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. 2011 Sep;138(18):4085-95.
doi: 10.1242/dev.066076. Epub 2011 Aug 10.

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation

Jennifer R Leslie  1 , Fumiyasu Imai, Kaori Fukuhara, Noriko Takegahara, Tilat A Rizvi, Roland H Friedel, Fan Wang, Atsushi Kumanogoh, Yutaka Yoshida
Affiliations

Ectopic myelinating oligodendrocytes in the dorsal spinal cord as a consequence of altered semaphorin 6D signaling inhibit synapse formation

Jennifer R Leslie et al. Development. 2011 Sep.

Abstract

Different types of sensory neurons in the dorsal root ganglia project axons to the spinal cord to convey peripheral information to the central nervous system. Whereas most proprioceptive axons enter the spinal cord medially, cutaneous axons typically do so laterally. Because heavily myelinated proprioceptive axons project to the ventral spinal cord, proprioceptive axons and their associated oligodendrocytes avoid the superficial dorsal horn. However, it remains unclear whether their exclusion from the superficial dorsal horn is an important aspect of neural circuitry. Here we show that a mouse null mutation of Sema6d results in ectopic placement of the shafts of proprioceptive axons and their associated oligodendrocytes in the superficial dorsal horn, disrupting its synaptic organization. Anatomical and electrophysiological analyses show that proper axon positioning does not seem to be required for sensory afferent connectivity with motor neurons. Furthermore, ablation of oligodendrocytes from Sema6d mutants reveals that ectopic oligodendrocytes, but not proprioceptive axons, inhibit synapse formation in Sema6d mutants. Our findings provide new insights into the relationship between oligodendrocytes and synapse formation in vivo, which might be an important element in controlling the development of neural wiring in the central nervous system.

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Figures

Fig. 1.
Fig. 1.
Proprioceptive axonal projections in the spinal cord of Sema6c–/–, Sema6d–/– and Plxna1–/– mice. (A) Illustration of the proprioceptive and cutaneous axons in the mouse spinal cord. (B-E) Positions of axonal shafts of proprioceptive sensory neurons determined by Pv expression in P0 spinal cords. (D,E) Defects in proprioceptive axonal shaft positioning (arrows) were detected in Sema6d–/– and Plxna1–/– mice. Scale bar: 50 μm.
Fig. 2.
Fig. 2.
Oligodendrocytes invade the superficial dorsal horn of Sema6d–/– mice. (A,B) Expression of Mag in the spinal cord of Sema6d+/– (A) and Sema6d–/– (B) mice at P10. The superficial dorsal horn is outlined in red; the blue dotted line indicates the border between the dorsal horn and the rest of the spinal cord. (C-E) Number of Mag-positive oligodendrocytes in the whole spinal cord (C), dorsal half (D) and superficial dorsal horn except dorsal funiculus (E) of Sema6d+/– and Sema6d–/– mice. *, P<0.05. Error bars indicate s.d. Scale bar: 100 μm.
Fig. 3.
Fig. 3.
Organization of vGlut1-positive synaptic terminals in Sema6d–/– mice. (A-L) vGlut1 (green) and Mag (red) expression in P0 and P14 Sema6d+/+ or Sema6d+/– (A-F) and Sema6d–/– (G-L) mice. Arrows indicate vGlut1-negative areas (J-L). (M) Quantification of vGlut1-positive synaptic terminals in the boxed regions (100 μm × 100 μm) in F and L. *, P<0.05. Error bars indicate s.d. (N-Q) vGlut1 (blue), Pv (green) and Mag (red) expression in P14 Sema6d–/– mice. Ectopic proprioceptive axons with oligodendrocytes cause the disruption of vGlut1-positive synaptic terminals. Scale bars: 100 μm in L; 10 μm in Q.
Fig. 4.
Fig. 4.
Various pre- and postsynaptic marker staining in Sema6d–/– mice. (A-L) Immunofluorescence for the indicated markers (vGlut1 and SV2, presynaptic; Shank1 and PSD95, postsynaptic; Mbp, oligodendrocytes), with merges in the right-hand column, in Sema6d–/– mice at P14. Scale bar: 10 μm.
Fig. 5.
Fig. 5.
NeuN-positive neurons and CGRP-positive axons enter the vGlut1-negative regions in Sema6d–/– mice. (A-D) NeuN-positive neurons enter the Mag-positive and vGlut1-negative regions in P14 Sema6d–/– mice. (E-J) CGRP-positive axons enter vGlut1-negative regions (arrows) in P14 Sema6d–/– mice. Scale bars: 50 μm in D; 100 μm in J.
Fig. 6.
Fig. 6.
Ectopically located proprioceptive axons and oligodendrocytes in Sema6d–/– mice do not inhibit axonal projections. (A-F) Both proprioceptive and cutaneous sensory fibers were detected by hPLAP activity in Sema6d+/–; Avil-hPLAP (A,C,E) and Sema6d–/–; Avil-hPLAP (B,D,F) mice at P0 (A,B), P8 (C,D) and P14 (E,F). Scale bar: 100 μm.
Fig. 7.
Fig. 7.
Aberrant proprioceptive axons and oligodendrocytes do not inhibit cutaneous axonal projections. (A-K) Dorsal root ganglia (DRG) neurons were labeled with f-Dex (Dextran) by injection from the dorsal nerve in Sema6d–/– mice at P7. (A-C) Confocal images of dorsal horn showing f-Dex (A) and Mbp immunostaining (B), with merge (C). (D-K) Confocal images of Mbp-positive and vGlut1-negative areas with f-Dex (D,H), Mbp (E,I), vGlut1 (F) and βIII tubulin (J), with merges (G,K). (L,M) Electron microscopy images from Sema6d+/– (L) and Sema6d–/– (M) mice. Synapses were observed in Sema6d+/– mice (arrowhead in L). Myelinated axons (m) and non-myelinated processes, but few synapses, were observed in Sema6d–/– mice (M). Scale bars: 100 μm in C; 10 μm in K; 500 nm in M.
Fig. 8.
Fig. 8.
Genetic deletion of oligodendrocytes from Sema6d–/– mice. (A-C) Dramatic reduction of Mag protein expression in the dorsal spinal cord of Sema6d–/–; Cnp-Cre; lox-stop-lox-DTA mice at P10. (D-O) vGlut1 (blue), Pv (green) and Mag (red) expression at P10. Defects in vGlut1-positive terminals exhibited by Sema6d–/– mice were not detected in Sema6d–/–; Cnp-Cre; lox-stop-lox-DTA mice. Scale bars: 100 μm in C; 10 μm in O.
Fig. 9.
Fig. 9.
Dorsal spinal cords of wild-type and Sema6d mutant mice used in this study. Transverse sections illustrate proprioceptive axons (red lines), oligodendrocytes (blue ovals), cutaneous axons (green lines) and cutaneous synapses (green wavy lines). Cutaneous synapses are disrupted when oligodendrocytes enter the dorsal horn of the Sema6d–/– spinal cord.
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