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Guidance of glial precursor cell migration by secreted cues in the developing optic nerve

Yoshihiko Sugimoto2, Masahiko Taniguchi4, Takeshi Yagi5, Yoshio Akagi2, Yoshiaki Nojyo3 and Nobuaki Tamamaki1,*

1 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto, 606-8501 Japan
2 Department of Ophthalmology and
3 Department of Anatomy, Fukui Medical University, Fukui 910-0063, Japan
4 Department of Biochemistry and Molecular Biology, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan
5 Institute for Molecular and Cellular Biology, Osaka University, Suita, Osaka 565-0871, Japan



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  		Fig. 1. GP cell migration in optic nerves in in vivo or in vitro cultures together with the optic chiasma and/or the eyeball. Migrating cells exiting from the UV-irradiated area were observed when the optic nerve was cultured with the optic chiasma. (A) schematic diagram showing the principle of the UV-TD labeling method used to study cell migration. The UV-irradiated site was identified by TD-positive stationary cells (see Tamamaki et al., 1999 or the text for details). (B) TD-labeled cells observed in a paraffin section of an optic nerve fixed immediately after UV irradiation. (C) TD-labeled cells observed in an optic nerve fixed after culture for five hours. (D) TD-labeled cells in an optic nerve cultured with the optic chiasma and eyeball for five hours. (E-G) reconstructed UV-irradiated area. Photographs of serial sections were taken on transparent paper and they were overlapped to confirm the UV-irradiation area. (H-I) Reconstructed pattern of the TD-labeled nuclei distribution shown in C and D. The labeled migrating cells (red dots) were traced onto the transparent photographs, which were then overlapped to show the distribution of the labeled migrating cells. Arrows in C, F and H, and D, G and I indicate the same cells. Arrowheads indicate the UV-irradiated area, as estimated from the distribution of TD-labeled stationary cells. (J) NG2-negative immunoreactive cells in the newborn rat optic nerve. (K) Double staining study for TD and NG2 immunoreactivity. The rectangular area in the figure was enlarged in L. (L) NG2-positive migrating cells (single arrows) and NG2-negative migrating cells (double arrows). (M) An optic nerve cultured with an eyeball and the chiasma in vitro. (N) Schematic diagrams summarizing the condition under which the GP cell migration was observed. The optic chiasma was necessary to guide the GP cell migration distally. Scale bar in B, 100 µm (B-I); in K is 100 µm; in J and L are 10 µm.

 


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  		Fig. 2. In situ hybridization to detect the expression of repulsive cues in the optic chiasma and that of their receptors in the optic nerves. (A) Netrin 1 expression in a frontal section of a postnatal day 1 (P1) rat brain. The ependymal layer of the third ventricle and the lateral edges of the optic chiasma were netrin 1 mRNA-positive. (B) In a horizontal section of the optic chiasma, a netrin 1 signal was found in the anterior pole and lateral edge of the optic chiasma. (C) A Sema3a signal was also found in the anterior pole and lateral edges of the optic chiasma in a horizontal section. Arrows indicate the significant accumulation of signals. (D) Many cell nuclei in the optic nerve were positive for Unc5h1 mRNA. The inset shows the sensing probe signal. (E) Many cell nuclei were also positive for Nrp mRNA. The inset shows the sensing probe signal. (F) Results of RT-PCR using mRNA obtained from a newborn rat optic nerve. Lane 1, Unc5h1; lane 2, molecular marker 0X174 HacIII digested; lane 3, Nrp. CH, optic chiasma; OP, optic nerve. The calibration bar in A-C is 100 µm and that in D-E is 10 µm.

 


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  		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).

 


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  		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).

 

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