Figure 6. VEGFR2 deficiency in hippocampal neurons promotes axonal branching by increasing filopodia formation.

(A) Representative images of 3 DIV hippocampal neurons isolated from control and Nes-cre;Kdr^lox/- embryos and stained beta-III tubulin. Axon branches are indicated by red arrowheads. Scale bars 50 µm. (B,C) Quantification of the axon branch number (B) and branch length (C) of control and Nes-cre;Kdr^lox/- neurons at 3 DIV. Data are represented as % of control. Mean ± SEM,>120 neurons from n = 6. ****p<0.0001; unpaired Student’s ttest. (D,E) Time-lapse movies over the course of 4 hr were recorded from 1 DIV hippocampal neurons of control and Nes-cre;Kdr^lox/- mice. The net axon branch growth was quantified over the course of the movies (D) and the growth rate of axon branch was calculated (E). Data represents mean ± SEM of at least three independent experiments. *p<0.05; unpaired Student’s ttest. (F) The number of newly forming actin patches during 2 min per 10 µm axon segment was counted in neurons of control and Nes-cre;Kdr^lox/- mice. Data represents mean ± SEM of at least three independent experiments. n.s. not significant; unpaired Student’s ttest. (G,H) The number (G) and the size (H) of newly formed protrusions and filopodia was analyzed during the course of 10 min in neurons of control and Nes-cre;Kdr^lox/- mice. Data represents mean ± SEM of at least three independent experiments. *p<0.05; **p<0.01; unpaired Student’s ttest.

Figure 6—figure supplement 1. α-VEGFR2 antibody treatment recapitulates the phenotype observed in Nes-cre;Kdr^lox/- mice.

(A,B) Data showing the number of retracting axon branches (A) as well as the retraction rate of axon branches (B) from control and Nes-cre;Kdr^lox/- mice shown in Figure 6. Data represents mean ± SEM of at least three independent experiments. n.s. not significant; unpaired Student’s ttest. (C) Representative images of beta-III tubulin stained 3 DIV hippocampal neurons treated with control or α-VEGFR2 AB. Axon branches are indicated by red arrowheads. Scale bars 50 µm. (D,E) Quantification of the branch number (D) and branch length (E) of control and α-VEGFR2 AB treated neurons at 3 DIV. Data are represented as % of control, mean ± SEM, from at least three independent experiments. *p<0.05; unpaired Student’s ttest. (F–H) Time-lapse movies over the course of 4 hr were recorded from 1 DIV hippocampal neurons of control and α-VEGFR2 AB treated neurons. The number of retracting axon branches was quantified over the course of the movies (G) and the net growth as well as the retraction rate of axon branch was calculated (F,H). Data represents mean ± SEM of at least three independent experiments. n.s. not significant; ***p<0.01; unpaired Student’s ttest. (I) The number of newly forming actin patches during 2 min per 10 µm axon segment was counted in neurons treated with control and α-VEGFR2 AB. Data represents mean ± SEM of at least three independent experiments. n.s. not significant; unpaired Student’s ttest. (J,K) The number and the size of newly forming protrusions and filopodia was analyzed during the course of 10 min in neurons treated with control and α-VEGFR2 AB. Data represents mean ± SEM of at least three independent experiments. *p<0.05; **p<0.01; unpaired Student’s ttest.