Fig. 4. GP cell migration in collagen gel co-cultured with BHK-cell-clusters secreting netrin 1 or Sema3a. (A) GP cell migration observed without the effect of a guidance cue. (B) GP cell migration observed under the effect of netrin 1 expressed by BHK cell clusters at a proximal cut end and at an additional point. (C) GP cell migration observed under the effect of Sema3a expressed by BHK cell clusters. (D) A schematic diagram showing the arrangement of BHK cell clusters secreting a guidance cue and a piece of optic nerve (ON) in a collagen gel culture. Gray indicates the guidance cue source. Pink indicates the sectors containing the guidance cue source and green indicates the sector with a smaller guidance cue effect. The sectors containing the guidance cue source were drawn 100 µm wider on both sides. (E-I) The distribution of NG2-positive cells in the co-culture with netrin 1 sources. (E) Low magnification photograph. (F) NG2-negative cells with large nuclei found near the netrin 1 source. (G) Bipolar NG2-positive cells. (H) Diagram of the culture in E. Red in H and I indicates the NG2-positive migrating cells. (I) Diagram of the culture in E showing the distribution of NG2-positive cells. (J-M) The distribution of O4-positive cells in the co-culture with netrin 1 sources. (J) Low magnification photograph. (K) Bipolar O4-positive cells. (L) Diagram of the culture in J. Red in L and M indicates the O4-positive migrating cells. (M) Diagram of the culture in J showing the distribution of O4-positive cells. (N-Q) The distribution of PLP-positive cells in the co-culture with netrin 1 sources. (N) Low magnification photograph. (O) Bipolar PLP-positive cells. (P) Diagram of the culture in N. Red in P and Q indicates the PLP-positive migrating cells. (Q) Diagram of the culture in N showing the distribution of PLP-positive cells. (R-U) The distribution of GFAP-positive cells in the co-culture with netrin 1 sources. (R) Low magnification photograph. (S) Bipolar GFAP-positive cells. (T) Diagram of the culture in R. Red in T and U indicates the GFAP-positive migrating cells. (U) Diagram of the culture in R showing the distribution of GFAP-positive cells. (V) Diagram of a co-culture of an optic nerve and Sema3a sources showing the distribution of NG2-positive cells (red). (W) Diagram of a co-culture of an optic nerve and Sema3a sources showing the distribution of O4-positive cells (red). (X) Diagram of a co-culture of an optic nerve and Sema3a sources showing the distribution of PLP-positive cells (red). (Y) Diagram of a co-culture of an optic nerve and Sema3a sources showing the distribution of GFAP-positive cells (red). N, netrin 1; ON, optic nerve; S, Sema3a. Scale bar in A is 500 µm, (A-C,E,J,N,R); in F is 50 µm (F,G,K,O,S).