Expression of Dystroglycan and the Laminin-α2 Chain in the Rat Peripheral Nerve during Development

Experimental Neurology 174, 109 –117 (2002) doi:10.1006/exnr.2001.7856, available online at http://www.idealibrary.com on

Expression of Dystroglycan and the Laminin-␣2 Chain in the Rat Peripheral Nerve during Development Toshihiro Masaki,* ,† Kiichiro Matsumura,‡ Akira Hirata,* Hiroki Yamada,‡ Asako Hase,‡ Ken Arai,‡ Teruo Shimizu,‡ Hiroshi Yorifuji,§ Kazuo Motoyoshi,* and Keiko Kamakura* *Third Department of Internal Medicine, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan; †Department of Neurology, National Institute on Alcoholism, Kurihama National Hospital, 5-3-1 Nobi, Yokosuka, Kanagawa 239-0841, Japan; ‡Department of Neurology and Neuroscience, Teikyo University School of Medicine, 2-11-1 Kaga, Itabashi-ku, Tokyo 173, Japan; and §Department of Anatomy, National Defense Medical College, 3-2 Namiki, Tokorozawa, Saitama 359-8513, Japan Received May 24, 2001; accepted November 19, 2001

In Schwann cells, the transmembrane glycoprotein ␤-dystroglycan comprises the dystroglycan complex, together with the extracellular glycoprotein ␣-dystroglycan, which binds laminin-2 (␣2/␤1/␥1), a major component of the Schwann cell basal lamina. To provide clues to the biological functions of the interaction of the dystroglycan complex with laminin-2 in peripheral nerves, we investigated the expression of ␤-dystroglycan and the laminin-␣2 chain in rat sciatic nerve during development by immunoblot, immunofluorescence, and immunoelectron microscopic studies. The expression of ␤-dystroglycan and the laminin-␣2 chain in the rat sciatic nerve was low and not confined to the Schwann cell outer membrane from embryonic day 18 to birth, when there was only an immature basal lamina assembly and no compact myelin formation by Schwann cells. However, the expression of these proteins increased markedly and became clearly localized to the Schwann cell outer membrane between birth and postnatal day 7, when both basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. From postnatal day 7 to adult, there was no remarkable change in the expression of these proteins. Our results support the hypothesis that the dystroglycan complex functions as an adhesion apparatus, binding the Schwann cell outer membrane with the basal lamina, and suggest that the dystroglycan complex plays a role in Schwann cell myelination through its interaction with laminin-2. © 2002 Elsevier Science (USA) Key Words: dystroglycan; laminin; basal lamina; myelin; Schwann cell; development.

INTRODUCTION

Dystroglycan (DG) 1 is encoded by a single gene and cleaved into ␣- and ␤-DG by posttranslational process1 Abbreviations used: DG, dystroglycan; E18, embryonic day 18; P0, P3, P7, and P28, postnatal day 0, 3, 7 and 28; ␤-DG-IR, ␤-dys-

ing (11, 17). ␣-DG is an extracellular glycoprotein anchored to the cell membrane by a transmembrane glycoprotein, ␤-DG. The complex comprising ␣-DG and ␤-DG is called the DG complex (7, 11, 12). In peripheral nerves, the DG complex is expressed in Schwann cells as well as in satellite cells of the dorsal root ganglia (20, 22, 31, 39). Recent data demonstrate that peripheral nerve ␣-DG binds laminin-2 (␣2/ ␤1/␥1), a major component of the Schwann cell basal lamina (18, 24, 28, 32, 39 – 41), and that the DG complex is involved in schwannoma cell adhesion to laminin (21). On the cytoplasmic side, ␤-DG is anchored to the dystrophin isoform, Dp116 (5, 20, 31). Importantly, there is now ample evidence that the components of the basal lamina, laminin-2 in particular, play crucial roles in Schwann cell process of myelination. The evidence includes data obtained in laminin-2-deficient dy mice in vivo as well as from neuron–Schwann cell coculture studies (1–3, 6, 10, 23, 25–27, 30, 33, 34, 37, 38). On the basis of these observations, we hypothesized that the DG complex might function as an adhesion apparatus that binds the Schwann cell outer membrane to the basal lamina and is associated with the process of Schwann cell myelination through interaction with laminin-2 (39 – 41). Recently, we have shown that the cytoplasmic tail of ␤-DG is localized selectively in the Schwann cell cytoplasm, just under the outer membrane, and that the expression of ␤-DG and the laminin-␣2 chain is downregulated during the axonal degeneration process. Conversely, it is upregulated during the process of regenerating Schwann cell ensheathment and myelination (19). In the in vivo study reported here, we investigated the expression of ␤-DG and the laminin-␣2 chain in rat peripheral nerve during develop-

troglycan immunoreactivity; laminin-␣2-IR, laminin-␣2 immunoreactivity; laminin-␤1/␥1-IR, laminin-␤1/␥1 immunoreactivity.

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0014-4886/02 $35.00 2002 Elsevier Science (USA) All rights reserved.

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FIG. 1. Immunoblot analysis of rat sciatic nerves. Protein samples (10 ␮g) were analyzed by 7.5% SDS–PAGE. In all of the developmental stages from P0 to adult, 8D5 recognized ␤-DG as a 43,000 MW band (A), and with 2D9, we identified a band higher than 213,000 MW (B). Considering the specificity of 2D9, the band seemed to correspond to the laminin-␣2 chain (320,000 MW). With L9393, we recognized a 200,000 MW band that represent the laminin-␤1/␥1 chain (C). ␤-DG and the laminin-␣2 chain showed a similar patterns of developmental expression: Both increased from P0 to P7. ␤-DG decreased slightly from P7 to adult. Whereas the laminin-␣2 chain was maintained from P7 to P28, it decreased from P28 to adult. The laminin-␤1/␥1 chain also showed a similar pattern but appeared to be expressed more consistently than the laminin-␣2 chain.

ment by immunoblot, immunofluorescence, and immunoelectronmicroscopic studies. We found that the expression of ␤-DG and the laminin-␣2 chain, along the Schwann cell outer membrane, was upregulated during the first week after birth, when basal lamina assembly and compact myelin formation by Schwann cells progressed rapidly. Our results indicate that the DG complex and laminin-2 are associated with these Schwann cell differentiation processes. MATERIALS AND METHODS

Tissue Preparation Wistar rats of various ages, ranging from embryonic day (E) 18, postnatal day (P) 0, 3, 7, 28 to young adult (12-week-old), were used. The experimental procedures followed the National Defense Medical College welfare rules and regulations for treatment of animals. The animals were deeply anesthetized by intraperitoneal injection with sodium pentobarbital (50 mg/kg body weight) or by ice cooling. For the immunofluorescence study, sciatic nerves were dissected out at the midthigh level, immediately frozen in isopentane cooled in liquid nitrogen, and stored at ⫺80°C. For the immunoelectron microscopic study, dissected nerve segments were immersed in 100 mM sodium phosphate buffer containing 4% paraformaldehyde at 4°C overnight. For electron microscopy, nerve segments were immersed in buffer containing 2.5% glutaraldehyde and 0.5% paraformaldehyde at 4°C overnight and then prepared for conventional electron microscopic study. For the immunoblot study, dissected sciatic nerves (P0 to adult) were homogenized in 5 vol of 50 mM Tris–HCl, pH 7.4, in 0.15 M NaCl containing 0.75 mM benzamidine and 0.1 mM phenylmethylsulfonyl fluoride and stored at

⫺80°C, as previously described (19, 20). Selective dissection of sciatic nerves of E18 rats for the immunoblot study was not possible. Antibodies Two mouse monoclonal antibodies, 8D5 against the carboxyl-terminus of ␤-DG and 2D9 against the proximal portion of the globular domain of the laminin-␣2 chain, have been previously described (39 – 41). Because ␤-DG is a type I transmembrane protein, the amino and carboxyl termini being extracellular and intracellular, respectively, the 8D5 epitope falls on the carboxyl-terminal tail of the cytoplasmic domain of ␤-DG. L9393, a rabbit polyclonal antibody against laminin, was purchased from Sigma Chemical Co. (St. Louis, MO). Immunoblot, Immunofluorescence, and Immunoelectron Microscopic Studies SDS–PAGE (7.5%) was performed as previously described (19, 20). Immunoblot and immunofluorescence analyses were performed using 8D5 and 2D9 as described previously (19, 20, 39 – 41). Immunoelectron microscopic analysis using 8D5 was performed, but immunoelectronmicroscopic analysis using 2D9, as described previously (19, 20, 31), was not possible. Immunoblot and immunofluorescence analyses were also performed using L9393 at a dilution of 1:2000 in these experiments. As secondary antibodies, we used biotinconjugated anti-rabbit IgG (1:200, Vector Laboratory, Burlingame, CA) in immunoblot analysis and rhodamine conjugated anti-rabbit IgG (1:200, AP182R, Chemicon International Inc., Temecula, CA) in immunofluorescence analysis.

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FIG. 2. Immunofluorescence analysis of rat sciatic nerves. (A–E) Immunofluorescent images showing ␤-DG-IR from E18 to adult. At E18 (A) and P0 (B), ␤-DG-IR was faintly and diffusely detected in the endoneurium. At P7 (C), ␤-DG-IR increased and small ring-shaped structures were clearly identified, indicating that ␤-DG was integrated in the Schwann cell outer membrane. At P28 (D) and adult (E), ␤-DG-IR was associated with the Schwann cell outer membrane but appeared weaker than at P7. (F–K) Immunofluorescence images showing laminin-␣2-IR from E18 to adult. The pattern of laminin-␣2-IR was similar to that of ␤-DG-IR. At E18 (F) and P0 (G), laminin-␣2-IR in the endoneurium was faint and diffuse. After P7 (H, J, K), laminin-␣2-IR increased and was associated with the Schwann cell outer membrane. (L–Q) Immunofluorescent images showing laminin-␤1/␥1-IR from E18 to adult. At E18 (L), laminin-␤1/␥1-IR was clearly associated with the Schwann cell outer membrane. At P0 (M), laminin-␤1/␥1-IR was weakly detected in the endoneurium and appeared to be associated with the Schwann cell outer membrane. After P7 (N, P, Q), the Schwann cell outer membrane-associated laminin-␤1/␥1-IR became more intense. The arrowheads indicate the boundary between the sciatic nerve and the surrounding connective tissue. Bar, 20 ␮m.

Quantitative Analysis for Electron Microscopy Images at a magnification of 2000⫻ were captured by electron microscopy and converted into digital images with an image scanner (GT-9600, Epson, Nagano, Japan). The number of Schwann cells, the total length of Schwann cell outer membranes (␮m), and the area of endoneurial space (␮m 2) in the images were calculated using Mac Scope 2.51 software (Mitani Corporation, Fukui, Japan). In addition, we calculated the total length of Schwann cell outer membranes per square micrometer of endoneurial space (␮m/␮m 2) and defined this value as the OM index. To calculate the total length of the basal lamina, we captured images (magnification 20,000⫻) containing the Schwann cell outer

membrane region, and converted them into digital images for analysis using Mac Scope 2.51 software. Quantitative Analysis for Immunofluorescence Images of the immunostained samples were captured on a microscope equipped with a laser scanning confocal attachment (LSM 410 invert; Carl Zeiss Inc., Jena, Germany). As negative controls, tissue sections were incubated with normal mouse or rabbit IgG (1: 200, Vector), followed by secondary antibodies. As described under Results, the immunoreactivities for ␤-dystroglycan (␤-DG-IR) and the laminin-␣2 chain (laminin-␣2-IR) were both localized to the Schwann cell outer membrane, except at E18 and P0. We de-

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TABLE 1 Quantitative Analysis of ␤-DG Expression by Immunofluorescence Study Age of rats (n)

IR index (mean ⫾ SD)(% of P7)

IR index/OM index (mean ⫾ SD)(% of P7)

E18 (5) P0 (5) P7 (5) P28 (5) Adult (5)

9.27 ⫾ 4.10 21.15 ⫾ 7.38 100.00 ⫾ 21.12 58.36 ⫾ 16.93 42.72 ⫾ 3.19

— 19.65 ⫾ 7.24 100.00 ⫾ 23.49 80.20 ⫾ 20.50 84.90 ⫾ 15.11

Note. IR index/OM index: P0 vs P7, P0 vs P28, P0 vs adult, P ⬍ 0.001. Sciatic nerves from five rats at each developmental age from E18 to adult were examined. One image per rat (total, 25 images) was analyzed.

signed a method of quantitative analysis for these immunoreactivities by calculating a value per unit length of the Schwann cell outer membrane in images of transverse sections. Digital photomicrographs (1024 ⫻ 1024 pixels, 8-bit gray scale) of FITC fluorescence were taken using a 40⫻ objective lens, representing an area of 0.28 ⫻0.28 mm, and stored on a disk as 1-Mb TIFF files. The images were analyzed for the mean pixel intensity of ␤-DG-IR or laminin-␣2-IR using Mac Scope 2.51 software. The mean pixel intensity was a mean of the intensities of all pixels within the sciatic nerve, immunostained with 8D5 or 2D9. We defined the IR index as the mean pixel intensity of ␤-DG-IR or laminin-␣2-IR from which the background (i.e., the mean pixel intensity of the corresponding negative controls) was subtracted. In addition, nerve segments adjacent to those used for immunofluorescence analysis, were processed to obtain the OM index, as described above. The value of the IR index divided by the OM index thus represents Schwann cell outer membrane-associated expression of ␤-DG or the laminin-␣2 chain. Quantitative Analysis for Immunoelectron Microscopy Images at a magnification of 4000⫻ were captured by electron microscopy, converted into digital images, and analyzed using Mac Scope 2.51 software. Because compact myelin was the most electron-dense structure in the endoneurium, any pixels that which had an intensity stronger than that of compact myelin were considered to have significant ␤-DG-IR. For each Schwann cell, we calculated the value of the total amount of the ␤-DG-IR pixel intensities per total length of the Schwann cell outer membrane as a representation of Schwann cell outer membrane-associated expression of ␤-DG. Statistical Analysis The quantitative ␤-DG-IR and laminin-␣2-IR data in the immunofluorescence and immunoelectron micro-

scopic studies were analyzed by Kruscal–Wallis analysis, followed by Sheffe’s post hoc tests. Differences were considered to be significant when the P value was less than 0.05. RESULTS

Immunoblot Study From P0 to adult, 8D5 recognized a 43,000 MW protein or glycoprotein as a band representing ␤-DG (Fig. 1A), and 2D9 consistently showed a band of molecular weight higher than 213,000 (Fig. 1B). Considering the specificity of 2D9 (39, 40), the band seemed to correspond to the laminin-␣2 chain (320,000 MW) in rat sciatic nerves. L9393 recognized a 200,000 MW protein represented as a band that corresponded to the laminin-␤1 and/or -␥1 chain, as previously described (39, 40) (Fig. 1C). ␤-DG and the laminin-␣2 chain showed a similar pattern of developmental expression. Both increased from P0 to P7. ␤-DG decreased slightly from P7 to adult. Whereas expression of the laminin-␣2 chain was maintained from P7 to P28, it decreased from P28 to adult (Figs. 1A and 1B). The laminin-␤1/␥1 chain also showed a similar expression pattern, but it appeared to be expressed more consistently from P0 to adult than was the laminin-␣2 chain (Fig. 1C). Immunofluorescence Study

␤-DG-IR was detected as only faint and diffuse in rat sciatic nerves at E18 and at P0 (Figs. 2A and 2B). At P7, the ring-shaped pattern of ␤-DG-IR along the Schwann cell outer membrane became clear and intense (Fig. 2C). From P7 to adult, the intensity of the ␤-DG-IR appeared to decrease slightly but Schwann cell outer membrane-associated expression was maintained (Figs. 2C–2E). Throughout these periods, laminin-␣2-IR showed course of developmental expression similar to that of ␤-DG-IR (Figs. 2F–2K); it increased from P0 to P7 and was maintained until adult. In

TABLE 2 Quantitative Analysis of Laminin ␣2 Chain Expression by Immunofluorescence Study Age of rats (n)

IR index (mean ⫾ SD)(% of P7)

IR index/OM index (mean ⫾ SD)(% of P7)

E18 (5) P0 (5) P7 (5) P28 (5) Adult (5)

23.20 ⫾ 3.94 18.26 ⫾ 3.39 100.00 ⫾ 36.53 78.31 ⫾ 2.99 76.16 ⫾ 14.11

— 17.58 ⫾ 3.78 100.00 ⫾ 32.20 108.90 ⫾ 6.40 154.29 ⫾ 42.85

Note. IR index/OM index: P0 vs P7, P0 vs P28, P0 vs adult, P ⬍ 0.001. Sciatic nerves from five rats at each developmental age from E18 to adult were examined. One image per rat (total, 25 images) was analyzed.

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TABLE 3 Schwann Cell Differentiation Process Percentage of Schwann cells in three differentiation stages Age of rats (n)

I

II

III

E18 (5) P0 (5) P7 (5) P28 (5) Adult (5)

100.00 66.43 12.20 14.60 10.63

0.00 33.33 8.57 2.20 3.90

0.00 0.23 79.20 83.20 85.47

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natal period, as reported previously (29, 35). For clarification, we defined three stages of Schwann cell differentiation: stages I, II, and III. At E18, all of the Schwann cells were immature, each enveloping a large population of axons and lacking compact myelin (stage 1). By P0, 33.33% of the Schwann cells enveloped only

Note. Sciatic nerves from five rats at each developmental age from E18 to adult were examined. Five images per rat (total, 125 images) were analyzed.

contrast to the ␤-DG and laminin-␣2-IRs, the laminin␤1/␥1-IR was clearly detectable surrounding Schwann cells at E18 (Figs. 2L–2Q). However, its course of developmental expression after P0 was similar to that of the former two antigens: low at P0, increasing from P0 to P7, decreasing slightly from P7 to adult. Whereas the laminin-␤1/␥1-IR was consistently localized all around Schwann cells, the ␤-DG and laminin-␣-IRs were localized in only parts of the Schwann cell outer membranes even after P7. The apparent intensity of ␤-DG-IR and laminin␣2-IR mentioned above is dependent not only on the intensity of immunoreactivity itself but also on the amount of Schwann cell outer membrane per unit area of endoneurial space. To determine the authentic intensity of immunoreactivity in the Schwann cell outer membrane, we therefore calculated the IR index/OM index of ␤-DG-IR and laminin-␣2-IR and performed statistical analysis. The IR index/OM index of ␤-DG and the laminin-␣2 chain increased strikingly between P0 and P7 (Tables 1 and 2), confirming the upregulation of ␤-DG and laminin-␣2 expression along the Schwann cell outer membrane during this period. On the other hand, the IR index/OM index of ␤-DG-IR and laminin-␣2-IR did not change significantly from P7 to adult (Tables 1 and 2). This suggests that the aforementioned apparent decrease of ␤-DG and laminin-␣2 expression from P7 to adult in the immunoblot study is due to the relative decrease of the OM index, as described below, and thus does not reflect authentic downregulation. At E18, ␤-DG-IR and laminin-␣2-IR were very faint and not confined to the Schwann cell outer membrane (Fig. 2). In addition, the OM index at E18 was far lower than that after P0. E18 data, therefore, could not be analyzed for the IR index/OM index. Electron Microscopic and Immunoelectron Microscopic Study The electron microscopic study demonstrated that Schwann cells change morphology rapidly in the peri-

FIG. 3. Electron microscopic analysis of rat sciatic nerves. (A, B) Stage I Schwann cells at E18. (C) Stage I Schwann cells at P0. (D) Stage III Schwann cells at P3. (E) Stage III Schwann cells at P7. At E18, a basal lamina-like structure was detectable only in a patchy and irregular pattern, partially surrounding stage I Schwann cells. No mature basal lamina, composing both lamina lucida and lamina densa, was observed. At P0, basal lamina was detectable surrounding most of stage I Schwann cells, but there was a considerable variation in its density and thickness. At P3, mature basal lamina (composed of lamina densa and lamina lucida) was observed surrounding stage III Schwann cells. At P7, the outer membrane of stage III Schwann cells was completely covered by the mature, two-layer basal lamina of uniform density and thickness. (a) Axon, (c) Schwann cell cytoplasm, (m) compact myelin, (n) nucleus, and (p) perineurial cell. Bar, 500 nm.

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TABLE 4 Quantitative Analysis of Basal Lamina Assembly

Age of rats E18 P0 P3 P7

Schwann cell differentiation stage Stage Stage Stage Stage

I I III III

n

Total length of identifiable basal lamina/total length of outer membrane ⬎50% (% of total Schwann cells)

Total length of basal lamina with lamina densa and lamina lucida/total length of outer membrane ⬎50% (% of total Schwann cells)

Total length of basal lamina with lamina densa and lamina lucida/total length of outer membrane ⬎80% (% of total Schwann cells)

5 5 5 5

56 92 100 100

0 32 68 100

0 8 58 98

Note. Fifty images, each containing one Schwann cell, were analyzed for each developmental age from E18 to P7 (total, 200 images).

one axon but lacked compact myelin (stage II) (see Table 3). Between P0 and P7, the Schwann cells began rapid formation of compact myelin (stage III; myelinforming Schwann cells). At P7, stage III cells were predominant (79.2%), while stage II cells accounted for only 8.57% of all Schwann cells (Table 3). There was no remarkable change in the proportions of stage I, II, and III Schwann cells from P7 to adult (Table 3). In addition, the OM index was 0.375 ⫾ 0.038 (␮m/␮m 2; mean ⫾ SD) at E18, 1.727 ⫾ 0.120 at P0, 1.518 ⫾ 0.200 at P7, 1.139 ⫾ 0.096 at P28, and 0.825 ⫾ 0.116 in adults. To acertain whether the upregulation of ␤-DGIR and laminin-␣2-IR during the P0 to P7 period was due to Schwann cell differentiation between stage I or II and stage III, we calculated the indices for stage I and II Schwann cells at P7 as 0.143 ⫾ 0.0062 and 0.048 ⫾ 0.0077, respectively. At E18, a basal lamina-like structure was barely detectable, appearing as only a patchy and irregular pattern surrounding stage I Schwann cells (Figs. 3A and 3B; Table 4). No mature basal lamina, which comprises two layers (lamina lucida and lamina densa), was present at this time (Figs. 3A and 3B; Table 4). By P0, the basal lamina was detectable surrounding most stage I Schwann cells, but there was considerable variation in its density and width (Fig. 3C; Table 4), as reported (35). The mature basal lamina, in which the lamina lucida and lamina densa layers were both discernible, appeared for the first time in the E18 –P0 period (Table 4). At P3, the mature basal lamina was clearly observed surrounding most stage III Schwann cells (Fig. 3D; Table 4). At P7, stage III Schwann cells were completely covered by a mature basal lamina comprising two layers of uniform density and thickness (Fig. 3E; Table 4). In addition, at P7, stage I Schwann cells were consistently surrounded by a mature basal lamina (data not shown). Immunoelectronmicroscopic analysis showed no significant ␤-DG-IR in rat sciatic nerves at E18 (Fig. 4), while laminin-␤1/␥1-IR was selectively associated with the Schwann cell outer membrane (data not shown). From P0 to adult, ␤-DG-IR was detected in the

Schwann cell cytoplasm immediately underlying the outer membrane. Throughout P0 –adult period, stage III Schwann cells always showed strong and continuous ␤-DG-IR in the outermost cytoplasm, but in the stage I and II Schwann cells, ␤-DG-IR was weak and irregular (Fig. 4). Quantitative analysis of ␤-DG-IR at P3 demonstrated a statistically significant increase in Schwann cell outer membrane-associated ␤-DG-IR from stage I to state II as well as from stage II to stage III (Table 5). DISCUSSION

Role of DG Complex and Laminin-2 in Schwann Cell Ensheathment and Myelination In this study, we used immunoblot, immunofluorescence, and immunoelectron microscopic studies to investigate the expression of ␤-DG and the laminin-␣2 chain in rat peripheral nerves during development. Although we detected little ␤-DG in the Schwann cell outer membrane from E18 to P0, it was markedly upregulated between P0 and P7. Whereas it seemed to decrease slightly from P7 to adult, the decrease was not statistically significant. The expression of the laminin-␣2 chain roughly paralleled that of ␤-DG during these periods. Electron microscopy showed that at P0 almost all Schwann cells were stage I or II. The rapid progression of Schwann cell differentiation from stage I or II to stage III occurred during the first week after birth, resulting in predominantly stage III Schwann cells by P7. From P7 to adult, there was no remarkable change in the proportions of stage I, II and III Schwann cells. These results showed that the period of upregulation of ␤-DG and the laminin-␣2 chain from P0 to P7 coincided with the rapid progression of Schwann cell differentiation from stage I or II to stage III. Also, important is the immunoelectron microscopic study’s demonstration that the expression of Schwann cell outer membrane-associated ␤-DG depends not simply on the developmental age of the animal but on the stages of

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FIG. 4. Immunoelectron microscopic analysis of rat sciatic nerves. Immunoelectron photomicrographs showing ␤-DG-IR. (A) At E18, immature stage I Schwann cells showed no ␤-DG-IR. Bar, 500 nm. (B) At P0, stage I Schwann cells ensheathing multiple axons showed faint and irregular ␤-DG-IR associated with the Schwann cell outer membrane. Bar, 500 nm. (C) At P7, myelin-forming stage III Schwann cells showed intense and continuous ␤-DG-IR in the cytoplasm just underlying the outer membrane. Bar, 500 nm. (D) At P3, stage I Schwann cells ensheathing multiple axon bundles (1) showed faint and irregular ␤-DG-IR, while stage III Schwann cells (3) showed intense and continuous ␤-DG-IR in the cytoplasm underlying the outer membrane. Stage II Schwann cells (2) showed an intermediate pattern of ␤-DG-IR. Bar, 1 ␮m. (A or *) An axon or axon bundle, (C) Schwann cell cytoplasm, (M) compact myelin, (OM) outer membrane.

Schwann cell differentiation, (designated as stages I, II, and III in this study). Together, the results of the study suggest that the upregulation of ␤-DG and the laminin-␣2 chain from P0 to P7 is associated with the

process of myelin formation by Schwann cells. Anchorage of the Schwann cell outer membrane to the basal lamina appears to play crucial roles in the myelination process (4). It has been suggested that, as laminin

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TABLE 5 Quantitative Analysis of ␤-DG Expression by Immunoelectron Microscopic Study Differentiation stage of Schwann cells (n)

Total amount of pixel intensity of ␤-DG-IR/outer membrane length (mean ⫾ SD)(% of stage III)

I (79) II (218) III (187)

18.99 ⫾ 17.18 40.17 ⫾ 54.57 100.00 ⫾ 59.72

Note. Stage I vs stage II, stage I vs stage III, stage II vs stage III, P ⬍ 0.001. P3 samples were analyzed, because of the abundance of all stages (I, II, and III) of Schwann cells. A total of 484 Schwann cells (79 stage I, 218 stage II, and 187 stage III) were analyzed.

receptors, the ␣6␤1 and ␣6␤4 integrins are involved in this anchorage of the Schwann cell outer membrane to the basal lamina (8, 13–15). Considering the functional role of the integrins, it is plausible that the binding of the DG complex in the Schwann cell outer membrane to laminin-2 in the basal lamina also plays a role in initiating the process of myelination. Further study will be needed to ascertain whether these hypotheses hold true. The results of this study did not provide firm evidence concerning whether the DG complex and laminin-2 are associated with the process of ensheathment of unmyelinated axons. Considering the far lower contribution of stage I Schwann cells (most of which should become non-myelin-forming cells) to the OM index at P7, compared with the contribution of stage III cells, it is not likely that the stage I cells significantly contributed to the upregulation of ␤-DG and the laminin-␣2 chain from P0 to P7, as observed by immunofluorescence. Role of the DG Complex and Laminin Isoforms in Schwann Cell Basal Lamina Assembly The molecular mechanism of basal lamina assembly around Schwann cells is not well known. In this study, we found that the assembly of the Schwann cell basal lamina began in the perinatal period and that the mature basal lamina composing the lamina densa and lamina lucida was established between P0 and P7. Thus, the basal lamina assembly coincided with the expression of ␤-DG and the laminin-␣2 chain along the myelin-forming Schwann cell outer membrane. This is not surprising, considering the primary role of the DG complex in the basal lamina assembly (16, 36). As for non-myelin-forming Schwann cells, however, this study did not show convincing evidence of the association of basal lamina assembly with the DG complex and laminin-2, because we did not analyze precisely whether those proteins in non-myelin-forming Schwann cells were upregulated between P0 and P7, when basal lamina maturation occurred.

The initial phase of Schwann cell basal lamina deposition might be associated with molecules other than DG or laminin-␣2 chain, because we observed the deposition of electron-dense material corresponding to the immature basal lamina as early as E18, when the expression of ␤-DG and laminin-␣2 chain was still low. In this respect, it is noteworthy that intense laminin␤1/␥1-IR was observed to be associated with the Schwann cell outer membrane at E18. It is possible that the laminin-␣ chain other than ␣2 is complexed with laminin-␤1/␥1 and that it plays a role in the initial deposition of the basal lamina in the late embryonic stage. This putative conversion of laminin-␣ chain isoform may play a role in the Schwann cell differentiation, as has been suggested in epithelial differentiation of kidney (9). ACKNOWLEDGMENTS Monoclonal antibodies 8D5 and 2D9 were kindly provided by Drs. Louise V. B. Anderson (Newcastle General Hospital) and Hisae Hori (Tokyo Medical and Dental University), respectively. This work was supported by a Research Grant from the Genome Research Center, Teikyo University, by Research on Brain Science and Research Grants 10B-3 and 11B-1 for Nervous and Mental Disorders from the Ministry of Health and Welfare, and Research Grants 09470156, 09770460, 09877121, 10044319, 11470151, 11670644, and 12470143 from the Ministry of Education, Science, Sports, and Culture.

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