Fig 2: Ndfip1 and Ndfip2 Expression in the Developing Spinal Cord(A and B) mRNA in situ hybridization reveals clear expression of Ndfip1(A) and Ndfip2 (B) in E10.5 and E11.5 mouse spinal cord. mRNA probes to the sense strand serve as controls for the specificity of Ndfip1 (A) and Ndfip2 (B) expression. Yellow arrows in the E11.5 images show expression in regions of dorsal commissural axon cell bodies.(C) Representative confocal images of transverse sections of wild-type mouse spinal cord from E10.5 to E12.5 labeled with anti-Ndfip1 antibody. Ndfip1 is expressed at the floor plate, in the motor column, and in DRGs.(D) Anti-GFP immunostaining of E10, E10.5, and E11.5 of embryos reveals the pattern of Ndfip2 expression. Embryos are heterozygous for an allele of Ndfip2 where the coding sequence has been replaced by a GFP reporter. Commissural axons are clearly labeled by E11.5.(E–G) Higher magnification images of E10.5 and E11.5 spinal cord sections illustrate co-labeling of Ndfip1 and TAG1 (E and F) or Robo1 (G) in the ventral commissure. Co-localization of Ndfip1 with TAG1-positive commissural axons demonstrates the commissural axonal expression of Ndfip1.(H–J) Higher magnification of anti-GFP immunostaining of E10.5 and E11.5 of Ndfip2-GFP heterozygous embryos reveals co-labeling of Ndfip2 and TAG1 (H and I) or DCC (J) in the ventral commissure.(K and L) Double immunostaining of Ndfip1 (K and L, green) and DCC (K, red) or TAG1 (L, red) in dissociated commissural neurons showing the expression of Ndfip1 in the cell body, axon and growth cone of commissural neurons.Scale bars represent 50 µm in (A)–(D), 20 µm in (E)–(J), and 10 µm in (K) and (L).

Fig 3: Ectopic Expression of Ndfip1 or Ndfip2 Decreases Robo1 Surface Levels In Vitro(A–C') Confocal micrographs of COS-7 cells expressing N-Myc-Robo1 with empty HA-vector (A and A') or with Ndfip1-HA (B and B') or with Ndfip2-HA (C and C'). Surface expression of Robo1 was visualized by staining the N-terminal Myc tag before fixation and permeabilization (A–C, green). The HA staining reveals the expression of Ndfip1 (B', red) and Ndfip2 (C', red). DRAQ-5 is a nuclear marker. Co-expression of Ndfip1 or Ndfip2 with Robo1 leads to a significant decrease in Robo1 at the cell surface.(D) The fluorescent intensity of surface Robo1 is measured as a mean gray value. Error bars represent SEM. Control, n = 8; Ndfip1-HA, n = 10; Ndfip2-HA, n = 12 (n, number of cells scored for each transfection) (Ndfip1, 29.8 ± 5.74; Ndfip2, 17.6 ± 1.33; p < 0.001). Significance was assessed using Student’s t test (**p < 0.001).(E) HeLa cells transiently transfected with Myc-Robo1 and Ndfip1-HA or Ndfip2-HA plasmids. 48 h after transfection, cell surface proteins isolated using biotinylation were analyzed using western blot using anti-Myc antibody (top panel). Levels of total Robo1 and the expression of Ndfip proteins were analyzed using western blot using anti-Myc and anti-HA antibodies, respectively. An anti-Tubulin antibody was used to control for equal protein loading. Biotinylated surface Robo1 levels are strongly reduced in Ndfip1 and Ndfip2 transfected cells (Ndfip1: 0.60 ± 0.103, p = 0.022; Ndfip2: 0.006 ± 0.004; p = 0.0014).(F) Quantitative representations for biotinylated surface Robo1 band intensities in control vector and Ndfip1-HA- and Ndfip2-HA-transfected cells. Data were normalized to control.Error bars represent SEM. Significance was assessed using Student’s t test (*p < 0.05 and **p = 0.001). Scale bar represents 10 µm.

Fig 4: Robo1 Expression Is Increased in Ndfip Mutants(A–C') Immunohistochemistry against Robo1 protein labels post-crossing axons and pre-crossing (arrow mark) and crossing commissural axons (arrowhead). At E11.5, in Ndfip1 mutant spinal cord, Robo1 levels are increased in pre-crossing commissural axons (B and C, arrow with asterisk) compared with wild-type (A, arrow). Robo1-positive axons are observed crossing the midline in Ndfip1 mutant embryos (B' and C', arrowhead with asterisk) (n = 4; n, number of embryos). The variability of Robo1 expression in Ndfip1 mutant embryos is represented in (B) and (C).(B' and C') Higher magnification images at the floor plate region.(D) Quantitative representation of Robo1 pixel intensity at the commissure in control, Ndfip1-/-, Ndfip2-/-, and Ndfip1-/-; Ndfip2-/- spinal cord sections.(E and F) Brain extracts (E) and spinal cord extracts (F) from wild-type and Ndfip1 mutant adult mice were immunoblotted with anti-Robo1 and anti-Robo2 antibodies. Anti-Tubulin antibody was used as a loading control. Robo1 levels are increased in both Ndfip1 mutant brain or spinal cord lysates compared with wild-type, whereas Robo2 levels are unaltered.(G and H) Quantitative representation of band intensities of Robo1, Robo2, and DCC in brain lysates (G) or Robo1 and Robo2 in spinal cord lysates (H) that were normalized with tubulin levels.Error bars represent SEM. Significance was assessed using Student’s t test (*p < 0.05 and **p < 0.01); ns, non-significant. Scale bars represent 50 µm in (A)–(C) and 20 µm in (A')–(C').

Fig 5: Ndfip PY Motifs and E3 Ligase Activity Are Required for Robo1 Degradation(A–C) Confocal micrographs of COS-7 cells expressing Myc-tagged Robo1 and HA-tagged Ndfip1 or Ndfip2 with mutations in the PY motifs.(A) Robo1 (in green) is localized mainly at the plasma membrane and Golgi apparatus in cells that co-expressed a vector control.(B and C) Co-transfection of Robo1 with either Ndfip1 (B) or Ndfip2 (C) (red) re-localizes Robo1 into endosomes.(D and E) Co-transfection of Robo1 with either Ndfip1PY (D) or Ndfip2PY (E) does not alter Robo1 localization.(F and G) COS-7 cells were transfected with plasmids expressing Myc-tagged Robo1 and either HA-tagged Ndfip1 or Ndfip1PY (F), or HA-tagged Ndfip2 or Ndfip2PY (G) as indicated. PY mutant indicates Ndfip versions in which each PY motif was mutated from PxY to PAG. 48 h after transfection, cell surface proteins isolated using biotinylation were analyzed using western blot using anti-Myc antibody. Co-expression of Ndfip1 or Ndfip2 strongly reduces both surface and total Robo1 protein levels, but co-expression of either Ndfip1 PY or Ndfip2PY does not (Ndfip1: 0.3 ± 0.01, p < 0.001; and Ndfip1-PY: 0.94 ± 0.19, p < 0.05; Ndfip2: 0.33 ± 0.10, p = 0.012; Ndfip2-PY: 0.75 ± 0.05, p < 0.05).(H and I) COS-7 cells were transiently co-transfected with Myc-Robo1, FLAG-Ub, HA-Ndfip1, and HA-Ndfip2 expression constructs as indicated. After 48 h of transfection, cells were treated with 100 µM Heclin for 2 h.(H) Cell lysates were immunoprecipitated with anti-Myc antibody, and immunoprecipitates were western-blotted with anti-FLAG antibody. Ubiquitylated Robo1 is strongly reduced upon Heclin treatment. Ubiquitylated forms appear as smears.(I) Robo1 protein is stabilized in Ndfip1 and Ndfip2 transfected cells that were treated with Heclin (Ndfip1 with Heclin: 0.57 ± 0.004 versus Ndfip1: 0.3 ± 0.01, p < 0.05; Ndfip2 with Heclin: 0.63 ± 0.01 versus Ndfip2: 0.33 ± 0.10, p < 0.05). The expression levels of both Ndfip proteins and Robo1 were analyzed using western blot using anti-HA and anti-Myc antibodies. An anti-Tubulin antibody was used to control for equal protein loading.(J and K) Quantification of total Robo protein levels in cells expressing Ndfip1 (J) or Ndfip2 (K) proteins with mutations in the PY motifs or in cells treated with Heclin. Data were normalized to tubulin levels. Error bars represent SEM. Significance was assessed using Student’s t test (*p < 0.05 and **p < 0.001).(L and M) Schematic illustrations demonstrating the mechanism and the effect of PY mutations in Ndfip proteins (L) or Heclin treatment (M) on Robo protein levels.Scale bars in (A)–(E) represent 10 µm.