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doi: 10.1242/10.1242/dev.00424

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Netrin 1 mediates spinal cord oligodendrocyte precursor dispersal

Hui-Hsin Tsai1, Marc Tessier-Lavigne2 and Robert H. Miller1,*

1 Department of Neurosciences, Case Western Reserve University, School Of Medicine, Cleveland, OH 44106, USA
2 Department of Biological Sciences, Stanford University, Herrin Lab 150, 385 Serra Mall Stanford CA, 94305, USA



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  		Fig. 1. Generation and migration of oligodendrocyte precursor depends on the spinal cord region. (A) The spinal cord shows three distinct domains used in this study. The spinal cord was opened at the roof plate and cutting at the sulcus limitians generates dorsal explants. Ventral regions were then bisected into intermediate and ventral explants. (B,C) Dorsal explants from stage 29 chick embryos did not give rise to oligodendrocyte precursors after 3 days in culture, consistent with previous findings (Warf et al., 1991Go), although numerous axon bundles were seen. (D,E) Ventral and intermediate explants generated O4-positive oligodendrocyte precursors that differed in number and migrational distances. (F,G) Ventral explants generated higher numbers of oligodendrocyte precursors that migrated farther than those from intermediate explants. P<0.001. Scale bar: 250 µm.

 


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  		Fig. 4. Netrin is a chemorepellent for purified chick spinal cord oligodendrocyte precursors in chemotaxis assays. (A) Ethidium bromide labeled migrating oligodendrocyte precursors on the lower surface of the filter after 20 hours in control conditions. (B) The number of migrating cells decreased when netrin 1 was added to the lower well, suggesting that cells are repelled by netrin 1. (C) The number of migrating cells increased when PDGF was added to the lower well, suggesting that cells are attracted by PDGF. (D) Boyden chamber assays. Two chambers are separated by a filter, cells are placed in the top chamber and putative chemotactic molecules placed in the upper or lower chamber. Migrating cells are counted on the lower surface of the filter. (E) Quantitation of the migration. Addition of 100 ng/ml netrin 1 to the lower chamber inhibited oligodendrocyte precursor migration, whereas addition of 20 ng/ml PDGF promoted migration (Armstrong et al., 1990Go). The chemoattraction of PDGF was blocked by addition of netrin 1 to the lower chamber in a dose-dependent manner. (F) Addition of netrin 1 to the upper chamber increased the number of oligodendrocyte precursors migrating through the filter. Scale bar in A: 100 µm for A-C.

 


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  		Fig. 6. Netrin 1-induced chemorepulsion of oligodendrocyte precursors is dependent on DCC-like signaling. (A) Netrin 1-positive cells mimic the repulsive activity exerted by ventral explant cells. The majority of intermediate explant-derived oligodendrocyte precursors migrated away from netrin 1-positive cells. The biased migration of oligodendrocyte precursors from intermediate explants was blocked by anti-DCC antibodies in co-cultures with (B) netrin 1-positive cells or (C) ventral explants, demonstrating that a DCC-like receptor mediates netrin 1 signaling. Cell aggregates and ventral explants are outlined. (D) Schematic representation of the quantitative analyses of oligodendrocyte precursors from intermediate explants. The counting criteria are described in the Materials and Methods. (E) Quantitative analysis of cell distribution in migration assays. In isolation, equal proportions of cells migrate in all directions. In co-culture with ventral explants or netrin-secreting cells, a higher proportion of cells are distal to the chemorepulsive cue and this is negated by anti-DCC but not control antibodies. (F) Scatter plot demonstrates the ratio of oligodendrocyte precursors migrating out from the distal to proximal side. Each dot represents one co-culture pair. A ratio larger than 1 represents repulsion, whereas a ratio smaller than 1 is attractive. VSC, ventral spinal cord; ISC, intermediate spinal cord; N293, netrin-producing 293 cells; C293, control 293 cells. *P=0.003, **P<0.001. Scale bar: 100 µm.

 


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  		Fig. 2. Ventral explants provide a chemorepulsive cue for intermediate explant derived migratory oligodendrocyte precursors. (A) The migration pattern of O4-positive cells from isolated stage 29 intermediate explants was uniformly radial after 3 days in culture. (B) Co-culture of ventral and intermediate explants resulted in non-radial migration of O4-positive cells. More cells migrated away from than toward to the ventral explant. Asterisk indicates position of the ventral explant. (C,D) The morphology of O4-positive cells proximal and distal from the ventral explant was subtly different. Distally (D), O4-positive cells had longer processes, whereas proximally (C) they had shorter and frequently multiple processes. Asterisks in C,D indicate location of intermediate explants. Scale bars: in A, 100 µm for A,B; in D, 20 µm for C,D.

 


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  		Fig. 3. Netrin 1 mRNA is present in the ventral spinal cord during the period of oligodendrocyte precursor migration. At stage 29 (E6), RT-PCR analyses revealed a band of the expected size (300 bp) in brain (B) and ventral spinal cord (VSC). Expression was retained until E9.

 


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  		Fig. 5. Chick and rodent oligodendrocyte precursors express netrin receptors. (A-D) Pan-purified O4-positive chick oligodendrocyte precursors. (A) Oligodendrocyte precursors from E10 or E12 chick spinal cords showed alternatively spliced forms (427 and 266 bp) of neogenin by RT-PCR. (B) The majority of O4-positive purified oligodendrocyte precursors (>90%) were labeled with anti-neogenin antibody, showing punctate labeling on the cell processes and brighter labeling over the cell body (C). A similar proportion of O4-positive cells were labeled with anti-DCC antibody (D). (E-H) Pan-purified A2B5-positive rat spinal cord oligodendrocyte precursors. (E) RT-PCR analyses reveal bands with sizes correlating with UNC5h1 (643 bp), UNC5h2 (446 bp) and DCC (498 bp). The amplified DCC product was further confirmed by predicted internal restriction enzyme site (DCC/cut). Most A2B5-positive (G) rat oligodendrocyte precursors were double labeled with anti-DCC antibody (H). (F) Phase contrast view. Scale bar: 20 µm.

 


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  		Fig. 7. The dispersal of oligodendrocyte precursors from the ventral ventricular zone is dependent on netrin. (A) Stage 28 chick spinal cord slice labeled with O4 antibody 1 hour after dissection showed minimal staining in the ventral ventricular zone. (B) After 24 hours O4-positive oligodendrocyte precursors showed ventral radial migration, which increased over the next 24 hours (C) (48 hours total). (D) The majority of migrating cells have immature unipolar or bipolar cell morphology. (E) In the presence of anti-DCC antibodies, O4-positive cells have a smaller cell body and more processes. (F) The migration of O4-positive oligodendrocyte precursors is inhibited in the presence of anti-DCC antibody with the majority of O4-positive cells remaining in the ventral spinal cord. (G) Addition of exogenous chick netrin 1 to the slices also inhibits oligodendrocyte precursor migration. (H) Normal mouse IgG did not disrupt oligodendrocyte precursor migration, neither did anti-prion antibody (I). (J) Anti-NCAM antibody altered the pattern of migration, but did not mimic the changes seen with anti-DCC or netrin 1. (K-O) Stage 31 chick spinal cord slices. (K) Stage 31 spinal cord slice after dissection showed ventral radial migration. (L) Spinal cord slices grown in the presence of anti-DCC antibody or chick netrin 1 ligand (N) did not show an altered migration pattern compared with controls (M,O) suggesting that more mature oligodendrocyte precursors are less sensitive to netrin 1. Scale bars: in C, 100 µm for A-C,F-J; in E, 10 µm for D,E; in K, 100 µm for K-O.

 

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