arrowheads). bar, 500 μm; M, medial; P, posterior.
(F) Parasagittal section of the SC in (D) showing two broad TZs (arrowheads). insert, schematic of retinal injection site; bar, 500 μm; A, anterior; D, dorsal. Images in (D) and (E) are from the same SC.
(G) Parasagittal SC section after focal injection of DiI (red) and DiA (green) in V1. In EphA3^ki/ki/β2^−/− mice, each single injection results in a single, broad TZ, which are not interdigitated. insert, schematic of V1 injection sites; bar, 500 μm; *, pretectal nucleus; A, anterior; D, dorsal.
(H) Representative intensity profile plots from two EphA3^ki/ki/β2^−/− mice after focal injection of DiI (red) and DiA (green) in V1.