Draxin inhibits axonal outgrowth through the netrin receptor DCC

Abstract

Draxin, a recently identified axon guidance protein, is essential for the formation of forebrain commissures, and can mediate repulsion of netrin-stimulated spinal commissural axons. Here, we report that draxin binds multiple netrin receptors: DCC (deleted in colorectal cancer), Neogenin, UNC5s (H1, H2, H3), and DSCAM (Down's syndrome cell adhesion molecule). Since draxin and Dcc knockouts showed similar phenotype in forebrain commissures formation, we show here the functional importance of draxin/DCC interaction. Draxin interacts with subnanomolar affinity to the netrin receptor DCC, in a region of DCC distinct from its netrin-binding domain. In vitro, neurite outgrowth from cortical and olfactory bulb explants of Dcc knock-out mice is significantly less inhibited by draxin, when compared with neurites from explants of wild-type mice. Furthermore, in comparison with wild-type mice, the growth cone collapse in response to draxin is largely abolished in Dcc-deficient cortical neurons. In vivo, double heteros of draxin/Dcc mice show markedly higher frequency of complete agenesis of corpus callosum than either of the single hetero. These results identify DCC as a convergent receptor for netrin and draxin in axon growth and guidance.

Figure 1.

Draxin binds netrin-1 receptors. The 293 cells were transiently transfected with receptor cDNAs: A–D, Dcc; E, F, UNC5H1; G, H, UNC5H2; I, J, UNC5H3; K, L, Dscam; M, N, Nscam; O, P, Neogenin. After 40 h of post-transfection, cells were treated either with netrin-1 protein (A, B) or with draxin-AP-conditioned medium (C–L, N, P) and double immunostaining was performed using antibodies against respective receptors (A, C, E, G, I, K) and against bound netrin-1 (B) or draxin (D, F, H, J, L). Bound draxin and receptor expression were merged in a manner similar to netrin-1 binding (B) and DCC expression (A). In cases of NCAM-draxin (M, N) and Neogenin-draxin (O, P) binding assay, staining of NCAM or Neogenin protein and bound draxin were done separately, since all anti-NCAM, anti-Neogenin, and anti-draxin antibodies were rabbit polyclonal antibodies. A similar extent of draxin binding (P) and Neogenin expression (O) was observed, while draxin did not bind to the NCAM-transfected cells (N). Scale bars: A–D, M, N, 100 μm; E–L, O, P, 50 μm.

Figure 2.

Draxin interacts with DCC by distinct domain of netrin-1 binding. A, Dcc-transfected 293 cells were incubated with or without draxin-AP-conditioned medium, and later cell lysates were subjected to IP assay. DCC protein in cell lysates and immunoprecipitates was detected by anti-DCC antibody. IB, Immunoblot. B, Conditioned medium from Dcc-ecto-Myc-His-transfected 293 cells was mixed with or without draxin-AP-conditioned medium, and IP assay was performed using anti-draxin as an IP antibody. Asterisk indicates the band of DCC-ecto-Myc-His, which was precipitated by draxin-AP. C, DCC-ecto-Myc-His specifically pulled down draxin-AP when both proteins were mixed and subjected to pull-down assay. ProBond resin bound His-tag. D, Draxin-AP was not immunoprecipitated by Robo 1-ecto-Fc bound by Protein G beads. E, E17 mice brain lysates were incubated with or without draxin-AP, and IP assay was performed using anti-draxin as an IP antibody. Endogenous DCC protein from E17 mouse brain lysates was immunoprecipitated by draxin-AP. The right lane is the brain lysate input. F, DCC-expressing or vector-transfected 293 cells were incubated with different concentrations of draxin-AP containing medium, and free and bound AP activities were measured as described in Materials and Methods. The apparent K_d was estimated as 970 pm. G, The analysis of DCC deletion constructs [DCC-ecto-hGH-His, DCC-FN(1–6)-hGH-His, DCC-IgG(1–4)-His] in pull-down assay experiments with draxin-AP using ProBond resin showed that draxin-AP bound only to the ectodomain and IgG domain of DCC. H, Dcc-transfected 293 cells were incubated with 12 nm netrin-1 first followed by 10 nm draxin-AP. Double immunostaining with anti-draxin and anti-DCC as primary antibodies was performed to see the bound draxin in the DCC expressing cells. Scale bar, 50 μm.

Figure 3.

Dcc-deficient neurite outgrowth and growth cone collapse are significantly less affected by draxin. A–H, Cortical (A–D) and OB (E–H) explants from E17.5 WT (A, C, E, G) and Dcc KO (B, D, F, H) mice were cultured in collagen gel in the presence of either control-AP-conditioned (A, B, E, F) or draxin-AP-conditioned (C, D, G, H) medium for 48 h. Neurites were labeled using an antibody to class III β-tubulin, and results were quantified (I, J) by measuring the maximum neurite length per explant from cortex (A–D) and OB (E–H). The total number of explants, n in I and J, was derived from two independent experiments, and the average maximum neurite length was determined. Similar results were observed in the other two independent experiments. Neurites of cortical (I) and OB (J) explants from Dcc KO mice were significantly (**p < 0.0001, Student's t test) less inhibited by draxin-AP compared with those from WT mice. K, L, Specific DCC immunoreactivity was observed only in the coronal section of E17.5 cortex from WT mouse (K) but not from Dcc KO mouse (L). Cortical neurons from E16.5 wild-type mice embryos were cultured on poly-l-lysine/laminin-coated dishes either in the presence of control medium-conditioned (M) or draxin-AP-conditioned (N) medium for 40 h. Neurites were stained with anti-DCC antibody and inhibited markedly by draxin-AP. Growth cones of E16.5 cortical neurons from both WT and Dcc KO littermate mice were treated with either medium alone or draxin-AP 100 nm for 1 h at 37°C to induce the collapse and were stained with phalloidin-Al 568 to visualize the growth cones. Representative pictures were shown in O, and quantified results were shown in P. Data were obtained from two independent experiments, and growth cones of 40 neurons were counted in each group on average per experiment. Arrows and arrowhead in O indicate the intact growth cone and collapsed cone, respectively. Scale bars: A–H, 200 μm; K, L, 100 μm; M–O, 50 μm. Error bar indicates mean ± SE.

Figure 4.

Frequency of CC phenotype in single- and double-hetero draxin/Dcc receptor mice. A–C, Representative L1-stained CC in coronal section. A, Normal. B, Weak CC phenotype, which was defined as most CC axons that crossed the midline while partial axonal misprojection (arrow) near the midline was observed. C, Strong CC phenotype, which was defined as CC axons that failed to cross the midline and misprojected ventrally (arrow). D, Frequencies of CC phenotypes in single- and double-hetero draxin/Dcc receptor littermate mice at P1. Scale bars, 400 μm.