VEGF Signaling through Neuropilin 1 Guides Commissural Axon Crossing at the Optic Chiasm

Summary During development, the axons of retinal ganglion cell (RGC) neurons must decide whether to cross or avoid the midline at the optic chiasm to project to targets on both sides of the brain. By combining genetic analyses with in vitro assays, we show that neuropilin 1 (NRP1) promotes contralateral RGC projection in mammals. Unexpectedly, the NRP1 ligand involved is not an axon guidance cue of the class 3 semaphorin family, but VEGF164, the neuropilin-binding isoform of the classical vascular growth factor VEGF-A. VEGF164 is expressed at the chiasm midline and is required for normal contralateral growth in vivo. In outgrowth and growth cone turning assays, VEGF164 acts directly on NRP1-expressing contralateral RGCs to provide growth-promoting and chemoattractive signals. These findings have identified a permissive midline signal for axons at the chiasm midline and provide in vivo evidence that VEGF-A is an essential axon guidance cue.

(A) In situ hybridisation (ISH) of horizontal sections through E12.5 -E17.5 wild type eyes with probe specific for Vegfa; staining in the retina is indicated with arrows; N, nasal; T, temporal. (B) ISH of horizontal sections through E12.5 and E14.5 wild types with probes specific for Flt1/Vegfr1 or Flk1/Vegfr2 and histochemical detection of beta-galactosidase activity in horizontal sections of E14.5 eyes from Flt1/Vegfr1 LacZ or Flk1/Vegfr2 LacZ knock-in reporter mice. Solid arrowheads indicate hyaloid vessels, clear arrowheads choroidal vessels; brackets indicate the neuroblastic layer. (C) Immunofluorescence staining of coronal sections of E15.5 Vegfa +/+ and Vegfa 120/120 retinas with antibodies for phosphohistone H 3 (PH3) to detect mitotic cells (red) in the neuroblastic layer and the RGC marker BRN3A (green), or with antibodies for the RGCs/amacrine cell markers ISL1/2 and PAX6 (green). (D) Neurofilament stains of retina flatmounts from E14.5 littermate wild types and Vegfa 120/120 mutants. Scale bars: 100 µm (A, B), 50 µm (C, D). Figure 4, related to Figure 6. VEGF164 promotes RGC axon outgrowth independently of FLK1/VEGFR2. (A) Explants of E14.5 dorsotemporal wild type retina cultured for 24 h on laminin in control culture medium or in medium containing 10 ng/ml or 50ng/ml of VEGF164 or VEGF120 and then fixed and stained with an antibody for -tubulin. (B) Mean (± s.e.m.) total axon outgrowth from explants cultured in the presence of 10 or 50 ng/ml VEGF120 or VEGF164, normalised to outgrowth in control cultures containing no exogenous VEGF (indicated with a dashed line). Number of control explants, 19-21 per quadrant; number of explants cultured with VEGF is indicated on the bars. * = p < 0.05; ** = p < 0.01 compared to controls. (C) Retinal explants of E14.5 wild type dorsotemporal retina cultured for 24 hrs in control medium or medium containing 10 ng/ml VEGF164 plus control goat IgG (1 µg/ml) or FLK1/VEGFR2/KDR (0.3 µg/ml) and then stained with an antibody for -tubulin.

Mouse strains
All animal procedures were performed in accordance with institutional and UK Home Office guidelines. Mice were mated in the evening, and the morning of vaginal plug formation was counted as embryonic (E) 0.5 days. To stage-match embryos, we compared facial and limb development. Nrp1-null mutants were obtained by breeding heterozygous mice on a JF1 genetic background to heterozygous mice on a CD1 background to extend the viability of Nrp1-null mutants beyond E12.5 (Kitsukawa et al., 1997;Schwarz et al., 2004).
Slides were mounted in 90% glycerol/PBS or Vectashield (Vector Labs) and imaged using a Nikon SMZ1500 and DXM1200 camera or a Zeiss LSM510 confocal microscope.

Anterograde and retrograde DiI labelling
Anterograde DiI labelling was performed as described (Plump et al., 2002;Thompson et al., 2006a; Fig. S2A). NIH Image was used to measure the fluorescent intensity of the ipsilateral and contralateral optic tracts in non-saturated wholemount images (Fig. 2D).
The ipsilateral index was calculated by dividing the fluorescent intensity in the ipsilateral optic tract by the sum of the fluorescent intensity in both tracts (adaptation of the method in Herrera et al., 2003). Wild type samples from different genetic backgrounds yielded similar results, demonstrating reproducibility (compare Fig. 2D and 4B,D,F). Retrograde labelling of VEGF164 mutants yielded comparable results, further validating this approach (compare Fig. 4F and 5C). Retrograde DiI labelling was performed as described previously (Manuel et al., 2008). Briefly, DiI crystals were placed in a row over the dorsal thalamus on one side of the brain of formaldehyde-fixed embryos and incubated for 6-9 weeks at room temperature (Fig. 5A). In some experiments, retinas were flatmounted in Vectashield and photographed using a Nikon SMZ1500 microscope and DXM1200 camera. To quantify the relative size of the ipsilateral projection, we determined the number and distribution of labelled RGCs in all 200 µm horizontal sections through the entire contralateral and ipsilateral retina. Statistical comparisons were performed using the Mann Whitney U-Test.
After 24 h, the cultures were fixed and stained with mouse anti--tubulin antibody (1:500; Sigma-Aldrich) followed by Cy3-conjugated goat anti-mouse IgG (1:2000; Jackson ImmunoResearch) and photographed using a Nikon SMZ1500 microscope and DXM1200 camera. Image J (http://rsbweb.nih.gov/ij/) was used to quantify the area covered by the RGC axons as a measure of total axon outgrowth in a minimum of 3 independent experiments. For this analysis, we deleted the explant core from the images, converted the remainder of the image containing the axons that had grown out of the explant into binary mode and quantified the number of black pixels. 1 or 2 explants per experiment lacked outgrowth completely and were excluded from the analysis. We also measured the area of each explant to ensure that differences in explant size did not affect our quantiation, but found no significant differences between control and VEGF-treated cultures. Statistical comparisons were made using ANOVA or the Mann Whitney U-test.

Growth cone turning assay
Growth cone turning assays were performed using an adaptation of the method of Murray and Shewan (2008). Ventrotemporal or dorsotemporal retinal explants were cultured on laminin as above. After 24 h, the dish was flooded with medium warmed to 37˚C and overlaid with a thin layer of vegetable oil. Individual growth cones were positioned on a heated microscope stage at a 45˚ angle and 100 µm distance from a micropipette containing PBS, VEGF164 (50 µg/ml) or VEGF120 (50 µg/ml). They were imaged at 10 min intervals over a 30 min period using a Nikon Diaphot inverted microscope connected to a PC running QWin version 2.1 software (Leica). Reagent gradients were generated by continuous injection from the pipette by an air pulse of 3 psi at 2 Hz and 10 ms duration, applied with a Picospritzer III (Intracel). This resulted in a VEGF concentration of ~50 ng/ml at the growth cone (Lohof et al., 1992). In some experiments, we added 0.5 µg/ml function-blocking goat anti-rat NRP1 or control goat IgG to the culture medium, as in the outgrowth assays. The angle turned by the growth cone and the extent of axon outgrowth was calculated using Image J. Only growth cones that advanced more than 10 µm during the 30 min observation period were included in the analysis. For each condition, we collected data from a minimum of 9 growth cones from at least 3 independent experiments. Statistical comparisons were made using a Mann-Whitney U Test.