Fig. 5. GDNF and NRTN are secreted from eye tissues during and after target innervation. (A-D) E9 ciliary ganglion explants grown for 2 days in the presence of GDNF (A), GDNF + anti-GDNF antibody (B), NRTN + anti-GDNF antibody (C), or E5 ECM + anti-GDNF antibody (D). Note that anti-GDNF suppresses GDNF-induced neurite outgrowth, but not NRTN-induced neurite outgrowth. Anti-GDNF is able to suppress E5 ECM-induced neurite outgrowth. A and C were immunostained with a TUJ1 antibody and B and D were unstained phase-contrast micrographs. Scale bar, 200 µm. (E,F) Quantitative analysis of in vitro function-blocking effects by anti-GDNF antibody. (E) Anti-GDNF antibody specifically reduces GDNF-induced neurite outgrowth in a dose-dependent manner. E9 ciliary ganglion explants were grown for 2 days in medium containing 10 ng/ml GDNF and different concentrations of anti-GDNF antibody. The length of neurites (± s.e.) extending from treated and untreated ciliary ganglion explants was measured. In another group, explants were grown in medium containing NRTN or CNTF (10 ng/ml, each) with or without anti-GDNF antibody (0.5 µg/ml). The function-blocking effect by the GDNF antibody is specific to GDNF, and this antibody has no effect on NRTN- or CNTF-induced neurite outgrowth. (F) E5 ECM-induced neurite outgrowth is suppressed by co-incubation with anti-GDNF antibody. In contrast, the GDNF antibody has no effect on E12 ECM-induced neurite outgrowth. (G,H) Quantitative analysis of in vitro function-blocking effects by anti-NRTN antibody. (G) Anti-NRTN antibody reduces NRTN-induced neurite outgrowth in a specific and a dose-dependent manner. The graph shows the average length of neurite (± s.e.) extended from ciliary ganglion explants that were cultured for 2 days in medium containing 10 ng/ml NRTN and different concentrations of anti-NRTN antibody. The graph also shows the effects of anti-NRTN antibody (0.5 µg/ml) on neurite outgrowth induced by GDNF or CNTF (10 ng/ml, each). (H) Anti-NRTN antibody has little effect on E5 ECM-induced neurite outgrowth, but decreases E12 ECM-induced neurite outgrowth. E9 ciliary ganglion explants were grown in medium containing E5 ECM or E12 ECM with or without anti-NRTN antibody. Each bar represents data from 8-14 explants. **, Significantly different from the 0 µg/ml group (P<0.01).