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.