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First published online January 13, 2009
doi: 10.1242/10.1242/dev.018234

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Netrin 1 and Dcc regulate oligodendrocyte process branching and membrane extension via Fyn and RhoA

Montreal Neurological Institute, Department of Neurology and Neurosurgery, McGill University, Montreal, Quebec H3A 2B4, Canada.

View larger version (122K): [in this window] [in a new window]   Fig. 1. Expression of netrin 1 by oligodendrocytes in vivo. (A,A') Longitudinal section through a P8 rat spinal cord showing Mbp-immunopositive oligodendrocytes (red) that do not express netrin 1, intermingled with netrin-1-expressing cells (green). (B,B') At P8, netrin 1 immunoreactivity (green) was detected in the immediate environment surrounding the cell bodies of Cnp-positive oligodendrocytes (red), but netrin 1 was not expressed by the oligodendrocytes themselves. (C,C') An Mbp-positive netrin-1-negative process, surrounded by netrin-1-expressing neuroepithelial cells (arrows) in P8 spinal cord. (D,D') Longitudinal section through a P8 rat spinal cord showing axons positive for neurofilament medium polypeptide (Nfm), surrounded by netrin-1-expressing cells. (E,E') Cell bodies of netrin-1-immunopositive neurons. (F) Cross-section of a P12 rat dorsal spinal cord showing Cnp-positive, myelinating oligodendrocytes (red) expressing netrin 1 (green). (G-G'') Oligodendrocytes adjacent to the central canal (arrow) express netrin 1 in P12 spinal cord. (H,H') Netrin 1 immunoreactivity (green) associated with a Cnp-immunopositive (red) myelinating oligodendrocyte in the P12 rat spinal cord. (I-I'') By P12, netrin 1 expression (green) colocalizes with Nfm-immunoreactive axons (blue), but is not detected in Gfap-positive astrocytes (red). (J,J') Transverse section of a P22 thoracic spinal cord showing widespread expression of netrin 1 (green) by Cnp-positive, myelinating oligodendrocytes (red). (K-K'') Magnification of the boxed region from J showing Cnp-positive netrin-1-expressing cell bodies. A,F, 20x0.5 n.a. objective; G,J, 40x0.75 n.a. objective; D,E,I,K, confocal microscopy, 40x0.75 n.a. objective; B,C,H, confocal microscopy, 100x1.4 n.a. objective. Scale bars: 10 µm in B',C',H; 20 µm in A',D,E,G,K; 40 µm in F,I',J.

View larger version (124K): [in this window] [in a new window]   Fig. 2. Expression of netrin 1 and Dcc by oligodendrocytes in vitro. (A,A') Netrin 1 protein (green) on the surface of myelin-like membrane sheets of non-permeabilized mature rat oligodendrocytes in culture. Recombinant netrin 1, labeled using a Myc epitope tag, preferentially localizes to branches formed by the cells (arrow). (B,B') Dcc (green) distributed along oligodendrocyte processes and at puncta along the edge of Mbp-immunopositive (red) myelin-like membrane sheets (arrow). A,B, 40x0.5 n.a. objective. Scale bars: 40 µm in A'; 20 µm in B'.

View larger version (88K): [in this window] [in a new window]   Fig. 3. Netrin 1 and Dcc regulate process outgrowth by immature oligodendrocytes in vivo. (A-C) Transverse sections of brachial spinal cord from E18 Ntn1-/- mice stained with antibodies to Cnp (green) and Nfm (red) to label premyelinating oligodendrocytes and neuronal cell bodies and axons, respectively. (A) Arrows indicate Cnp-immunopositive premyelinating oligodendrocytes. (B) As premyelinating oligodendrocytes search for and initially contact axons, they extend one or two major processes (arrows). (C) Netrin 1 expression, determined by β-galactosidase (β-Gal) reporter expression in a transgenic Ntn1+/- mouse, shown at the E18 spinal cord floor plate (arrow). Premyelinating oligodendrocytes are not immunopositive for β-galactosidase, but are adjacent to cells expressing netrin 1. (D) Dashed lines illustrate the measurement of extending processes of premyelinating oligodendrocytes in Ntn1+/+ and Ntn1-/- embryos. (E,F) Oligodendrocytes in the spinal cords of Ntn1-/- or Dcc-/- mice exhibit significantly shorter processes than wild-type or heterozygous littermates. *P<0.05, ***P<0.0001 versus control. The number of cells analyzed in each condition is indicated in parentheses. Error bars indicate s.e.m. A, 20x0.5 n.a. objective; B-D, 40x0.75 n.a. objective. Scale bars: 40 µm in A; 20 µm in B-D.

View larger version (42K): [in this window] [in a new window]   Fig. 4. Netrin 1 induces Dcc-dependent process extension by immature oligodendrocytes, and Dcc-dependent process branching and myelin-like membrane sheet extension by mature oligodendrocytes, in vitro. (A) Addition of netrin 1 (100 ng/ml, 24 hours) to cultured rat oligodendrocytes results in a 4% decrease in the ratio of immature cells expressing Cnp only (arrowheads) as compared with more mature cells expressing Cnp and Mbp. Netrin 1 did not change the ratio of cells expressing Mbp only versus Mbp and Mag. (B) Cnp-positive immature oligodendrocytes are multipolar cells with one major process. Dashed red lines indicate examples of the processes measured. (C) The length of the major process increased following the addition of netrin 1 (100 ng/ml) for 24 hours. Application of Dcc function-blocking antibody (Dccfb) to immature oligodendrocytes blocks the netrin-1-induced increase in process extension, whereas Dccfb alone does not have a significant effect on process extension. (D) A mature Mbp- and RIP-positive oligodendrocyte extending both myelin-like membrane sheets and branched processes. Sheet area was quantified by tracing the Mbp-positive myelin-like membrane sheets (left, red outlines). Branching was quantified by measuring the number of intersections that processes made with concentric circles (right, red), which are numbered 1-5 to reflect increasing distance from the cell body (and as labeled on the x-axis of bar charts displaying branching complexity). (E) Netrin 1 (100 ng/ml, 24 hours) increased the area of Mbp-positive sheets compared with the control. This effect was blocked by Dccfb. (F) Processes of mature oligodendrocytes exposed to netrin 1 for 24 hours exhibited an increase in branching. Dose-response analysis indicated maximal branching at 100 ng/ml netrin 1. (G) Addition of Dcc function-blocking antibody together with netrin 1 prevented the netrin-1-dependent increase in branching, and decreased branching compared with controls. A, 20x0.5 n.a. objective; B,D,F, 40x0.75 n.a. objective. Scale bars: 40 µm in A; 20 µm in B,D,F. A.U., arbitrary units. *P<0.05, **P<0.005, versus control. Error bars indicate s.e.m.

View larger version (31K): [in this window] [in a new window]   Fig. 5. Oligodendrocytes from mice lacking netrin 1 or Dcc exhibit defects in process branching and in myelin-like membrane sheet formation. (A) Representative examples of the morphology of oligodendrocytes derived from wild-type and netrin-1-deficient or Dcc-deficient mice. 40x0.75 n.a. objective. Scale bar: 20 µm. (B) Application of netrin 1 increased branching by oligodendrocytes from Dcc+/+ and Dcc+/-, but not from Dcc-/-, mice. (C) Oligodendrocytes from Dcc-/- or Ntn1-/- mice exhibit no difference in branching complexity compared with cells from wild-type and heterozygous littermates. (D) Ntn1-/- oligodendrocytes exhibit a significant decrease in myelin-like membrane sheet area compared with cells derived from wild-type and heterozygote littermates. (E) Dcc-/- oligodendrocytes elaborate smaller myelin-like membrane sheets than wild-type and heterozygous littermates. A.U., arbitrary units. ***P<0.0001 versus control. Error bars indicate s.e.m.

View larger version (15K): [in this window] [in a new window]   Fig. 6. Netrin 1 regulates oligodendrocyte morphology independently of integrins. (A) Application of β1 integrin function-blocking antibody (anti-β1, 2 µM) or a peptide encoding the netrin 1 integrin-binding domain (peptide, 20 µg/ml) failed to block the increase in oligodendrocyte process branching induced by netrin 1 (100 ng/ml). (B) The β1 integrin function-blocking antibody blocks the fibronectin-induced increase in the area (cell spreading) of HEK293T cells, whereas an anti-IgM control antibody does not. A.U., arbitrary units. *P<0.05, ***P<0.0001, versus control. Error bars indicate s.e.m.

View larger version (41K): [in this window] [in a new window]   Fig. 7. Netrin 1 binding to Dcc recruits Fyn. (A) Increased interaction between Dcc and Fyn is detected upon addition of exogenous netrin 1 (200 ng/ml, 5 minutes) to cultures of rat oligodendrocytes. This interaction was detected in both immature (4 DIV) and mature (6 DIV) oligodendrocytes and was observed when co-immunoprecipitations (IP) were performed with antibodies against Dcc or Fyn. A constitutive interaction between FAK and Dcc was also detected. IgG indicates immunoprecipitation with species-matched non-immune control IgG. (B) Increased active Src family kinase [phospho (p) SFK (pY416)] associated with Dcc in oligodendrocytes following application of netrin 1 (200 ng/ml, 5 minutes). (C) No detectable change in SFK activity in whole-cell lysates of cultured oligodendrocytes treated with netrin 1 (200 ng/ml) for either 5 minutes or 24 hours. (D) Quantification of the interaction between Fyn and Dcc, and between active SFK and Dcc. *P<0.05 versus control. Error bars indicate s.e.m.

View larger version (52K): [in this window] [in a new window]   Fig. 8. SFK activity is required for netrin-1-induced changes in oligodendrocyte morphology. (A) Oligodendrocytes were obtained from mice lacking Fyn (Fyn KO) and treated with netrin 1 (100 ng/ml, 24 hours). Cells obtained from wild-type mice of a matched genetic background were used as controls. (B) Netrin 1 increased branching in control oligodendrocytes (F2 hybrid), but not in Fyn KO oligodendrocytes. (C,C') Phospho-SFK [P-SFK (pY416); red] immunoreactivity colocalizes (yellow) with Dcc (green) within the oligodendrocyte cell body and proximal branches. The confocal section shown is adjacent to the substrate and the appearance of staining throughout the cell body is not indicative of staining in the nucleus. (D,D') Portions of the images in C and C' have been magnified to illustrate punctate staining present within distal branches. Arrows indicate colocalized enrichment of phospho-SFK (red) and DCC (green) at puncta. (E) Binary images of phospho-SFK immunostaining were used to quantify the number of phospho-SFK-positive puncta. (F) The relative number of phospho-SFK-positive puncta increased in the presence of netrin 1 (100 ng/ml, 24 hour treatment). PP2 (2 µM) blocked the netrin-dependent increase in puncta, and PP2 alone showed a significant decrease in puncta compared with the control. PP3 (2 µM) did not block the effects of netrin 1 and had no independent effect on the cells. (G) The number of Fyn-positive puncta per unit area was not significantly (n.s.) changed upon addition of netrin 1. (H) The netrin-1-dependent increase in process length of Cnp-positive immature oligodendrocytes was blocked by the addition of PP2 (2 µM). Addition of PP3 did not disrupt the effect of netrin 1 on the cells. Oligodendrocytes treated with PP2 alone (2 µM), but not PP3 alone, exhibit a small but significant decrease in process extension compared with the control. (I) Disruption of SFK activity in mature Mbp-positive oligodendrocytes prevented the increased branching observed in the presence of netrin 1. PP2 and PP3 did not independently affect oligodendrocyte morphology. *P<0.05, **P<0.005, ***P<0.001, versus control. Error bars indicate s.e.m. A,E, 40x0.75 n.a. objective; C-D', confocal microscopy, 40x0.75 n.a. objective. Scale bars: 20 µm in A,E; 40 µm in C',D'.

View larger version (37K): [in this window] [in a new window]   Fig. 9. Treatment of mature oligodendrocytes with netrin 1 inhibits RhoA activity (A) Levels of GTP-bound RhoA in rat oligodendrocytes were assessed using GST-rhotekin pulldown. Treatment of mature oligodendrocytes with netrin 1 (100 ng/ml, 5 minutes) caused a significant decrease in RhoA-GTP compared with controls. (B,C) Similar treatment of cells resulted in no significant change in the levels of GTP-bound Cdc42 or Rac1, as assessed by GST-Pak-CRIB pulldown. (D) Addition of netrin 1 to mature oligodendrocytes (100 ng/ml, 5 minutes) did not alter the amount of N-WASP recruited to Dcc. *P<0.05 versus control. Error bars indicate s.e.m.

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