Figure 5.

NET1 mediates SVZ-neuroblast migration via NEO1

(A) Schematic of the neurosphere migration assay. Neurospheres were generated from the adult mouse subventricular zone (SVZ) subsequently plated on control or NET1 proteins.

(B) Immunocytochemisty for NEO1, DCC, and TUJ1 (to label SVZ-neuroblasts) in DIV5 SVZ-NSC cultures. SVZ-neurospheres (NSCs) and neuroblasts (arrowheads) express NEO1 and DCC. Boxed areas are shown at higher magnification on the right. Scale bar, 50 μm.

(C and D) Analysis of migrating neurons from SVZ-NSCs grown on full-length NET1 constructs. Ablation of either NET1-NEO1 interface-1 or -2 interactions causes loss of NET1-mediated neuron migration. Mean ± SEM of the relative number of Tuj1/DCX positive migrating neurons per neurosphere: vehicle = 100.00, NET1_FL-WT = 223.25 ± 27.51, NET1_FL-Interface-1 = 114.20 ± 6.07, NET1_FL-Interface-2 = 85.06 ± 13.02, n = 3-4 experiments. Brown-Forsythe to test significant difference between SDs (p < 0.05): ns. One-way ANOVA followed by Tukey’s multiple comparisons test: vehicle vs. NET1_FL-WT P = 0.0003, NET1_FL-WT vs. NET1_FL-Interface-1 P = 0.0007, NET1_FL vs. NET1_FL-Interface-2 p < 0.0001. Representative samples of mouse SVZ-NSCs grown on coverslips coated with indicated proteins are shown in (D). Boxed areas are shown at higher magnification on the right of each panel. Migrating neurons (white arrowheads) were identified via labelling with the microtubule markers TUJ1 (green) and DCX (red) as well as the nuclear marker DAPI (blue).

(E and F) Analysis and representative samples of migrating SVZ neuroblasts (white arrowheads) grown on NET1_ΔNTR. NET1 lacking the C-terminal NTR domain fails to increase neuron migration. Mean ± S.E.M of the relative number of TUJ1/DCX-positive migrating neurons per neurosphere: control (vehicle) = 100.00, NET1_ΔNTR = 86.18 ± 2.215, n = 2 individual experiments. Unpaired t test: p = 0.0247. See also Figure S5.