A. Immunofluorescence staining for NrCAM (red) in mouse retina at P0 and P6 demonstrated NrCAM localization in fibers of the retinal NFL, ON, IPL, and lens, as shown in confocal images. Double staining for L1 (green) showed partial co-localization with L1. GCL, ganglion cell layer; NFL, nerve fiber layer; IPL, inner plexiform layer; ON, optic nerve head; OPL, outer plexiform layer; L, lens; D, dorsal; V, ventral. B. In situ hybridization with antisense probes in the mouse retina (P0) showed NrCAM transcripts in cell bodies in the GCL. L1 transcripts were present in the GCL and inner nuclear layer (INL). Background labeling of retina is shown by hybridization with the NrCAM sense probe. C. Immunofluorescence staining for NrCAM in sagittal sections of SC at P0 showed partial co-localization with the axonal marker NF165 in a portion of RGC axons in the SGS and SO, as well as some additional labeling within the SC. SGS, stratum griseum superficiale; SO, stratum opticum. D. In situ hybridization with antisense probe showed NrCAM transcripts in cell bodies located within the SC (P0, sagittal sections). Background is indicated by hybridization with the NrCAM sense probe.

A. In WT mice, DiI injection into the temporal retina at P8 labeled a single dense termination zone (TZ) in the anterior SC at P10. B–D. In NrCAM null (KO) mice, DiI injection into the temporal retina labeled multiple laterally displaced ectopic TZs (eTZs) in the SC at P10. E–F. DiI injections into the nasal retina labeled a single TZ in the posterior SC that was normally positioned in WT and NrCAM KO mice at P10. G–H. DiI injections into the dorsal (G) and ventral (H) retina of NrCAM KO mice resulted in normally positioned single TZs in the lateral (G) and medial (H) SC, respectively. The location of DiI injections were shown in retinal flat mounts in the lower left of each image. L, lateral; M, medial; A, anterior; P, posterior; D, dorsal; V, ventral; T, temporal; N, nasal.

Left panel: The schematic diagram illustrates the location of TZs of temporal RGC axons in the NrCAM null mutant (KO) SC (P10). The centers of TZs and eTZs from NrCAM KO mice (n = 14) are marked and connected. The distribution of normal TZs of temporal RGC axons in WT mice are all found within the anterior region of the SC depicted by the oval. L, lateral; M, medial; A, anterior; P, posterior. Right panel: Percentage of abnormally distributed single and multiple eTZs of temporal RGC axons in the SC of NrCAM null mice (P10). In NrCAM KO mice 85% of mice showed abnormally located eTZs. A single laterally displaced eTZ is found in 14% of mutants, and multiple eTZs were found in 71% of the mutants.

A–C. DiI labeling of VT RGC axons in WT mice showed that most branches from VT axons in the lateral zone of the SC oriented medially to the future SC, as seen in a higher magnification of the boxed area in B (arrows). D–F. DiI labeling of VT axons in NrCAM null mutants (KO) revealed more laterally oriented branches in axons within the lateral zone of the SC, as seen in a higher magnification of the boxed area in E (arrows). M, medial; L, lateral; P, posterior. Scale bar: 200 µm in A,D; 100 µm in B,E; 50 µm in C,F.

A. Diagram of the location and branch orientation of DiI-labeled axons from the VT retina within the SC of WT mice at P3. The SC was divided into 3 bins along the medial-lateral axis as shown by the dashed lines: lateral to TZ (L), within TZ (TZ), and medial to TZ (M). The shaded circle represents the site of the forming TZ. Branches were scored and their orientations were analyzed within each bin. The arrows represent the preferred orientation of WT branches. B. Branch distribution of VT RGC axons in WT and NrCAM null mutant (KO) mice. The distribution of branches was expressed as a branch directional coefficient for each bin by subtracting the number of laterally oriented branches from medially oriented branches, then dividing by the total number of branches. In WT (n = 4) and NrCAM null (n = 4) mice, 184 and 205 branches were analyzed, respectively. Brackets indicate S.E.M., and asterisks show significant differences (ANOVA, p<0.05). C. Diagram of entry position of VT RGC axons in the SC of WT mice at P3. The SC was divided into 10 equal segments along the medial-lateral axis at its anterior border relative to the position of the forming TZ: lateral to TZ (L), within TZ (TZ), and medial to TZ (M). The position of labeled RGC axons in each segment was scored and expressed as the percent of total axons. D. Distribution of DiI-labeled VT RGC axons of WT and NrCAM null mutant (KO) mice entering the anterior SC (expressed as the percent of total axons) in bins along the mediolateral axis, as described in C. Shaded circle indicates region near the location of normal TZ. In WT (n = 4) and NrCAM null (n = 4) mice, 146 and 167 axons were analyzed, respectively. Error bars represent SEM. There were no significant differences between WT and mutant at any location (ANOVA, p<0.05).

A. NrCAM was expressed with or without EphB2 or EphB2 kinase dead mutant (EphB2 KD) in transfected HEK293 cells, followed by immunoprecipitation with NrCAM antibodies and immunoblotting with phospho-FIGQY antibodies. EphB2 induced phosphorylation of NrCAM (200 kD) at FIGQY (p-FIGQY) compared to NrCAM alone or to co-expression of the EphB2 kinase dead mutant. Blots were stripped and reprobed with antibodies to NrCAM to indicate relative levels of NrCAM in the immunoprecipitates. B. NrCAM was expressed with or without EphB1, EphB2, or EphB3 in transfected HEK293 cells, followed by immunoprecipitation with NrCAM antibodies and immunoblotting with phospho-FIGQY antibodies. EphB1 (HA-tagged) or EphB2 (not HA-tagged) induced effective phosphorylation of NrCAM at FIGQY, whereas EphB3 (HA-tagged) had a weak effect. Blots were stripped and reprobed with antibodies to EphB2 or to the HA-epitope on EphB1 and EphB3. Each EphB receptor was co-immunoprecipitated efficiently with NrCAM. C. Lysates of the superior colliculus from WT, EphB1/3 double null, EphB1/2/3 triple null and constitutively active EphB2 (F620D) homozygous mice (P2-P3) were immunoprecipitated with antibodies to NrCAM, and immunoblotted with phospho-FIGQY or NrCAM antibodies. Bands were quantified using the threshold function of ImageJ. The numbers under the blots represent the normalized average phospho-FIGQY/NrCAM band intensity from individual mice. Phosphorylation of NrCAM at FIGQY was reduced in the SC of EphB1/3 and EphB1/2/3 null mice compared to WT, while phospho-FIGQY on NrCAM was increased in the EphB2 F620D mice. D. Cytofluorescence assay for recruitment of ankyrinG-EGFP to NrCAM in the plasma membrane of transfected HEK293 cells was visualized by confocal imaging. Boxes above images indicate the expression plasmids used for transfection, while boxes at the left indicate fluorescence labeling of ankyrinG-EGFP or NrCAM in the same cells. Corresponding differential contrast (DIC) images are shown below. AnkyrinG-EGFP localized within the cytoplasm of cells expressing ankyrin alone, while co-expression of NrCAM led to the recruitment of ankyrinG-EGFP to the cell membrane, where NrCAM was localized. When NrCAM was co-expressed with EphB2, ankyrinG-EGFP remained largely present in the cytoplasm. When NrCAM was co-expressed with EphB2 KD, ankyrinG-EGFP was recruited to the plasma membrane where NrCAM was localized in most cells, although occasionally some cytoplasmic localization was seen. Scale bar = 15 µm. E. Quantification of the percentage of cells with ankyrinG-EGFP recruited to the plasma membrane of HEK293 cells transfected as in C. NrCAM expression significantly increased ankyrinG-EGFP recruitment to the plasma membrane in ankyrin/NrCAM-expressing cells as compared with ankyrin expression alone (Ankyrin/NrCAM: 84±3%; Ankyrin: 3±1.6%). In cases of ankyrin/NrCAM/EphB2 co-expression, ankyrinG-EGFP recruitment was strongly decreased (7±2%) compared to ankyrin/NrCAM expression. There was no significance decrease in ankyrinG-EGFP recruitment in cases of ankyrin/NrCAM/EphB2 KD co-expression (84±4%). Error bars show S.E.M. Asterisks indicate significant differences in means (one-way ANOVA, Tukey’s post-hoc test, *p<0.001).