Afadin is localized to the OS, OLM, OPL, INL, and IPL.

A. Immunostaining of the wild-type (WT) mouse retina (1M) with anti-afadin antibody. The afadin signals disappeared in the pre-absorbed (peptide+) sample. Nuclei were stained with DAPI (blue). B. Immunostaining of the WT retinal section (1M) using anti-afadin (green) combined with anti-Nectin-1 (red) antibody. Necin-1 was partially co-localized with afadin in the OLM, OPL, and IPL. Nectin-2 and nectin-3 were localized only in the OLM. C. Afadin was colocalized with nectin-1, nectin-2, and nectin-3 at the OLM. The 1M WT retinal sections were immunostained with anti-afadin (green), anti-nectin-1 (red, upper panels), anti-nectin-2 (red, middle panels), and anti-nectin-3 (red, lower panels) antibodies. D. Immunostaining of 1M WT retinal sections using anti-afadin (green), anti-SCGN (red), and anti-PKCα(white) antibodies. Afadin signals overlapped with SCGN and PKCα in the OPL and INL and PKCα in the IPL. OS, the outer segment; IS, the inner segment; OLM, the outer limiting membrane; ONL, the outer nuclear layer; OPL, the outer plexiform layer; INL, the inner nuclear layer; IPL, the inner plexiform layer; GCL, the ganglion cell layer.

Outer retinal lamination is severely disrupted in the afadin cKO retina.

A. Toluidine blue staining of the cHet and cKO mouse retinal sections at 1M. The retinal layer structure was disrupted in the cKO retina. Rod photoreceptor nuclei (insets) were deeply stained. The red asterisk indicates rod photoreceptor. The arrowhead indicates a rosette-like structure. Scale bar in the inset, 2.5 μm. B. Representative images of the cHet and cKO retinas (1M) stained with anti-Rom1 (green) and anti-Rhodopsin (Rho, red) antibodies and PNA-rhodamine (white). Rom1, Rhodopsin, and PNA signals were remarkably decreased and scattered in the cKO mice. The arrowhead indicates the rosette-like structure. C. Representative images of the cHet and cKO retinas (1M) stained with anti-S-opsin (green) and anti-M-opsin (white) antibodies and PNA-rhodamine (red). Cone OSs were aberrantly located in the cKO retina. The arrowhead indicates the rosette-like structure. D. Retinal flat-mount immunostaining of the cHet and cKO retinas immunostained with anti-Arr3 (green, left panels), anti-Rom1 (green, right panels), and anti-Rhodopsin (red, right panels) antibodies. The inset shows an area with a relatively high density of photoreceptors in the cKO retina. E. Immunofluorescent analysis of the cHet and cKO retinas (1M) using anti-Arr3 (cone marker, green in the left panels), anti-Otx2 (photoreceptor and BC marker, red in the left panels), anti-RNA binding protein with multiple splicing (RBPMS, RGC marker, green in second left panels), anti-Chx10 (BC marker, red in second left panels), anti-Lhx2 (Müller glia marker, green in third left and third right panels), anti-AP2α (AC marker, red in third left and second right panels), and anti-Calbindin (horizontal cell and AC marker, white in third left and right panels) antibodies. Arrowheads indicate RGCs and ACs near the rosette-like structure.

Photoreceptor-BC synapses were severely impaired and mislocalized in the afadin cKO retina.

A. Immunostaining of the cHet and cKO retinal sections at 1M with anti-Bassoon (green), anti-mGluR6 (red), and anti-PKCα (white) antibodies. The arrowhead indicates the synapse between rod and rod-BC. B. Immunostaining of the cHet and cKO retinas at 1M with anti-GluR5 (green), anti-PSD95 (red), and anti-PKARIIβ (white) antibodies. The arrowhead indicates the synapse between the cone and OFF-BC. C. 3D projection of confocal image immunostaining with anti-Bassoon (green), anti-mGluR6 (red), and anti-PKCα (white) using the cHet and cKO retina (1M). The inset shows the synapse between the rod and BC at high magnification. The arrowhead indicates the ribbon synapse between rod and rod-BC. D. 3D projection of confocal image immunostaining with anti-GluR5 (green), anti-PSD95 (red), and anti-PKARIIβ (white) antibodies using the cHet and cKO retina (1M). E. Quantification of the number of synapses between rod photoreceptor and rod-BC under 1mm2 of retinal surface in the cHet and cKO mice immunostained with anti-Bassoon, anti-mGluR6, and anti-PKCα antibodies (cHet; 599.6 ± 49.7, n = 3, cKO; 29.1 ± 12.75, n = 4, 10 images from each mouse. Error bars, mean ± SD. ***p < 0.001 by Student’s t test). The number of synapses decreased to about 5 % of the cHet in the cKO retina. F. Quantification of the number of synapses between photoreceptor cells and type 3 OFF-BC under 1mm2 of the retinal surface in the cHet and cKO mice immunostained with anti-GluR5, anti-PSD95, and anti-PKARIIβ antibodies (cHet; 24.0 ± 2.8, n = 3, cKO; 4.1 ± 1.3, n = 3, 10 images from each mouse. Error bars, mean ± SD. ***p < 0.001 by Student’s t-test). The number of synapses between the photoreceptor and Type 3 OFF-BC was decreased to about 15 % of the cHet in the cKO retina.

GluR5 is ectopically localized to ON and OFF BC processes.

A. Immunostaining of the cHet and cKO retinal sections at 1M with anti-GluR5 (green), anti-SCGN (red), and anti-PKCα antibodies. Obvious GluR5 signal was observed in the IPL of the cKO retina. Yellow and white arrowheads indicate overlap of GluR5 and PKCα, and GluR5 and SCGN, respectively. B. Representative images of immunostained cHet and cKO retinas (1M) with anti-GluR5 (green), anti-vGlut1 (red), and anti-SCGN (white) antibodies. C, D. The cHet and cKO retinas (1M) stained with anti-EAAT5 (green), anti-SCGN (red in C), and anti-PKCα (white in C), anti-PSD95 (red in D) antibodies and PNA-rhodamine (white in D). Glutamate transporter EAAT5 localization to ON and OFF BC axon terminals and photoreceptor axon terminals were unaffected in the cKO retina. E. Immunofluorescent analysis of the cHet and cKO retinas (1M) with anti-HPC-1 (AC marker, green in the left panels), anti-Calbindin (red in the left panels), anti-Tuj1 (RGC marker, green in the right panels), and anti-RBPMS (red in the right panels) antibodies. F. The number of each retinal cell types per 100 μm width of retinal section (cHet; rod 185.9 ± 17.5, cone 8.4 ± 1.4, BC 45.5 ± 5.2, horizontal cell 2.1 ± 0.4, AC 30.1 ± 3.2, RGC 7.4 ± 1.5, Müller glial cell 15.7 ± 2.3, n = 4, cKO; rod 57.3 ± 17.3, cone 3.5 ± 1.3, BC 64.6 ± 7.8, horizontal cell 1.9 ± 0.7, AC 27.6 ± 6.3, RGC 5.7 ± 0.9, Müller glial cell 12.4 ± 1.5, n = 4. Error bars, mean ± SD. Horizontal cell p = 0.58, AC p = 0.52, RGC: p =0.10. ***p < 0.001, **p < 0.001, *p < 0.05 by Student’s t test). Rod and cone photoreceptors and Müller glial cells were significantly decreased to about 30, 40, and 80% of the cHet retina, respectively, and BCs were significantly increased to about 150% of the cHet retina in the cKO retina.

Reduction of the a- and b-waves in the afadin cKO retina.

A. Left, Scotopic ERGs from anesthetized cHet (n = 3; black) and cKO (n = 3; magenta) mice. A flash (duration; 5 ms, white LED, intensity; 1.0 × 104 cd/m2) was applied to eyes in the dark three times. Traces: averaged (dark) and SD (pale). Right, Photopic ERGs. Anesthetized cHet (black) and cKO (magenta) mice were light (31.6 cd/m2) adapted for 10 min, and then, a flash (duration; 5 ms, intensity; 1.0 × 104 cd/m2) was superimposed on the adapted light to eyes 16 times. B. mERGs recorded by MEA from the cHet (black) and cKO (magenta) retinas. A flash (duration; 5 ms, green LED; λmax = 510 nm, intensity; 2.5 × 104 photon/s/µm2) was applied to the isolated whole retina 3 times every 10 s. Control (solid line), L-AP4 (10 μM) (pale), and after washout (dotted line). C. Left, Amplitude of the a-wave (cHet; 155 ± 58.7 μV, n = 292 electrodes, cKO; 44.1 ± 21.7 μV, n = 13 electrodes, Error bars, mean ± SD. ***p < 0.001, Mann-Whitney U test). Right, Amplitude of the b-wave (cHet; 163 ± 76.5 μV, n = 292 electrodes, afadin cKO; 28.4 ± 19.6 μV, n = 13 electrodes, p = 7.08 x 10-09, Mann-Whitney U test). D, Ratio of b-wave amplitude / a-wave amplitude (b / a) (cHet; 1.19 ± 0.67, cKO; 0.59 ± 0.23, Error bars, mean ± SD. ***p < 0.001, Mann-Whitney U test). E, Left, Implicit time of the a-wave (cHet; 62.6 ± 10.8 ms, n = 292 electrodes, cKO; 149 ± 16.0 ms, n = 13 electrodes, p = 5.34 x 10-10, Mann-Whitney U test). Right, Implicit time of the b-wave (cHet; 127 ± 25.3 ms, n = 292 electrodes, cKO; 229 ± 60.7 ms, n = 13 electrodes, Error bars, mean ± SD. ***p < 0.001, Mann-Whitney U test). F, Difference between implicit time of the b-wave and that of the a-wave (cHet; 64.4 ± 22.0 ms, n = 292 electrodes, cKO; 80.4 ± 50.9 ms, n = 13 electrodes, Error bars, mean ± SD. p = 0.40, Mann-Whitney U test).

RGC classification based on the light-evoked responses.

A,B. Light-evoked responses. A, cHet. B, cKO. A flash (duration; 2 s, intensity; 24.2 or 27.3 cd/m2) was applied seven times every 8 s. After spike sorting, the raster plots (top) and the PSTHs (bottom) were created (20 ms/bin). Based on the PSTHs, RGC responses were classified into “ON”, “ON-OFF”, “ON-OFF inhibition”, “OFF”, and “None” types. C. Ratio of responded to non-responded types (cHet; n = 434, n = 4 retinas, cKO; n = 388, n = 4 retinas). D. Rod and cone inputs to RGCs (cHet; n = 434, n = 4 retinas, cKO; n = 388, n = 4 retinas). 2-s green LED flash (λmax = 510 nm, intensity; 4.8 x 103 photons/s/µm2) and UV LED flash (λmax = 360 nm, intensity; 3.3 x 104 photons/s/µm2) were applied 7 times every 8 s to stimulate mainly rods and S cones, respectively.

Spatiotemporal properties of the RF of RGCs.

A. Left, An isolated retina on the MEA. Electrodes (black square) mapped receptive fields (colored oval), and the region (magenta square) where immunohistochemical examination was performed after recording. Scale bar 200 μm. Right, Immunohistochemical staining with anti-PKARIIβ (green), anti-PSD95 (red), and anti-PKCα (white) antibodies. IPL: the inner plexiform layer. B. Number of cells whose RF was clearly observed (cHet; 195/434 cells, n = 4 retinas, cKO; 69/388 cells, n = 4 retinas). C. The spatial profile of RFs in the cHet (Top) and cKO (Bottom) retinas. Color scale illustrates high (red) to low (blue) relative to the average (green). Scale bar 100 μm. D. RF size of the cKO retina was significantly smaller than that of the cHet retina (cHet; 0.0307 ± 0.0210 mm2 n = 195, n = 4 retinas, cKO; 0.0161 ± 0.00839 mm2 n = 69, n = 4 retinas, Error bars, mean ± SD. ***p < 0.001, Mann-Whitney U test). E. Short and long axes of the oval fitted to each RF. F. Clustering of RGCs based on the temporal profile of RFs. Five clusters were visualized (cHet; circle, cKO; x). G. Temporal profiles from each cluster were superimposed separately (cHet; black, cKO; magenta, mean; solid line, SD; pale line). H. Time-to-peak latency of the temporal profile (cHet; filled bar, cKO; open bar). Mean ± SD. (cHet; cluster 1; -113 ± 15 msec, n = 31, cluster 2; -111 ± 13 msec, n = 36, cluster 3; -200 ± 31 msec, n = 51, cluster 4; -171 ± 19 msec, n = 37, cluster 5; -178 ± 28 msec, n = 40, cKO; cluster 3; -163 ± 21 msec, n = 10, cluster 4; -238 ± 34 msec, n = 36, cluster 5; -217 ± 41 msec, n = 23).

Ectopic AJs are observed in the developing afadin cKO retina.

A. Immunostaining of the cHet and cKO retinas (1M) with anti-active caspase 3 (AC3, green) antibody. Arrowhead indicates AC3 positive cell. B. Quantification of apoptotic cells per 1mm2 in the cHet and cKO retinas at 1M (cHet; 0.6 ± 0.7, n = 4, cKO; 66.7 ± 18.2, n = 5). Apoptotic cells were significantly increased in the cKO retina. Error bars, mean ± SD. ***p < 0.001 by Student’s t-test. C. Immunostaining of the cHet and cKO retinas at postnatal day 0 (P0) with anti-phospho-histone H3S10 (pHH3, mitotic cell marker, green), anti-N-Cadherin (red in left, second left, and second right panels), anti-Nectin-1 (white in left, second left, and third right panels), anti-β-catenin (red in the right panels) antibodies. Ectopic N-cadherin, nectin-1, and β-catenin signals were observed inside the cKO retinas, and pHH3-positive cells were localized near these ectopic signals.

Afadin is localized to AJs in the developing and mature retinas.

A. Immunostaining of the WT retinal section (1 M) with anti-afadin (green) antibody combined with nectin-2 (red, upper panels) and nectin-3 (red, upper panels) antibodies. B. Immunostaining with anti-afadin (green), anti-nectin-1 (red), and anti-Arr3 (a cone marker, white) antibodies. Afadin was co-localized with nectin-1 in cone synapses. C. Immunostaining of the WT retinal section (P0) with anti-afadin (green) and anti-nnectin-1 (red) antibodies.

The OLM AJs are disrupted in the afadin cKO retina.

A. Western blot analysis of the cHet and cKO retinas using anti-afadin (upper panel) and anti-GAPDH (lower panel) antibodies. No significant afadin band was detected in the cKO retina. B. Electron microscopic analysis of the cHet and cKO retinas (1M). A few ectopic disc structures were observed in the cKO retina. The inset shows the ectopic disc structure at high magnification. The arrowhead indicates the outer segment disc structure. C. Immunostaining of the cHet and cKO retinal sections at 1M using anti-β-catenin (green, left panels), anti-nectin-1 (red, left panels), and anti-N-cadherin (green, right panels). Signals of these markers were not observed at the outer retinal surface in the cKO mice. D. Immunostaining of the cHet and cKO retinal sections at 1M using anti-glutamine synthetase (GS, green) antibody. Obvious GS signals were observed in the inner side of the cKO retina but not at the outer retinal surface in the cKO mice.

The OS and photoreceptor-BC synapses are affected in the developing afadin cKO retina.

A. Immunostaining of the cHet and cKO retinas at 1M with anti-PKCα (green), anti-PSD95 (red) antibodies. B. 3D projection of confocal image immunostaining with anti-PKCα (green), anti-PSD95 (red) antibodies. C. Quantification of the number of contacts between photoreceptor and rod ON-BC under 1mm2 of retinal surface in the cHet and cKO mice immunostained with anti-GluR5, anti-PSD95, and anti-PKARIIβ antibodies (cHet; 550.5 ± 29.1, n = 3, cKO; 65.2 ± 7.9, n = 3, 10 images from each mouse. Error bars, mean ± SD. ***p < 0.001 by Student’s t-test). The number of synapses between the rod and ON BC was decreased to about 10 % of the cHet in the cKO retina. D. Representative images of the cHet and cKO retinas (P11) stained with antibodies against Rom1 (green, left panels), Rhodopsin (white, left panels), PKARIIβ (green, middle panels), Arr3 (red, middle panels), Bassoon (green, right panels), mGluR6 (red, right panels), PKCα (white, right panels). The inset shows the synapse between Cones and Type 3 OFF-BCs (middle panels) and ribbon synapses (right panels) at high magnification. E. Immunostaining of the cHet and cKO retinal sections at 1M with anti-ChAT (green), anti-Calretinin (red), and anti-Calbindin (white) antibodies. Obvious ChAT bands, Calretinin bands, and Calbindin bands were observed in the IPL of the cKO mice. F. The number of PKCα+ and SCGN+ cells per 100 μm width of retinal section (cHet; PKCα+ 15.8 ± 2.6, SCGN+ 24.7 ± 4.8, n = 4, cKO; PKCα+ 23.6 ± 5.2, SCGN+ 37.4 ± 7.7, n = 4. Error bars, mean ± SD. *p < 0.05 by Student’s t-test). PKCα+ cells and SCGN+ cells were significantly increased to 150% of the cHet. G. The number of PKCα+ cell processes in the IPL per 100 μm width of retinal section (cHet; 18.6 ± 0.5, n = 4, cKO; 14.7 ± 1.2, n = 4. Error bars, mean ± SD. ***p < 0.01, *p < 0.05 by Student’s t-test). The number of PKCα+ cell processes was significantly decreased to 150% of the cHet. H. Expression of photoreceptor and BC marker genes in the cHet and cKO at P14 (Nrl and Rhodopsin; rod marker, Opnsw and Opnmw; cone maker, Trpm1 and mGluR6; ON BC marker, Chx10; BC marker). Each gene expression was normalized by GAPDH, a house keeping gene. Expression of Nrl, Rhodopsin, Opnsw, and Opnmw were significantly decreased and those of mGluR6, Trpm1, and Chx10 were significantly increased in the cKO retina. Error bars, mean ± SD. ***p < 0.001, ***p < 0.01 by Student’s t-test.