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First published online 1 March 2006
doi: 10.1242/dev.02312

Development 133, 1379-1387 (2006)
Published by The Company of Biologists 2006
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Dorsally derived netrin 1 provides an inhibitory cue and elaborates the `waiting period' for primary sensory axons in the developing spinal cord

Keisuke Watanabe1,2, Nobuaki Tamamaki3,4, Takahiro Furuta4, Susan L. Ackerman5, Kazuhiro Ikenaka1,2 and Katsuhiko Ono1,2,*

1 Department of Physiological Sciences, School of Life Science, The Graduate University for Advanced Studies (Sokendai), 5-1 Higashiyama, Myodaiji, Okazaki 444-8787, Japan.
2 Division of Neurobiology and Bioinformatics, National Institute for Physiological Sciences, Okazaki 444-8787, Japan.
3 Department of Morphological Neural Science, Graduate School of Medical Sciences, Kumamoto University, Kumamoto 860-8556, Japan.
4 Department of Morphological Brain Science, Graduate School of Medicine, Kyoto University, Kyoto 606-8501, Japan.
5 Howard Hughes Medical Institute and The Jackson Laboratory, Bar Harbor, MN 04609, USA.


Figure 1
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  		Fig. 1. Expression of netrin 1 and its receptors in the developing spinal cord and DRG. (A-E) Expression of netrin 1 mRNA visualized by in situ hybridization on transverse sections of the mouse spinal cord. At E11.5 (A), netrin 1 is expressed in the floor plate and the ventral-most part of the ventricular zone of the neural tube. At E12.5 (B,D), netrin 1 mRNA is localized in the dorsolateral region of the cord (arrowheads in B,D) as well as the floor plate and the ventral ventricular zone. The boxed area is magnified in D. At E13.5 (C,E), netrin 1 expression in the dorsal spinal cord is decreased (arrowheads in C,E). The boxed area is magnified in E. (D) Netrin 1-expressing cells are localized near the DREZ (arrow). fp, floor plate. (F-I) In situ hybridization for Dcc (F), Unc5a (G), Unc5b (H) and Unc5c (I) in the E12.5 mouse DRG. Unc5a and Unc5c are expressed in the DRG, whereas Dcc and Unc5b cannot be detected. Sections were counterstained with Nuclear Fast Red. Scale bar: 200 µm in A-C; 100 µm in D-I.

 

Figure 2
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  		Fig. 2. The dorsal funiculus is disorganized in the developing spinal cord of netrin 1 mutant mice. Whole-mount preparations (A,B) and transverse sections (C-H) stained with anti-neurofilament antibody from wild-type (A,C,G), netrin 1 heterozygous (E) and homozygous (B,D,F,H) mice. (A,B) E11.5. In both animals, DRG axons correctly project from the DRG to the spinal cord to form dorsal rootlets (arrowheads) and extend longitudinally in the dorsal spinal cord. DF, dorsal funiculus; SPC, spinal cord. (C-F) E12.5. DRG axons grow to the dorsolateral margin of the spinal cord forming the dorsal funiculus, and very few axons invade the dorsal mantle layer of wild-type and netrin 1 heterozygous mice (arrowheads in C,E). By contrast, the dorsal funiculus is severely disorganized with the loss of a clear inner border in netrin 1 mutant mice (arrowheads in D). Many axon bundles directly enter the dorsal mantle layer (arrows in D,F). Sections in E and F are processed for lacZ in situ hybridization followed by neurofilament immunohistochemistry. In netrin 1 mutants, direct invasion of axons into the dorsolateral part of the spinal cord in which netrin 1 expression is lost (demarcated by lacZ expression) is observed (arrows in F). (G,H) E13.5. In wild-type mice (G), the dorsal funiculus extends to the most dorsomedial region of the spinal cord (arrowheads in G), and some collaterals enter the dorsal spinal cord from the dorsomedial region of the dorsal funiculus (arrows in G). In netrin 1 mutant mice (H), the dorsal funiculus in the marginal zone is thinner than that in wild-type mice (arrowheads in G,H), and the inner border is less sharp. Moreover, thick axon bundles are observed within the dorsal mantle layer of netrin 1 mutants (open arrowheads in H). As observed in wild-type animals, some neurofilament-positive fibers extend ventrally from this ectopic funiculus (G,H arrows). Scale bar: 250 µm in A,B; 200 µm in C,D; 100 µm in E-H.

 

Figure 3
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  		Fig. 3. Both cutaneous and proprioceptive afferents directly invade the dorsal mantle layer in the absence of netrin 1. (A,C,E,F) Wild-type mice. (B,D,G,H) Netrin 1 mutant mice. DRG axons in the lateral views of whole-mount E12.5 spinal cord preparations (A,B) and on transverse sections of the E13.5 spinal cord (C,D) labeled by DiI, and immunohistochemically labeled with anti-TrkA (E,G) or TrkC (F,H) antibodies at E12.5. (A,B) In both animals, DRG axons bifurcate at DREZ (arrow in insets) and extend longitudinally more than three segments (arrowheads). An epifluorescent picture is merged onto a differential interference contrast (DIC) picture. Insets indicate the site of DiI injection in the DRG. Asterisks indicate DRG. (C,D) In E13.5 wild-type animals, the dorsal funiculus has been formed in the marginal zone, which is composed of DiI-labeled fibers (arrowheads in C). However, in netrin 1 mutant mice, most DRG axons directly invade the dorsal mantle layer forming the ectopic dorsal funiculus (arrowheads in D). (E-H) In wild-type mice, TrkA-(E) and TrkC-positive (F) fibers project to the marginal zone, and their projection into the dorsal mantle layer is not observed. In netrin 1 mutants, TrkA-positive axon bundles stay near the DREZ (G), and many TrkA- and TrkC-positive (H) fibers aberrantly project into the dorsal mantle layer. White broken lines indicate the boundary between the marginal zone and the dorsal mantle layer. Sections were counterstained with Hoechst 33342 (blue). Scale bar: 250 µm in A,B; 200 µm in C,D; 100 µm in E-H.

 

Figure 4
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  		Fig. 4. Netrin 1 suppresses axon outgrowth from DRG explants in vitro. E13.5 mouse DRG explants were co-cultured for 24-48 hours with aggregates of BHK cells expressing EGFP (A) or netrin 1 (B,C) in collagen gel. Explants were stained with anti-neurofilament antibody. Axon outgrowth from DRG explants co-cultured with netrin 1-expressing cells is inhibited (B) compared with that of control DRG explants cultured with EGFP-expressing cells (A). (C) The inhibitory effects are neutralized by the application of 2 µg/ml anti-netrin 1 rabbit antibodies. Scale bar: 250 µm. (D) Quantification of axon growth from DRG. The areas covered by neurofilament-positive axons were measured. *P<0.05.

 

Figure 5
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  		Fig. 5. Dorsally derived netrin 1 is important for the projection of sensory afferents. In situ hybridization for netrin 1 (A,B) and neurofilament immunohistochemistry (C,D) in transverse sections of the E12.5 spinal cord from wild-type (A,C) and Gli2 mutant (B,D) mice. (A) Netrin 1 is expressed in the floor plate and the ventral ventricular zone of the neural tube and at low levels in the dorsolateral spinal cord (arrowheads) in wild-type mice. (B) Netrin 1 expression in the ventral-most spinal cord is lost in Gli2 mutant mice (arrow), while the dorsolateral spinal cord expresses netrin 1 similarly to wild-type mice (arrowheads). (C,D) In both animals, the dorsal funiculus is correctly established in the marginal zone of the spinal cord with a sharp inner border (arrowheads). Scale bar: 200 µm in A,B; 100 µm in C,D.

 

Figure 6
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  		Fig. 6. Dorsal neural cell patterning is largely normal in netrin 1 mutant mice. Cellular patterning in the dorsal spinal cord demonstrated by Nissl staining (A,B) and in situ hybridization for Pax3 (C,D) and Lmx1b (E,F) on transverse sections from the E12.5 spinal cord. (G-K) BrdU labeling experiment. (A,C,E,G,I) Wild-type mice. (B,D,F,H,J) Netrin 1 mutant mice. (A) In wild-type mice, Nissl staining reveals a three-layered structure, and few cells are observed in the marginal zone (dorsal funiculus) which shows a sharp inner border (arrowheads). (B) In netrin 1 mutant mice, an ectopic small island is observed in the marginal zone (arrows in B,D,F), although a three-layered structure is formed. VZ, ventricular zone; ML, mantle layer; MZ, marginal zone. (C-F) Pax3 labels dorsal ventricular cells (C,D), and Lmx1b expression is found in cells in the dorsal mantle layer (E,F), both of which show few differences between wild-type and netrin 1 mutant mice. (G-J) The distribution of E10.5 (G,H) or E11.5 (I,J) BrdU-labeled cells in the E12.5 spinal cord. Many BrdU-labeled cells are observed in the dorsal mantle layer. (K) Quantification analysis of BrdU-labeled cells between the dorsal half and the ventral half of the dorsal spinal cord. Scale bar: 100 µm in A,B; 200 µm in C-J.

 

Figure 7
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  		Fig. 7. Mutation of Unc5c results in the disorganization of the dorsal funiculus. Cytoarchitecture and axonal patterning of the E12.5 Dcc mutant and Unc5crcm mutant dorsal spinal cord revealed by double staining with Nissl method and neurofilament immunohistochemistry (A,B), and by immunohistochemistry for TrkA (C,E) and TrkC (D,F). (A,C,D) Dcc mutant mice. (B,E,F) Unc5crcm mutant mice. (A) The dorsal funiculus is normally formed in Dcc mutant mice with a sharp inner border (arrowheads). (B) The Unc5crcm mutant dorsal spinal cord contains an ectopic cell island in the marginal zone (arrow), and neurofilament-positive axons form a triangle dorsal funiculus with an irregular inner border at the ventral part of the dorsal funiculus. Insets indicate higher magnification of the dorsal funiculus. (C,D) Both TrkA- and TrkC-positive afferents grow into the marginal zone without invading the dorsal gray matter, both of which show a sharp inner border (arrowheads). (E,F) TrkA- and TrkC-positive axons in Unc5crcm mutant mice form an aberrant dorsal funiculus, slightly spreading into the dorsal gray matter (arrows). The medial region of the dorsal funiculus has a sharp border to the dorsal mantle layer (arrowheads in F). Scale bar: 100 µm.

 

Figure 8
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  		Fig. 8. Schematic diagram summarizing the function of dorsally derived netrin 1 on primary sensory afferents. (Left) In the E12.5 wild-type spinal cord, primary sensory axons have formed the dorsal funiculus in the dorsolateral margin of the spinal cord and extend longitudinally without extending collaterals into the dorsal spinal cord. At this stage, netrin 1 is expressed in the dorsal spinal cord and prevents the axons from entering the mantle layer. At E13.5, netrin 1 expression is decreased, allowing some collaterals (probably proprioceptive afferents) to enter the dorsal spinal cord. (Right) In the absence of netrin 1, the `waiting period' is abolished at this developmental stage (E12.5), and many sensory axons directly enter the dorsal mantle layer.

 




© The Company of Biologists Ltd 2006