Fig 2: 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 3: Robo2 but not Robo3 is enriched in di1 ipsilateral neurons. (A,B) Schematic representation of a transverse section of the spinal cord illustrating the expression of the Barhl2GFP transgene used in this study to genetically delineate dI1 neurons (gray) at E11.5 (A) and E12. (B) dI1 commissural (dI1c) and dI1 ipsilateral (dI1i) cell body and axonal projections are depicted. (C–E) Photomicrographs of Barhl2GFP E12.5 mouse embryonic spinal cord brachial transverse sections. The images show immunohistochemical labeling with GFP (green) to delineate dI1 neurons and with Robo1 (red) (C), Robo2 (red) (D), or Robo3 (red) (E), respectively. The single-channel GFP images and the Robo1/GFP, Robo2/GFP and Robo3/GFP merged image are shown. The white arrow points to commissural axons and the arrowhead to dI1i axons and soma. The asterisk * indicates the dorsoventral position of dI1c cell bodies. The position of motor neurons (MN) is indicated. At least three embryos were analyzed for each condition. Representative images are shown. (F–H) Photomicrographs of brachial spinal cord sections from E12.5 Barhl2GFP embryos labeled by in situ hybridization for Robo1 (F) Robo2 (G) or Robo3 (H). At least three embryos were analyzed for each condition. (I) Example photomicrographs of the quantification method used illustrated for the sample shown in C. Brachial E12.5 spinal cord samples were labeled with GFP and Robo1, Robo2, or Robo3. The GFP labeling used to anatomically define dI1c or dI1i neurons (outlined in I). (J) Quantification of the relative expression of Robo1 (n = 5), Robo2 (n = 3), and Robo3 (n = 5) expressed as a ratio of labeling in dI1c vs. dI1i neurons. Mean, standard errors, and statistical significance are shown. One way ANOVA Kruskal–Wallis test followed by Dunn’s multiple-comparison analysis was performed. Scale bars in (C–I) are 50 μm and represent the images within the same panel.

Fig 4: Quantification of di1 cell body migration in Robo2–/–:Barhl2GFP and control embryos. (A,B) Example photomicrographs of mouse embryonic spinal cord tissue at E11.5 (A) and E12.5 (B) immunohistochemically labeled with the dI1 transcription factor Barhl2 in control Barhl2GFP (E11.5 n = 5, E12.5 n = 6) and Robo2 mutant Robo2–/–:Barhl2GFP (E11.5 n = 3, E12.5 n = 8) embryos. (C) Schematics representing an E12.5 spinal cord hemisection with the overlying grid used for demarking bins for counting Barhl2+ nuclei for quantification are depicted. The mean number of Barhl2+ cells shown for control (n = 6) and in Robo2–/– (n = 8) is depicted by a heat map (green). The bold boxed regions are enlarged to show the mean number of Barhl2+ neurons per bin. Values in bold are the statistically significant increase (up arrow) or decrease (down arrow), after two-way ANOVA statistical test. Each individual box mean and statistical significance are shown in Supplementary Table 1. Scale bar in (A,B) are 50 μm and represents images within the same panel.

Fig 5: Quantification of dI1 axonal misprojections within the mantle zone of Robo2–/–:Barhl2GFP and control embryos. (A) Example photomicrographs of mouse E11.5 (left panels) and E12.5 (right panels) spinal cord tissue immunohistochemically labeled with GFP (expressed in dI1 neurons, white) in control Barhl2GFP (E11.5 n = 6, E12. 5 n = 7), heterozygote Robo2+/–:Barhl2GFP (E11.5 n = 6, E12. 5 n = 6), and mutant Robo2–/–:Barhl2GFP (E11.5 n = 4, E12. 5 n = 10) embryos. The area demarked by the red dashed lines represents the area quantified region of interest (ROI). The white dotted boxed area in the E12.5 images depict the dorsal and ventral boundaries used for quantification (described in the methods). (B,C) Quantification of the GFP+ axons misprojecting into the ROI is expressed as pixel density per area is shown. This was measured for all genotypic groups, at brachial level for E11.5 and E12.5 in control Barhl2GFP (black circle, E11.5 n = 6, E12.5 n = 7 embryos), heterozygote Robo2+/–:Barhl2GFP (black square, E11.5 n = 6, E12.5 n = 6 embryos), and mutant Robo2–/–:Barhl2GFP (black triangle, E11.5 n = 4, E12.5 n = 10 embryos). This measurement is referred to on the graph as % signal per ROI. Mean, standard errors, and statistical significance of Kruskal–Wallis test are shown. ** represent a p < 0.01. Scale bars in (A) are 50 μm and represents the images within the same embryonic ages, E11.5 and E12.5, respectively.