Figure S5.

Silencing of RGMA-mediated growth cone collapse by NET1 is DCC-independent, related to Figures 3, 5, 6, and 7

(A) Immunocytochemistry of NEO1, deleted in colorectal cancer (DCC) and TUJ1 in P0 mouse cortical neurons at DIV3. Scale bar = 50 μm. (B) Mean ± S.E.M. of the percentage of collapsed growth cones following exposure to RGMA or RGMA + NET1_FL in cortical neurons from Emx1-Cre^-/-;Dcc^fl/+ (control) or Emx1-Cre^+/-;Dcc^fl/fl (knockout) mice. Emx1-Cre^-/-;Dcc^fl/+ (mean ± S.E.M.): vehicle = 18.83 ± 2.17, RGMA = 49.40 ± 3.61, RGMA + NET1_FL = 19.47 ± 1.47. Emx1-Cre^+/-;Dcc^fl/fl (mean ± S.E.M.): vehicle = 22.35 ± 1.48, RGMA = 45.28 ± 3.15, RGMA + NET1_FL = 27.88 ± 2.53. n = 6 experiments, two-way ANOVA with Tukey’s multiple comparisons test, ^∗∗∗ p < 0.0001. (C-G) Quantification of migration distance in SVZ-NSC assays and analysis of GFP⁺ neurons following IUE. Migration distance (per 50 μm bin) of SVZ-neuroblasts related to (C) Figures 5C and 5D, (D) Figures 5E and 5F, (E) Figures 6A and 6B, (F) Figures 6I and 6J, and (G) Figures 6G and 6H. (H-J) Cortical migration of GFP⁺ electroporated neurons. (H) At E14 embryos were in utero electroporated (IUE) with expression vectors for GFP, RGMA, and/or NET1 (each condition has GFP). Embryos were harvested two days later at E16. Migration distance from the VZ to the MZ was measured per bin (1-8) (i.e. the number of GFP⁺ cells per bin/total GFP⁺ cells). (I-J) Electroporation of RGMA or NET1 caused an increase in the number of GFP⁺ in bins near the VZ, indicating reduced migration towards the MZ. Simultaneous overexpression of RGMA and NET1 in part rescued this inhibitory effect. The reduction in the number of GFP⁺ cells in more upper layers was visible in the images but did not reach statistical significance due to the low numbers of these more superficially located neurons. One-way ANOVA followed by Sidaks multiple comparisons test: RGMA vs. GFP bin 1 p < 0.0001, RGMA vs. GFP bin 2 p = 0.0305, NET1 vs. GFP bin 1 p = 0.379, NET1 vs. GFP bin 2 p = 0.362. GFP: n = 6 animals, RGMA: n = 6 animals, NET1: n = 4 animals, NET1+RGMA: n = 5 animals. Marker: 100 μm. VZ, ventricular zone; SVZ, subventricular zone; IZ, intermediate zone; CP, cortical plate; MZ, marginal zone.