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Comparative Study
. 2007 Jan 24;27(4):957-68.
doi: 10.1523/JNEUROSCI.4616-06.2007.

p130CAS is required for netrin signaling and commissural axon guidance

Guofa Liu  1 , Weiquan Li, Xue Gao, Xiaoling Li, Claudia Jürgensen, Hwan-Tae Park, Nah-Young Shin, Jian Yu, Ming-Liang He, Steven K Hanks, Jane Y Wu, Kun-Liang Guan, Yi Rao
Affiliations
Comparative Study

p130CAS is required for netrin signaling and commissural axon guidance

Guofa Liu et al. J Neurosci. .

Abstract

Netrins are an important family of axon guidance cues. Here, we report that netrin-1 induces tyrosine phosphorylation of p130(CAS) (Crk-associated substrate). Our biochemical studies indicate that p130(CAS) is downstream of the Src family kinases and upstream of the small GTPase Rac1 and Cdc42. Inhibition of p130(CAS) signaling blocks both the neurite outgrowth-promoting activity and the axon attraction activity of netrin-1. p130(CAS) RNA interference inhibits the attraction of commissural axons in the spinal cord by netrin-1 and causes defects in commissural axon projection in the embryo. These results demonstrate that p130(CAS) is a key component in the netrin signal transduction pathway and plays an important role in guiding commissural axons in vivo.

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Figures

Figure 1.
Figure 1.
Expression of p130CAS in primary culture neurons. a–f, Expression of DCC (a, d) and p130CAS (b, e) in primary cortical neurons from E15 mice. c is the merged image of a and b. f is the merged image of d and e. d, e, and f are higher-magnification images of the boxed regions in a, b, and c, respectively. Neurons from E15 mice cortex were cultured overnight and immunostained with anti-DCC (G97–449) and anti-p130CAS (SC-860). Scale bar, 10 μm. g–m, Expression of DCC (g, j) and p130CAS (h, k) in E13 primary spinal cord neurons. i is the superimposed images of g and h. l is the superimposed image of j and k. m is the phase image of j, k, and l. j–m are higher-magnification images showing DCC and p130CAS expression in the growth cone of embryonic spinal cord neurons. Scale bar, 10 μm. n–o, Expression of p130CAS in E13 mice spinal cord. o is the higher-magnification image of the boxed region of image n. p130CAS is strongly expressed in the commissural axons as they project toward the floor plate (white arrow), the ventral commissural region (VC; arrowhead), and lateral fasciculus (LF; green arrow). Scale bar, 100 μm.
Figure 2.
Figure 2.
Increase of p130CAS tyrosine phosphorylation by netrin-1. a–c, Induction of p130CAS tyrosine phosphorylation in dissociated cortical neurons by netrin-1. The anti-p130CAS antibody (a) or the anti-phosphotyrosine (pY) antibody (b) was used to immunoprecipitate proteins from cortical neurons treated with the sham-purified control (left lane) or netrin-1 (right lane), and the blot was analyzed with the anti-phosphotyrosine antibody (a) or the anti-p130CAS antibody (b). Tyrosine phosphorylation of p130CAS tyrosine residues 165 and 249 was increased in dissociated cortical neurons by netrin-1 (c). d, e. Netrin-1 increased tyrosine phosphorylation of p130CAS at tyrosine residues 165 and 249 in dissociated E13 dorsal spinal cord neurons (d, e), which was blocked by the anti-DCC antibody (d). f, Interaction of endogenous p130 CAS and FAK in E15 cortical neurons. Cortical neurons were treated with the 250 ng/ml purified netrin-1 for 5 or 10 min. Extracts from 2.5 × 107 cortical cells were immunoprecipitated with an anti-p130CAS antibody and analyzed with an anti-FAK antibody. g, Interaction of endogenous p130CAS and Fyn in cortical neurons. Left lane, Immunoprecipitation (IP) with IgG; middle lane, IP with an anti-Fyn antibody; right lane, IP with an anti-Fyn antibody. h, P3 domain in DCC is required for induction of p130CAS tyrosine phosphorylation by netrin-1. HEK 293 cells were transfected with a DCC construct and a construct encoding p130CAS tagged by HA (HA-CAS) and treated with netrin-1 or the sham-purified control for 20 min. Tyrosine phosphorylation of p130CAS was determined by IP with the anti-HA antibody and analyzed by probing the blots (IB) with the anti-phosphotyrosine antibody. DCC-ΔP1, ΔP2, ΔP3, and Δ1/2P3 correspond to deletion of residues of DCC intracellular domains 1147–1171, 1335–1356, 1412–1447, and 1426–1447, respectively. i, The C-terminal half of P3 domain of DCC is essential for netrin induction of tyrosine phosphorylation of p130CAS. 1420Y/F and 4Y/F indicate mutations in the tyrosine(s) of the intracellular domain of DCC, Y1420, and Y1261, Y1272, Y1363 and Y1420, respectively.
Figure 3.
Figure 3.
Induction of p130CAS tyrosine phosphorylation by netrin-1 required FAK and Fyn. a, The C-terminal domain of FAK (FRNK) and Src family kinase-specific inhibitor PP2 inhibited netrin-induced p130CAS tyrosine phosphorylation in HEK cells. HEK293 cells were transfected with DCC, HA-CAS, and Myc-FRNK. Tyrosine phosphorylation of p130CAS was determined by immunoprecipitation with an anti-HA antibody and immunoblotting with the anti-phosphotyrosine antibody. b, PP2, but not PP3, blocked netrin-stimulated tyrosine phosphorylation of endogenous p130CAS in primary neurons. Cortical cells (6 × 106 plate/well) were stimulated with netrin-1. The anti-p130CAS antibody was used to immunoprecipitate proteins, and the blot was analyzed with the anti-phosphotyrosine antibody (top) or anti-p130CAS antibody (bottom). c, A p130CAS mutant, myc-p130CAS (F15), could not block netrin-induced FAK phosphorylation. HEK cells were transfected with DCC, HA-FAK, and myc-p130CAS (F15). The anti-HA antibody was used to immunoprecipitate the extracts, and anti-phosphotyrosine was used to probe the immunoblots. The bottom panel showed Western blot analysis with the anti-FAK antibody to control for amount of input proteins. d, The p130CAS mutant, CAS-SH3 tagged by HA, also could not inhibit netrin-stimulated FAK phosphorylation. HEK cells were transfected with DCC, myc-FAK, and HA-CAS-SH3. FAK phosphorylation was detected by immunoprecipitation with anti-myc antibody immunoprecipitate and immunoblot with the anti-phosphotyrosine antibody. e, p130CAS (F15) could not block Fyn tyrosine phosphorylation. HEK293 cells were transfected with DCC, Fyn, and myc-p130CAS (F15). Fyn tyrosine phosphorylation was analyzed by immunoprecipitation with an anti-Fyn antibody and immunoblotting with an anti-phospho-Src antibody. f, Netrin-1 increased FAK phosphorylation at tyrosine residue 576 in the p130CAS−/− mouse embryonic fibroblasts, which were blocked by PP2.
Figure 4.
Figure 4.
Involvement of p130CAS in netrin-induced activation of Rac1 and Cdc42. HEK293 cells were transfected with DCC and either the full-length p130CAS (HA-CAS) or the dominant-negative p130CAS mutant myc-p130CAS (F15). Cell lysates were incubated with GST-PAK1-CRIB fusion protein and GTP-bounded Rac1, or GTP-bounded Cdc42 was detected by Western blot analysis with the anti-Rac1 and anti-Cdc42 antibodies, respectively. a shows an example when Rac1 and Cdc42 activation by netrin-1 was completely inhibited by p130CAS (F15). b and c show statistics of Rac1 and Cdc42 activation from three independent experiments. In b, netrin could not induce Rac1 activation in the presence of p130CAS (F15): the difference between the netrin (+) and netrin (−) groups in the presence of p130CAS (F15) is not statistically significant (p > 0.1). In c, p130CAS (F15) blocked netrin-induced Cdc42 activation; the difference between the netrin (+) and netrin (−) groups in the presence of p130CAS (F15) is not statistically significant (p = 0.06). Error bars are SEM from three independent experiments.
Figure 5.
Figure 5.
The induction of neurite outgrowth of dissociated cortical neurons by netrin-1 was blocked by p130CAS RNAi. Cortical neurons from E15 mouse embryos were cotransfected with Venus YFP and the p130CAS shRNA (c, d, g, h) or the control shRNA vector (a, b, e, f) and plated on coverslips coated with PDL and laminin. Neurons were stained with rhodamine-phalloidin (red) and Hoechst. Only the neurites of YFP-positive neurons not in contact with other cells were measured and used in the statistical analyses. Data are mean ± SEM from three separate experiments. Scale bar, 20 μm. a–d, Neurite outgrowth from YFP-positive E15 cortical neurons transfected with control vectors (a, b) or p130CAS shRNA (c, d) in the presence of purified netrin-1 (b, d) or in the sham-purified control (a, c) after culturing for 20 h. Transfected neurons were labeled by YFP, and filamentous actin was visualized by staining with rhodamine-coupled phalloidin (red). p130CAS shRNA inhibited the promotion of neurite outgrowth by netrin (b, d, i, j). e–h, Similar to a–d except that neurons were cultured for 40 h. Scale bar, 20 μm. i, j, Quantification of netrin-1-induced neurite outgrowth of cortical neurons cultured for 20 h. Both the length of the longest neurite from each neuron (i) and the total length of all neurites from each neuron (j) were inhibited by p130CAS shRNA. The p values are <0.0001 between RNAi-treated neurons and control neurons. The length on the y-axis is in micrometers. k, Quantification of the length of the longest neurite from each cortical neuron cultured for 40 h. Group I, 53.26 ± 4.15 μm; Group II, 113.08 ± 5.44 μm; Group III, 56.67 ± 4.46 μm; Group IV, 53.31 ± 4.53 μm. The difference between Groups I and II is very significant (p value <0.0001); the difference between Groups II and IV is very significant (p < 0.0001); the difference between Groups III and IV is not significant (p = 0.577). These results indicate that netrin promoted neurite outgrowth (compare Groups I and II) and that p130CAS shRNA significantly inhibited the effect of netrin (compare Groups II and IV). l, Quantification of the total length of all neurites from each cortical neuron cultured for 40 h. Group I, 73.55 ± 4.45 μm; Group II, 173.00 ± 6.58 μm; Group III, 78.15 ± 4.66 μm; Group IV, 91.6 ± 5.53 μm. m, p130CAS shRNA significantly reduced p130CAS protein level in E15 primary cortical neurons (left) 3 d after shRNA transfection (left). p130CAS protein level is reduced in dissociated cortical neurons 3 d after siRNA transfection (right).
Figure 6.
Figure 6.
Inhibition of netrin-induced turning of spinal cord axons by the p130CAS (F15) mutant. Electroporation of Venus GFP into the neural tube of chick embryos allowed visualization of axons. Netrin attraction was indicated by the turning of axons toward aggregates of netrin-1 secreting HEK cells (Liu et al., 2004). In a, c, e, and g, neural tube explants were cocultured with control HEK cells, and axons do not turn toward these cells. In b, d, f, and h, neural tube explants were cocultured with aggregates of HEK cells secreting netrin-1. In a and b, Venus GFP alone was electroporated into the neural tube. In c and d, Venus GFP was electroporated into the neural tube together with the control vector. In e and f, Venus GFP was electroporated into the neural tube together with the wild-type p130CAS. In g and h, Venus GFP was coelectroporated with the p130CAS (F15) mutant. Only the p130CAS (F15) mutant inhibited axon turning toward the netrin source. Scale bar, 100 μm. i, Quantification of axon turning. Turning percentage was calculated from the number of axons extended toward the netrin source divided by the total number of axons within 300 μm from the edge of the HEK cell aggregates. Only axons that had an angle of turning >5° were counted. Data are mean ± SEM. Group I (example shown in a), 5.7 ± 1.7%; Group II (exemplified in b), 90.6 ± 2.1%; Group III (exemplified in c), 5.8 ± 2.0; Group IV (as in d), 91.3 ± 1.7%; Group V (as in e), 6.2%±2.0%; Group VI (as in f), 88.7 ± 4.1%; Group VII (as in g), 4.1 ± 1.6%; Group VIII (as in h), 12.4 ± 3.6%. The numbers on the top (n) indicate the numbers of explants tested. Net, Netrin-1-secreting HEK cell aggregates. p values are as follows (Student's t test): <0.0001 between Groups I and II; <0.0001 between Groups III and IV; <0.0001 between Groups V and VI; <0.0001 between Groups VIII and II, IV, or VI. Error bars are SEM.
Figure 7.
Figure 7.
Requirement of p130CAS for netrin-induced attractive turning of spinal cord axons. Venus YFP was electroporated into the chick neural tube with the control vector or p130CAS shRNA or wild-type p130CAS plus shRNA or siRNA. a, c, g, Aggregates of control HEK cells could not attract the commissural axons. b, e, An aggregate of netrin-secreting HEK cells attracted the commissural axons transfected with Venus YFP only (e) or with Venus YFP and the control vector (b). d, p130CAS shRNA blocked the attraction of the commissural axons by netrin-1. f, The attractive effect on the commissural axons by netrin-1 was also blocked by p130CAS siRNA. h, Cotransfected p130CAS shRNA with wild-type p130CAS rescued the attraction of netrin-1. Scale bar, 100 μm. i. Quantification of axon turning. The turning percentages were as follows: 5.4 ± 1.0% (Group I, not shown); 90.1 ± 2.0% (Group II, as exemplified in e); 6.1 ± 1.3% (Group III, example shown in a); 90.3 ± 2.1% (Group IV, as exemplified in b); 5.0 ± 1.4% (Group V, not shown); 28.5 ± 3.9% (Group VI, f); 6.3 ± 1.6 (Group VII, as exemplified in c); 16.8 ± 3.4% (Group VIII, as exemplified in d); 6.0 ± 1.5% (Group IX, g); 67.3 ± 5.2% (Group X, h). WT, Wild type of p130CAS. p values are as follows (Student's t test): <0.0001 between Groups I and II, Group III, and IV; <0.0001 between Groups II and VI, Group IV and VIII; <0.0001 between Group VIII and X. Error bars are SEM.
Figure 8.
Figure 8.
Inhibition of commissural axon projection in vivo by p130CAS (F15) mutant. a, Schematic diagram showing the experimental design. DNA was injected into chick neural tube in ovo at stage 12, and samples were isolated and fixed at stage 23. Anti-axonin-1 immunostaining shows the commissural axons (Stoeckli and Landmesser, 1995; Stoeckli et al., 1997). b–d show neurons electroporated with Venus YFP only. e–g are neurons electroporated with Venus YFP and the control vector. h–j are neurons electroporated with Venus YFP and p130CAS (F15). b, e, and h show YFP images. c, f, and i show anti-axonin-1 antibody immunostaining. d, g, and j are merged images. Scale bar, 100 μm. k, Quantification of the percentage of axons reaching the floor plate. Group I (Venus YFP alone), 79.6 ± 2.0%; Group II (YFP coelectroporated with the vector), 76.5 ± 3.0%; Group III (YFP coelectroporated the wild-type p130CAS), 75.4 ± 2.0%; Group IV (YFP coelectroporated with p130CAS F15 mutant), 26.8 ± 3.0%. Group IV is significantly different from any other group (p values <0.0001; Student's t test). Numbers of chick embryos examined are shown as n in each column. WT, Wild-type p130CAS; F15, p130CAS (F15). l, Quantification of the average distance of axons away from the floor plate. Group I, 7.2 ± 1.1 μm; Group II, 7.1 ± 2.0 μm; Group III, 7.4 ± 1.0 μm; Group IV, 45.1 ± 5.76 μm. p value <0.0001 between group IV and group I, II, or III. Error bars are SEM.
Figure 9.
Figure 9.
Inhibition of spinal cord commissural axon projection in vivo by p130CAS RNAi. a–c, Venus YFP was coelectroporated with the control shRNA vector into the chick neural tube. d–f, Venus YFP was coelectroporated with p130CAS shRNA. g–i, p130CAS siRNA was coelectroporated with Venus YFP. j–l, p130CAS shRNA was coelectroporated with wild-type p130CAS and Venus YFP. a, d, g, and j are YFP images; b, e, h, and k are images of anti-axonin-1 antibody immunostaining; c, f, i, and l are merged images. g, Quantification of the percentage of axons reaching the floor plate: 79.6 ± 2.0% for commissural axons expressing Venus YFP only; 54.5 ± 4.1% for siRNA group; 80.8 ± 2.6% for commissural axons expressing Venus YFP and the control vector; 29.9 ± 4.1% for commissural axons expressing Venus YFP and p130CAS shRNA; 71.6 ± 4.0% for shRNA and wild-type group (p < 0.001 between Venus YFP and siRNA group; p < 0.0001 between vector and shRNA group; p < 0.0001 between shRNA and shRNA plus wild-type group. Student's t test). h, Quantification of the average distance of axons away from the floor plate: 7.2 ± 0.9 for Venus YFP; 20.9 ± 3.3 for siRNA; 7.6 ± 1.5 μm for the control vector; 44.3 ± 5.4 μm for p130CAS shRNA; 11.4 ± 2.3 for shRNA plus wild type (p < 0.01 between Venus YFP and siRNA group; p < 0.0001 between vector and shRNA group; p < 0.0001 between shRNA and shRNA plus wild-type group). The numbers on the top (n) indicate the number of embryos tested. Scale bar, 100 μm. Error bars are SEM.
Figure 10.
Figure 10.
Wild-type p130CAS rescue of commissural defects caused by p130CAS RNAi. The chick neural tube was electroporated with Venus YFP only (a, a′), Venus YFP plus shRNA vector (b, b′), Venus YFP plus shRNA (c, c′), or Venus YFP plus shRNA plus wild-type p130CAS (d, d′). a′–d′ are the monochrome images of a–d. p130CAS shRNA not only inhibited the commissural axon extension but also caused aberrant pathfinding (c, c′). Overexpression of wild-type p130CAS rescued the defect of p130CAS shRNA on commissural axon projection and turning (d, d′). The effect of p130CAS shRNA on the spinal cord commissural axon projection was also observed in embryonic mice. E10.5 mouse neural tube was electroporated with Venus YFP only (e, e′), Venus YFP plus shRNA vector (f, f′), or Venus YFP plus shRNA (g, g′). e′, f′, and g′ are images of e, f, and g. The arrow shows a misguiding axon, and the arrowhead indicates the short commissural axon. Scale bar, 100 μm.
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