Fig. 3. GP cell migration in an optic nerve co-cultured with COS1 cell clusters secreting netrin 1 and /or Sema3a. The cell migration was shown by reconstructed distribution of TD-labeled migrating cells 5 hours after UV irradiation. The left sides of all the figures represent the distal direction of the optic nerve. (A-I) Small arrowheads indicate the UV-irradiation area. Large arrowheads indicate the direction of diffusion of guidance cue. (A) Cell migration in an optic nerve co-cultured with COS1 cell clusters secreting netrin 1. The COS1 cell clusters were placed at the proximal cut end of an optic nerve. (B) Cell migration in an optic nerve co-cultured with COS1-cell-clusters secreting Sema3a. The COS1 cell clusters were placed at the proximal cut end of an optic nerve. (C) Cell migration in an optic nerve co-cultured with COS1 cell clusters secreting both netrin 1 and Sema3a. (D-F) The same conditions as in A-C, except that the COS1 cell clusters were placed at the distal cut end of the optic nerves. (G) Cell migration in an optic nerve co-cultured with two COS1 cell clusters, one of which was secreting netrin 1 and the other was secreting Sema3a, placed at opposite cut ends of an optic nerve. (H) Cell migration in an optic nerve treated with anti-neuropilin blocking serum and co-cultured with COS1 cell clusters secreting Sema3a. The number of migrating cells significantly decreased. (I) Cell migration in an optic nerve treated with the anti-Nrp serum and co-cultured with COS1 cell clusters secreting netrin 1. The number of migrating cells was similar to that in A. (J) Migrating cells seen with Nomarski optics. Large TD-positive migrating cells responsive to Sema3a had a leading process (arrowhead) directed away from the Sema3a source. (K) Small TD-positive migrating cells responsive to netrin 1 had a leading process (arrowhead) directed away from the netrin 1 source. (L) Cell migration in an optic nerve obtained from a Sema3a mutant mouse and co-cultured with COS1 cell clusters secreting Sema3a. (M) The smallest TD-positive migrating cells responsive to Sema3a (indicated by an arrow in L) had a leading process (arrowhead) directed away from the Sema3a source. (N) Schematic diagram of the experimental arrangement. Scale bar in A is 100 µm (A-I,L); in J is 5 µm (J,K,M).