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First published online February 22, 2006
doi: 10.1242/10.1242/jcs.02796

Journal of Cell Science 119, 933-942 (2006)
Published by The Company of Biologists 2006
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Upregulation of chondroitin 6-sulphotransferase-1 facilitates Schwann cell migration during axonal growth

Jun Liu1, Chi-Ho Chau1,2, Hengying Liu1, Benjamin R. Jang1, Xiaoguang Li1,3, Ying-Shang Chan2 and Daisy K. Y. Shum1,*

1 Department of Biochemistry, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China
2 Department of Physiology, Faculty of Medicine, The University of Hong Kong, 21 Sassoon Road, Pokfulam, Hong Kong, China
3 Department of Neurology, Peking Union Medical College Hospital, Beijing, China


Figure 1
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  		Fig. 1. Recombinant rat C6ST-1 catalyzes sulphation of chondroitin at C-6 of GalNAc residues. (A-C) The sulphotransferase reaction was carried out with chondroitin as the acceptor substrate under conditions described in the Materials and Methods with constructs containing C6ST (A), His-C6ST (B) and His-LacZ (C). The 35S-labelled product was digested with chondroitinase ACII ({triangleup}) or chondroitinase ACII + chondro-6-O-sulphatase (*). The digests were subjected to Partisil-10 SAX HPLC and eluted fractions were monitored for radioactivity. Arrows indicate elution positions of standard disaccharides: (1) 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-D-galactose ({Delta}Di-0S); (2) 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose ({Delta}Di-6S); (3) 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose ({Delta}Di-4S); (4) inorganic sulphate; and (5) 2-acetamide-2-deoxy-3-O-(ß-D-gluco-4-enepyranosyluronic acid)-4,6-bis-O-sulpho-D-galactose ({Delta}Di-diSE). The broken line depicts the eluting salt gradient.

 

Figure 2
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  		Fig. 2. C6st1 mRNA expression is upregulated in Schwann cells emigrating from sciatic nerve explants but not in Schwann cells at confluence. Schwann cells emigrating from explants of sciatic nerves (adult rats) are compared with confluent cultures of purified Schwann cells. (A) Phase-contrast (A1) and immunofluorescence (A2) views of emigrating Schwann cells doubly immunopositive for the S100 and CS56 epitopes. Emigrating Schwann cells reveal colocalisation of fluorescence in situ hybridisation signal for C6st1 mRNA (C6ST) and immunofluorescence for either S100 (A3) or GFAP (A4). The GFAP-positive migrating Schwann cells show negligible hybridisation signal when tested with the sense riboprobe (A5). (B) Phase-contrast (B1) and combined hybridisation- and immunofluorescence (B2) views of confluent cultures of purified Schwann cells, showing insignificant expression of C6st1 mRNA in spindle-shaped, bipolar, S100-positive Schwann cells. Similarly, these Schwann cells were S100-positive but CS56-negative, as observed with double immunofluorescence (B3). (C) Histogram showing prevalence of C6ST-expressing, S100-positive Schwann cells (SC) in the sciatic nerve (SN) explant over those in confluent cultures of purified Schwann cells (P<0.001). Total Schwann cell counts are listed under the histogram. Error bars represent s.e.m. of three independent experiments. Bars, 25 µm (B, upper panels in A); 50 µm (lower panels in A).

 

Figure 3
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  		Fig. 3. C6st1 mRNA expression is upregulated in migrating Schwann cells and not in Schwann cells that are engaged with axonal fascicles. DRG explants (E14 rats) were cultured on laminin-coated coverslips for 2 days in 10% FBS-DMEM-F12 supplemented with NGF (20 ng/ml). (A) Left panel, phase-contrast view of axons (small arrows) and cells radiating from the explant. Right panel, the same view showing C6st1 mRNA (C6ST) hybridisation signals in cells at the frontier (box); the enlarged inset image of the boxed region reveals the various morphologies of C6st1-expressing cells. (B) In situ hybridisation for C6st1 mRNA combined with SMI31 immunocytochemistry for axons. Left panel, SMI31-positive fibres (small arrows) radiate as networks and fascicles from a DRG explant. C6st1 hybridisation signals were hardly visible in cells associated with axonal fascicles. Towards the frontier (≥1090 µm from DRG centre), C6st1-expressing were visible. Right panel, an enlarged view of the frontier zone shows scattered C6st1-expressing cells (arrowheads) apparently in advance of SMI31-positive axons (small arrows). (C) Double immunofluorescence for SMI31-positive axons and S100-positive Schwann cells. S100-positive cells can be seen crowded around the DRG explant (upper right), aligned as chains along SMI31-positive axon fascicles (small arrows), and as scattered cells (arrowheads) in the frontier. The large arrow in each panel indicates the direction of projection of growing axons and emigrating cells from the DRG centre. Bars, 200 µm (A,B), 100 µm (C).

 

Figure 4
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  		Fig. 4. Chondroitinase digestion of pericellular proteoglycans of migrating Schwann cells at the DRG frontier results in a shift to lower mobilities. (A-D) Extended treatment with chondroitinase ABC (40 hours) revealed cells radiating from the DRG centre (towards lower right, phase-contrast view in A), many of which were S100-positive Schwann cells (B) bearing pericellular 3B3-immunopositivity (C). (D) Merged image of B and C. (E-H) Although control treatment with the heat-inactivated enzyme similarly revealed cells radiating from the DRG centre (towards mid-right, phase-contrast view in E), none of the S100-positive cells (F) showed pericellular 3B3-immunopositivity (G). (H) Merged image of F and G. The bar chart (I) shows the shift towards lower mobility among Schwann cells at the migration front during time-lapse video recording of cultures undergoing a further 3-hour treatment with (+) chondroitinase ABC compared with those without (-) the enzyme. Mobility ranges are classified high at >2.3 µm/minute, intermediate at 1.5-2.3 µm/minute and low at <1.5 µm/minute. Results are the mean ± s.e.m. of triplicate DRG experiments prepared for time-lapse video recording (*P=0.46; **P=0.29). (J,K) One of these DRGs is shown both under light microscopy to reveal C6st1 mRNA signals (J) and under phase-contrast optics to reveal cell profiles (K). Cells that had been mapped for mobility measurements were numbered as indicated. The corresponding video clip and profile of cell mobility are shown in supplementary material Movie 2 and Fig. S3. Bars, 100 µm (A-H) and (J,K).

 

Figure 5
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  		Fig. 5. Upregulated C6st1 mRNA in Schwann cells during an early phase of tissue reorganisation after nerve crush. (A) In situ hybridisation patterns of C6st1 mRNA revealed with DIG-labelled antisense probes on transverse cryosections of normal and post-crush sciatic nerves (at 1, 3, 7, 14 and 28 days as indicated). Arrows in the zoom views (insets) indicate significant C6st1 transcripts in cells circumferential to axons (1 dpc), in irregularly shaped cells profusely distributed about Schwann tubes undergoing reorganisation (3 dpc and 7 dpc). By 14 days post crush, signals were detectable only in the small-diameter Schwann tubes. Signals approach the normal pattern by 28 dpc. Occasional C6st1 transcripts are detectable around blood vessels (arrowheads in normal and 28-day images). The DIG-labelled sense probe yielded negligible hybridisation signal against the background as exemplified with a section of a 14 dpc sciatic nerve. (B) RT-PCR analysis of C6st1 mRNA expression for the profile of change in post-crush sciatic nerves (days 1-28 as indicated) against the expression of GAPDH as an internal reference. Gel bands are shown directly above the corresponding histogram of C6ST: GAPDH intensity ratio. Results are the mean ± s.e.m. of three independent sets of analyses. Bars, 200 µm.

 

Figure 6
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  		Fig. 6. Expression of C6st1 mRNA in Schwann cells in 7 dpc nerves. Fluorescence in situ hybridisation for C6st1 mRNA in combination with fluorescence immunohistochemistry for either S100 (A-H) or GFAP (I-P). Images represent both transverse (A-D and I-L) and longitudinal (E-H and M-P) cryosections of 7 dpc sciatic nerves at the crush site. Phase-contrast views of the tissues are shown in the right-hand column. Merges of immunocytochemistry and hybridisation images are shown (C,G,K and O) with enlarged views (insets) of the boxed areas (C and K). Only some of the S100-positive Schwann cells expressed the C6st1 transcript (arrows) but all C6st1-expressing Schwann cells were GFAP-positive. Bars, 100 µm (A-P); 50 µm (insets).

 

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© The Company of Biologists Ltd 2006