a–a”, b–b”, Double-immunostaining using antibodies directed against the C-terminus of rat melanopsin (a and b, green) and against TH (a’ and b’, red) of wild-type (a–a”) and PlexA4^−/− (b–b”) adult retina sections (merged in a” and b”). Ectopic dendritic processes of M1-type ipRGCs were observed in the S4/S5 sublaminae of PlexA4^−/− retinas, as were aberrant dopaminergic amacrine processes (yellow arrows, b–b”, n=4 mutant animals). Wild-type M1-type ipRGC dendritic processes and dopaminergic amacrine cell processes are only observed in S1 (a–a”).
c, High-magnification of S4/S5 in PlexA4^−/− retinas double-immunostained with anti-C-terminal melanopsin and anti-TH. Most TH-positive puncta are co-localized with melanopsin-positive puncta (white arrowheads).
d, Quantification of ectopic TH-positive puncta co-localized with the ectopic M1-type melanopsin puncta in S4/S5 of PlexA4^−/− retinas. Nearly 76% (194 TH-positive puncta among a total of 254 puncta) of the ectopic TH-positive puncta were co-localized with ectopic M1-type melanopsin puncta in S4/S5 (76.4±1.2% co-localization). In wild-type retinas, almost no TH-positive puncta were observed in S4/S5. Error bar indicates SEM (n=3 animals per genotype). Scale bars: 50 µm in a for a–a” and b–b”, and 5 µm in c.

a–f, Wild-type (a–c) and Sema6A^−/− (d–f) adult retina sections were immunostained with antibodies against TH (a, d), C-terminal melanopsin (b, e), and calbindin (c, f). Sema6A^−/− retinas recapitulate the lamina-specific neurite arborization defects of dopaminergic amacrine cells, M1-type ipRGCs, and calbindin-positive cells (yellow arrows) observed in PlexA4^−/− retinas (n=8 Sema6A^−/− animals).
g–j, Wild-type (g), PlexA4^+/− (h), Sema6A^+/− (i), and Sema6A^+/−; PlexA4^+/− (j) adult retina sections were immunostained with anti-TH.
k, Quantification of ectopic TH-positive puncta detected in the S4/S5 sublaminae in wild-type (g), PlexA4^+/− (h), Sema6A^+/− (i), and Sema6A^+/−; PlexA4^+/− (j) sections (n=4 animals for each genotype). An increased number of TH-positive puncta was observed in S4/S5 in Sema6A^+/−; PlexA4^+/− retinas (10.1±1.7 puncta per 100 µm, yellow arrow, j) as compared to the other three genotypes (0.1±0.1 puncta per 100 µm, wild-type 2.6±0.3 puncta per 100 µm, PlexA4^+/−; 2.3±0.4 puncta per 100 µm, Sema6A^+/−; g–i). Error bars are SEM. *** indicates P < 0.01 by Tukey’s multiple comparison test.
Scale bars: 50 µm in f for a–f, and in g for g–j.

a–h, Wild-type (a, c, e) and PlexA4^−/− (b, d, f) adult retina sections were immunostained with antibodies against TH (a, b), calbindin (c, d), and calretinin (e, f). In PlexA4^−/− retinas, TH-positive dopaminergic amacrine cells and calbindin-positive cells exhibit defects in lamina-specific neurite arborization (yellow arrows, b and d, n=10 PlexA4^−/− animals). In wild-type retinas, dopaminergic amacrine cell processes are observed predominantly in the S1 sublamina of the IPL (a). In contrast, aberrant punctate immunostaining is detected in the S4/S5 sublaminae, in addition to S1, in all PlexA4^−/− retinas examined (b). The normal stratification of calbindin-positive cells in the IPL (c) is disrupted in PlexA4^−/− retinas, resulting in aberrant processes in S4/S5 (d). Calretinin-positive cells show normal sublaminar stratification in the IPL of PlexA4^−/− retinas (e–f).
g and h, PlexA4^−/− adult retina sections double-immunostained with anti-calretinin (white bars) and anti-TH (g), or anti-calretinin and anti-calbindin (CB) (h). Aberrant processes in PlexA4^−/− retinas from dopaminergic amacrine cells, and also from calbindin-positive cells, are found adjacent to and closer to the GCL than the calretinin-positive processes that lie between S3 and S4 in the IPL (yellow arrows). Scale bars: 50 µm in h for a, b, g, h, and in f for c–f.

a–a”, P14 retina section double-immunostained with anti-PlexA4 (a, red) and anti-Sema6A (a’, green) (merged in a”). Strong Sema6A immunoreactivity in the IPL was observed in ~one-half of S3 and throughout S4 and S5, whereas PlexA4 expression is stratified in two distinct layers in S1 and S2.
b, P14 retina section double-immunostained with anti-PlexA4 (green) and anti-calbindin (CB, red) shows PlexA4 protein localization in S1/S2 sublaminae relative to calbindin-positive neuronal processes.
c, P14 retina section double-immunostained with anti-Sema6A (green) and anti-calbindin (CB, red) shows Sema6A protein localization in the S3–S5 relative to calbindin-positive neuronal processes.
d–d”, P14 retina section double-immunostained with anti-PlexA4 (d, green) and anti-TH (d’, red), revealing co-localization of PlexA4 and TH immunoreactivity in S1 of the IPL (merged in d”).
e–e”, P14 retina section hybridized with PlexA4 antisense probe (e, green) followed by anti-TH immunolabeling (e’, red, merged in e”). PlexA4 mRNA is localized to the cell body of dopaminergic amacrine cells (of 26 TH-positive amacrine cells scored, all were positive for PlexA4 mRNA).
f and f’, P14 retina section double-immunostained with anti-PlexA4 (green) and anti-N-terminal melanopsin (red), which labels multiple ipRGC subtypes18 (f; high magnification of the area in the white square shown in f’). PlexA4 immunoreactivity was not observed in the cell bodies or dendrites of ipRGCs.
g and h, P14 retina sections double-immunostained with anti-Sema6A (green) and anti-TH (g, red) or anti-N-terminal melanopsin (Me) (h, red). Sema6A protein was not observed in cell bodies, or processes, of dopaminergic amacrine cells and ipRGCs (M1-type ipRGC dendritic processes in the S1 indicated by yellow arrow in h).
Scale bars: 50 µm in c for a–a”, b, c, 50 µm in h for d–d”, f, g–h, 20 µm in e” for e–e”, and 20 µm in f’.