The transmembrane semaphorin Sema4D/CD100, an inhibitor of axonal growth, is expressed on oligodendrocytes and upregulated after CNS lesion

Abstract

Semaphorins are a family of secreted and membrane-bound proteins, known to regulate axonal pathfinding. Sema4D, also called CD100, was first isolated in the immune system where it is involved in B and T cell activation. We found that in the mouse, Sema4D is expressed in cells throughout the CNS white matter, with a peak during the myelination period. Double-labeling experiments with different markers of oligodendrocyte lineage such as olig1, olig2, platelet-derived growth factor receptor alpha, and proteolipid protein showed that Sema4D was expressed selectively by oligodendrocytes and myelin. The presence of Sema4D in myelin was confirmed using Western blot. Sema4D expression in myelinating oligodendrocytes was further observed using neuron-oligodendrocyte cocultures. Moreover, using stripe assay, we found that Sema4D is strongly inhibitory for postnatal sensory and cerebellar granule cell axons. This prompted us to examine whether Sema4D expression is modified after CNS injury. At 8 d after spinal cord lesions, Sema4D expression was strongly upregulated in oligodendrocytes at the periphery of the lesion. Sema4D-positive cells were not colabeled with the astrocyte marker GFAP, with the microglial and macrophagic marker isolectin B4, or with NG2, a marker of oligodendrocyte precursors. This upregulation was transient because from 1 month after the lesion, Sema4D expression was back to its normal level. These results indicate that Sema4D is a novel inhibitory factor for axonal regeneration expressed in myelin.

Figure 1.

Expression of Sema4D mRNA in oligodendrocytes of the postnatal mouse brain. In situ hybridization was performed on coronal sections with digoxigenin-labeled (A, C) or ³⁵S-labeled (B) riboprobes for Sema4D. Double-labeling in situ hybridization was performed with a fluorescein-labeled riboprobe for Sema4D and with digoxigenin-labeled riboprobes for PDGFRα (D), Olig1 (E), and Olig2 (F), respectively. A, At P0, Sema4D expression in the brainstem is restricted to few dispersed cells in the gray matter (inset, arrows). B, C, At P10, Sema4D-expressing cells (arrowheads) in the corpus callosum (cc) display a tissue distribution similar to that of oligodendrocytes. D, Double-labeling in situ hybridization of P10 cerebellum with Sema4D (red) and PDGFRα (blue) probes shows that the cells expressing these two genes are distinct. Arrowheads indicate examples of Sema4D-labeled cells. E, F, At P10, in the corpus callosum, double-labeling in situ hybridization with Sema4D (red) and oligodendrocyte lineage markers Olig1 (E) (blue) and Olig2 (F) (blue) shows that Sema4D-expressing cells represent a subset of Olig1- or Olig2-expressing cells. Examples of double-labeled cells are indicated by arrowheads, and single olig1- or olig2-labeled cells are indicated by arrows. Scale bars: A, 400 μm; B, 900 μm; C, 120 μm; D, E, 60 μm; F, 20 μm.

Figure 2.

Expression of Sema4D mRNA and Sema4D protein in mouse oligodendrocytes. A, At P10, PLP antibody immunostaining (brown) combined with in situ hybridization for Sema4D (blue) shows that a subpopulation of oligodendrocytes expressing PLP coexpress Sema4D.B, At P10, Sema4D antibody labels in the CNS white matter, cells that have the morphology of oligodendrocytes. C, D, At P30, myelin in the corpus callosum (cc) and in the striatum (long arrow) is strongly labeled by Sema4D antibody. Dispersed oligodendrocytes labeled by anti-Sema4D are also observed in the corpus callosum (C, D, short arrows). E, In P10, PLPlacZ brain, Sema4D-expressing cells (red) represent a subpopulation of β-galactosidase (β-gal)-positive (green) oligodendrocytes. Double-labeled cells are indicated by arrowheads, and single β-galactosidase-positive cells are indicated by arrows. F, Higher magnification shows examples of double-labeled cells (arrowheads). Scale bars: A, B, F, 20 μm; C, 140 μm; D, E, 35 μm.

Figure 4.

Sema4D expression in the adult spinal cord is upregulated after CNS injury. Control (A, B) or lesioned (C-F) parasagittal sections of spinal cord were immunostained with Sema4D antibody (A-D). A, B, In the adult spinal cord of adult control mice, a weak expression of Sema4D is observed in some oligodendrocytes in the white matter and on myelin. C, D, One week (1w) after spinal cord injury, a strong upregulation of Sema4D expression is observed in the vicinity of the lesion that appears as a dark area. The number of cells expressing Sema4D (D, arrows) near the lesion is increased compared with intact regions. E, Immunoblot analysis of Sema4D expression in myelin extracts. Myelin proteins from 3-week-old mice were analyzed with antisera to Sema4D, Nogo-A, or MAG. As described previously, a band at 150 kDa was detected with anti-Sema4D, a band at 220 kDa for Nogo A, and a band ∼100 kDa for MAG. Scale bars: A, C, 200 μm; B, 50 μm; D, 100 μm.

Figure 3.

Sema4D expression in myelinating cultures. Neurons-oligodendrocytes myelinating cocultures were derived from E15 mouse cerebral hemispheres and stained at 2 (A, B), 15 (C, D), or 23 (E, F) DIV with anti-Sema 4D antibody (B, D, F) and A2B5 (A), anti-MBP (C), or anti-MOG (E) mAbs. A, B, At 2 DIV, Sema4D was detected on cells labeled with the A2B5 mAb. These cells could be either neurons or oligodendrocyte precursor cells. C, D, At 15 DIV, mature nonmyelin-forming oligodendrocytes identified by their expression of MBP (C) also expressed Sema4D. E, F, Under our culture conditions, myelin began to be deposited around axons at 18-21 DIV. Sema4D was strongly expressed on myelinated internodes, co-labeled by anti-MOG mAb.

Figure 5.

Characterization of Sema4D-expressing cells after adult spinal cord injury. Parasagittal sections of spinal cord 1 week after lesion were immunostained with Sema4D (B, C, E, F, H, I), GFAP (A, C), and NG2 (G, I) antibodies or isolectin B4 (D, F). A, After spinal cord injury, the glial scar contains a dense network of hypertrophic GFAP-immunostained processes oriented perpendicularly to the cut. B, Sema4D expression is upregulated in cells (arrowheads) present around the lesion site and oriented in a parallel direction to the astrocytic processes. C, Double labeling with GFAP (FITC) and Sema4D (Cy3) antibodies shows that these Sema4D-positive cells (arrowheads) are not astrocytes. D, F, FITC-conjugated isolectin B4 reveals the presence of macrophages or activated microglial cells, or both, in a 1-week-old lesion. The cells are round (F, arrow) or ramified according to their state of activation. E, F, Double labeling with isolectin B4 (FITC) and Sema4D (Cy3) shows that Sema4D-expressing cells (F, arrowheads) are not macrophages or activated microglial cells (F, arrow). G, One week after injury, many NG2-positive oligodendrocyte precursors accumulate at the lesion. H, I, Double labeling with NG2 (FITC) and Sema4D (Cy3) antibodies indicates that Sema4D-positive cells (I, arrowheads) are not oligodendrocyte precursors (I, arrows). Scale bars: A-C, 40 μm; D-I, 80 μm.

Figure 6.

DRG and granule cell axons avoid Sema4D. P6 DRG (A, B) or P5 granule cell (C, D) explants were cultured on substrates patterned with alternating stripes of Sema4D (asterisk) or laminin (L). Explants were immunolabeled for class III β-tubulin, and the Sema4D stripes were labeled with fluorescein-conjugated BSA. A, B, DRG and cerebellar axons grow homogeneously when confronted with laminin only (A, C), whereas when given a choice between Sema4D and laminin, they strongly avoid Sema4D (B, D). Scale bars, 60 μm.

Figure 7.

Expression of Sema4D receptors in the CNS. Coronal sections were hybridized with digoxigenin-labeled riboprobe (A-C) or with ³⁵S-labeled riboprobes (D-G) for plexin B1 (A-D) and CD72 (E-G). A, At E13, plexin B1 mRNA is detected in the ventricular zone (arrowheads) from the rhombencephalon (Rho) to the telencephalon (tel). B, Coronal section at the level of the spinal cord of an E15 embryo. Plexin B1 transcripts are found in the ventricular zone lining the central canal (arrowheads). C, At P0, plexin B1 is still expressed in the telencephalon and in the ventricular zone (arrowhead) but also in cells leaving the subventricular zone (arrow). D, In adult mouse, plexin B1 is detected exclusively in the subventricular zone (svz) and rostral migratory stream (arrowheads) to the olfactory bulb (OB). Purkinje cells (arrows) in the cerebellum also express plexin B1 mRNA. E, F, Coronal sections of adult mouse brain hybridized with CD72 riboprobe. CD72 mRNA is strongly and homogeneously expressed in all neurons, with the highest levels in the hippocampus (hi) and neocortex (Cx). F, High magnification of CD72 expression in the neocortex. G, Sagittal section through the cerebellum of a P16 mouse hybridized with CD72 riboprobe. CD72 is highly expressed in the granule cell layer (GCL) and in the external granule cell layer (arrowheads). Scale bars: A, 300 μm; B, 1000 μm; C, 500 μm; D, 1600 μm; E, 1000 μm; F, 300 μm; G, 200 μm.