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Expression and Function of Ganglioside 9-O-Acetyl GD3 in Postmitotic Granule Cell Development
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Molecular and Cellular Neuroscience 17, 488 – 499 (2001) doi:10.1006/mcne.2000.0943, available online at http://www.idealibrary.com on
Expression and Function of Ganglioside 9-O-Acetyl GD3 in Postmitotic Granule Cell Development Marcelo F. Santiago, Marcia Berredo-Pinho, Marcos R. Costa, Mario Gandra, Leny A. Cavalcante, and Rosalia Mendez-Otero Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ 21941-590, Rio de Janeiro, Brazil
We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-Oacetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dosedependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.
INTRODUCTION During development of the central nervous system, postmitotic neurons undergo a marked migration toward their final positions. This active displacement of immature neurons is essential for the establishment of normal cytoarchitecture and synaptic connectivity in the vertebrate brain (Caviness and Rakic, 1978; Hatten, 1990; Rakic, 1990). In the cerebellar cortex, for example, granule cell precursors migrate from the external granular cell layer where they are generated, to reach their final positions in the internal granular layer (Ramo´n y Cajal, 1911; Miale and Sidman, 1961). Several studies have supported the hypothesis that
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functional interactions between postmitotic granule cells and Bergmann glial fibers are essential for this migration (Rakic and Sidman, 1973a, b; Rakic, 1976, 1981; Hatten et al., 1984, 1986; Gao and Hatten, 1993). Although the molecular mechanisms influencing granule cell migration are not yet fully understood, it has become clear from various in vitro studies that a number of molecular and cellular mechanisms, including cell– cell recognition, cell– cell adhesion, cell motility, intracellular Ca 2⫹ dynamics, and activity of NMDA receptors are involved in the control of the migration program (Antonicek et al., 1987; Edmondson and Hatten, 1987; Fishell and Hatten, 1991; Komuro and Rakic, 1993; Cameron and Rakic, 1994; Rakic et al., 1994; Komuro and Rakic, 1995). The possibility that cell surface carbohydrates and/or lipids mediate cellular interactions within the vertebrate nervous system has been considered on numerous occasions although most of the studies in cell migration have focused on protein–protein interactions. Gangliosides (sialic acid-containing glycosphingolipids) constitute a major group of cell surface carbohydrate-containing molecules. They have been implicated in numerous cellular functions in the developing and adult mammalian nervous system, since they are enriched in the outer surface of neural membranes (Ledeen, 1985; Hakomori, 1990; Yu, 1994). In previous works we observed that the ganglioside 9-O-acetyl GD3 recognized by the Jones monoclonal antibody (Jones mAb—Constantine-Paton et al., 1986; Bonafede et al., 1989) is developmentally regulated and expressed in regions of cell migration and neurite outgrowth (Mendez-Otero et al., 1988; Mendez-Otero and Ramon-Cueto, 1994; Men1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Heterogeneous patterns of immunoreactivity to 9-O-acetyl GD3 in P7 rat cerebellum. (A) Low power photomicrograph, (B) confocal image, and (C) electromicrograph. Observe the heterogeneous binding of Jones mAb on different folia and the apparently compact labeling of one caudal folium (A). In the confocal image (B) notice the labeling of cell bodies and processes in the external granular cell layer and presumptive molecular layer. In the electronmicrograph (C), notice the selective labeling of few granular-Bergmann membranes. (A) Silverenhanced colloidal gold labeling of the secondary antibody. (B) Cy3-labeling of the secondary antibody. (C) PAP procedure. Calibration bars: (A) 375 m, (B) 25 m, and (C) 3 m.
dez-Otero and Cavalcante, 1996). We have also observed that cultured neurons from rat cerebella when in contact with glial cells that support cell migration (radial glial cells), express the Jones-reactive ganglioside, as do the glial cells. On the other hand, glial cells with a stellate morphology, even when in contact with granule neurons do not express this antigen (Mendez-Otero and Constantine-Paton, 1990). In the present study, we have chosen the rat cerebellum to further elucidate the functional role of ganglioside 9-O-acetyl GD3 in neuronal migration. We chose this system because it has been thoroughly characterized histologically and correlation with neurogenesis, migration and differentiation are easy to perform (Altman, 1972a, 1972b, 1972c). Furthermore, migration of neuronal precursors occurs postnatally and is therefore more available to experimental analysis. Initially, the localization of 9-O-acetyl gangliosides in explants of rat cerebellum was determined using cellular markers. We also report that the antibody against 9-O-acetyl GD3
inhibits migration of granule neuron precursors along glial fibers in a dose-dependent manner in this model system. This is the first time in which a specific ganglioside has been implicated as a signaling molecule in neuronal migration.
RESULTS Expression of 9-O-Acetyl GD3 in Postnatal Cerebellum The ganglioside recognized by the Jones mAb was expressed in a stacked pattern in the external granule cell layer (EGL) during the first postnatal week (Fig. 1A). The intensity of expression of this antigen differed in the various folia of the cerebellum, which may reflect different maturational stages of these structures. Granular cell precursors leaving the EGL with a typical migratory morphology express 9-O-
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FIG. 2. Pattern of expression of gangliosides in cerebellar explants. Phase contrast (A and C) and fluorescence photomicrographs of cerebellar explants labeled with Jones (B) or A2B5 (D) antibodies. Observe labeling of cell bodies and neurites (insets) with either antibody. Calibration bar, 20 m.
acetyl GD3 (Fig. 1B). At the EM level, Jones immunoreactivity in the presumptive molecular layer was detected mainly at sites of contact between migrating granule cells and Bergmann glial fibers (arrows - Fig. 1C). Labeling of granular cells and/or adjoining Bergmann fibers is heterogeneous with many unlabeled sets of granule–Bergmann elements in the close vicinity of labeled cells.
Expression of 9-O-Acetyl GD3 in Cerebellar Microexplants In cerebellar microexplants, several cells were observed migrating out of the explant 24 h after plating. Based on their size (8 –10 m) and nuclear morphol-
ogy, these cells are most likely granule cells which constitute the majority of the neurons in cerebella explant cultures (Hockberger et al., 1987). Figures 2A and 2C show the typical morphology of explants with granule cells migrating in a radial orientation from the core (bottom-left in all pictures) after 72 h in vitro. These cells when stained with Jones mAb show immunoreactivity for 9-O-acetyl GD3 on their cell bodies and neurites (Fig. 2B). Migrating cells also bind other antibodies against gangliosides. For instance, immunoreaction with the A2B5 monoclonal antibody (A2B5 mAb) also labels cell bodies and neurites in these explants (Fig. 2D). Both antibodies stain the cell body very strongly and delineate the membrane of the neurites (insets in Figs. 2B and 2D).
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Characterization of the Jones-Positive Cells on Cerebellar Microexplants To further characterize the cell types present in these cultures we double labeled them with antibodies against MAP-2 to identify neurons and GFAP to identify glia (Figs. 3A–3C). In Fig. 3A we observe a large population of radial glial fibers labeled with GFAP (green). Closely associated with these glial cells are a great number of granular neurons labeled with MAP-2 (red). These neurons seem to migrate out of the explant closely apposed to the radial glial fibers (arrows, Fig. 3B) in a fashion very similar to that shown in vivo by electron microscopy (see also Fig. 1C) and in vitro with video-enhanced differential contrast microscopy (Rakic, 1971; Hatten, 1990). Besides this glial-guided neuronal migration, we could also observe neurons labeled with MAP-2 (red) migrating without any apparent association with glial fibers (Fig. 3C). These cells probably migrate associated with the neurites of other neurons similarly to the chain migration that occurs in the rostral migratory stream of the developing and adult telencephalon (Wichterle et al., 1997; Mendez-Otero and Cavalcante, 1996). In order to determine which cell types expressed Jones antigen in these cultures we performed double label experiments. Figure 3D shows a double staining for MAP-2 (green) and 9-O-acetyl GD3 (red). The colocalization of the neuronal marker with the 9-O-acetyl GD3 (yellow) shows that these migrating cells are neurons (arrows). In our model system, the expression of Jones antigen is not restricted to granule neurons. Double labeling of GFAP (green) and Jones mAb (red) reveals that radial glial cells also express 9-O-acetyl GD3 (yellow, Fig. 3E). Nevertheless, the expression of this antigen is restricted to glial cells that contact migrating granule neurons and is not observed on glial cells that are not associated with neurons (outline in Fig. 3E). These results confirm our previous suggestion from cerebellar dissociated microcultures that there exists an inducible effect from the apposition of these two cells (Mendez-Otero and Constantine-Paton, 1990).
Jones mAb Inhibits the Migration of Cerebellar Neurons in Microexplant Cultures The influence of Jones mAb on neuronal migration in microexplant cultures prepared from P4 –P7 rat cerebellum was tested. In this in vitro system, migration of granule cells occurs over a period of 3 days. The effects on neuronal migration were therefore assessed after 3
491 days following the addition of Jones mAb and A2B5 mAb at the first day. Similar-sized explants (600 –700 m in diameter) were analyzed for each experimental group. Neuronal cells were labeled with the nucleophilic dye DAPI and then measurements were made in eight sites located at 45° angles to each other at the periphery of each explant. The distance reached by the wavefront of cells was measured from the border of the explant. Using this methodology we found a 66% decrease in the distance from the explant reached by granule cells in the presence of Jones mAb (214 ⫾ 34 m, n ⫽ 13; Figs. 4C and 4D) as compared to those of control cultures in which no antibody was added (638 ⫾ 64 m, n ⫽ 18; Figs. 4A and 4D). When the same test was performed in the presence of A2B5 mAb, granule cell migration was not modified (614 ⫾ 28 m; n ⫽ 21; Figs. 4B and 4D) when compared with control cultures. This experiment showed that only Jones mAb specifically interferes with migration of granule cells (Fig. 4D). Using different dilutions of Jones mAb we verified that the immunoblockade effect occurs in a dose-dependent manner and that it has a plateau of activity around the 1:100 dilution (Table 1). Since granule neurons and radial glial cells express the Jones antigen, the decrease in migration distance could be due to interference of this monoclonal antibody on the extension of the glial cells and/or on the migration of the granule neurons. To answer this question, Jones blocked explants were double labeled with GFAP (green) and MAP-2 (red). Figure 3F shows a phase contrast image of an immunoblocked explant with a small number of cells migrating out of it (arrowheads) and some fibers that may be neurites and/or glial fibers (arrows). A similar explant was double labeled for GFAP and MAP-2 (Fig. 3G), and it showed that even when the explant grows in the presence of the Jones mAb, glial cells are able to extend their process. The staining for MAP-2 reveals that some neurons migrate a short distance from the border of the explant in this condition. These results suggest a specific effect of Jones mAb on the migration of granule cells along glial fibers. Posmitotic Granular Cell Migration Is Arrested by Jones mAb in Cerebellum Slice Cultures To investigate whether the immunoblockage obtained with the explant cultures could be also extended to organotypic slice cultures we labeled proliferating cells with a 15-min pulse of 5-Bromo-2⬘-deoxyuridine (BrdU) and mapped their migratory movement throughout the laminar structure of the cerebellar slice
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FIG. 3. Characterization of 9-O-acetyl GD3 immunoreactive cells in cerebellar explants. (A–C) Cerebellar explants, double-labeled with GFAP (green) and MAP-2 (red), showing the presence of radial glial fibers and their close association with neurons. In (B), neurons (arrows) closely associated with glial fibers are shown and neurons that migrate outside the region of glial fibers are illustrated in (C). (D) Explant double-labeled for 9-O-acetyl GD3 (red) and MAP-2 (green). Notice that all MAP-2-positive cells (arrows) are also labeled for 9-O-acetyl GD3 (yellow). (E) Explants double-labeled for GFAP (green) and 9-O-acetyl GD3 (red). Some of the glial cells are double-labeled (yellow) and others are only stained for GFAP (outline). Most of the neurons (red) are positive for 9-O-acetyl GD3. (F and G) Explants blocked with Jones mAb showing that radial glia cells are present in these explants but neurons do not migrate very far out of the explant. Calibration bar, 20 m.
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FIG. 4. Immunoblocking of cerebellar explants with different antibodies. (A) Phase contrast photomicrograph of an explant in the absence of antibodies. (B) Similar explant in the presence of A2B5 mAb or (C) Jones mAb. Observe the blockage of cell migration out of the explant in the presence of Jones mAb (C and D). (D) Quantitative analysis of several explants in the presence or absence of different ganglioside-reactive antibodies. *P ⬍ 0.01. Calibration bar, 40 m.
cultures. In the control group, the slices were incubated with fresh medium after the BrdU pulse (Fig. 5B). In two other sets of cultures Jones mAb was added at the same time as BrdU (Figs. 5D and 5E). After 15 min this
medium was removed and changed for fresh culture medium without Jones mAb (Fig. 5D) or with Jones mAb (Fig. 5E). In another set of slices A2B5 mAb was used instead of Jones mAb and maintained throughout
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TABLE 1 Dose-Dependent Effect of Jones mAb in Neuronal Migration Jones mAb concentration
Migrated distance (m) Percentage
Control
1:800
1:400
1:200
1:100
1:50
638 ⫾ 64 (n⫽18) 100
512 ⫾ 34 (n⫽7) 80
270 ⫾ 27 (n⫽5) 42
225 ⫾ 32 (n⫽5) 35
214 ⫾ 34 (n⫽13) 33.5
216 ⫾ 24 (n⫽6) 33.8
Note. The quantitative analysis of neuronal migration was performed as described under Experimental Methods. The number of explants analyzed is shown in parentheses. The mean ⫾ SEM of the migrated distance after each treatment is shown in the first line. The percentage in relation to the control is shown in the second line.
culture period (Fig. 5C). After 72 h in vitro the cultures were fixed and an antibody against BrdU was used to trace the destination of the postmitotic cells in the different conditions. In the control experiments (Fig. 5B) the BrdU-labeled cells migrated well throughout the cerebellar layers. Comparing with a similar slice stained with DAPI (Fig. 5A), we could observe BrdU-positive cells in all layers, including the internal granular layer (Fig. 5B). Similar results were obtained with A2B5 mAb. The pattern of BrdU staining was quite similar to the
control group with positive cells throughout cerebellar layers (Fig. 5C). The presence of Jones mAb for 15 min in the culture medium did not arrest cells in the external granule cell layer and BrdU positive cells were observed migrating throughout all layers in a pattern similar to the control cultures (Fig. 5D). In addition, this experiment also suggests that Jones mAb did not interfere with cell proliferation since the number of BrdUpositive cells is similar to that found in the control cultures (Figs. 5B–5D). However, the presence of Jones
FIG. 5. Immunoblocking of 9-O-acetyl-GD3 arrests cells in the external granular cell layer. Cerebellar slices were pulse labeled with BrdU and maintained in vitro for 72 h in the presence or absence of antibodies. (A) DAPI labeling to show the cerebellar layers. (B–E) Confocal immunofluorescence micrographs of slices immunolabeled for BrdU. In (B) observe the migration of cells from the external granular cell layer, where they had incorporated BrdU, to the molecular and internal granular cell layer. (C) A similar pattern is observed in the presence of A2B5 mAb during the period in culture. In (D) the slices received Jones mAb for 15 min at the same time they were pulse labeled with BrdU. Notice that the number of BrdU-positive cells is similar to (B) and (C). (E) In the presence of Jones mAb during the 72-h period, cells were arrested in the external granular cell layer and in the superficial portion of the molecular layer. EGL, external granular cell layer; ML, molecular layer; PCL, Purkinje cell layer; IGL, Internal granular cell layer. Calibration bar, 25 m.
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mAb during the culture period arrests BrdU-positive cells in the external granular cell layer and in the superficial portion of the molecular layer (Fig. 5E).
DISCUSSION During development of the central nervous system, widespread cell migration takes place ubiquitously. Most of the neuronal precursors migrate from the proliferative zones along processes of radial glia. Several molecules have been implicated in the directed migration of neurons along astroglial substrates (for review see Pearlman et al., 1998; Hatten, 1999). The developing cerebellar cortex has been used as a model for studies of glial-guided migration since the postmitotic granule cells migrate to the presumptive granule cell layer apposed to Bergman fibers. This system has been widely described and several molecules have been involved in its neuronal migration program (see Hatten, 1999, for review). Among these, several components of the extracellular matrix (ECM) have been proposed to influence granule cell migration (O’Shea et al., 1990; Husmann et al., 1992). In this work, we have shown that the ganglioside 9-O-acetyl GD3 is expressed in the developing cerebellum and that an antibody against this ganglioside perturbs the migration of granule cell on cerebellar explants and slices. The results obtained from the functional studies are compatible with the developmental profile of this ganglioside in the cerebellar cortex and further support our view that this molecule is involved in cell migration (Mendez-Otero et al., 1988; MendezOtero and Ramon-Cueto, 1994; Mendez-Otero and Cavalcante, 1996). We specifically tested the role of this ganglioside in gliophylic radial migration of granule cells but the expression of this molecule is also associated with Purkinje cell migration that takes place at embryonic ages and with the tangential migration of precursors from the rhombic lip to form the external granule cell layer (Mendez-Otero et al., 1988). The differential distribution of 9-O-acetyl GD3 in the developing nervous system is a strong indication that it plays specific functions in this tissue. Cleavage of 9-Oacetyl groups by transgenic expression of influenza C virus hemaglutinin caused abnormalities in the development of mice retina (Varki et al., 1991). However, the specific functions of 9-O-acetyl GD3 are not known. Association of this ganglioside with cell migration has been well characterized in cancer research where it was found that tumors arising from neural crest-derived tissues express high levels of 9-O-acetyl GD3. These
495 gangliosides are concentrated in adhesion plaques and are involved in cell adhesion (Cheresh et al., 1984, see also Lloyd and Furukawa, 1998, for review). Using the melanoma cell line abc-1, Birkle and colleagues (1999, 2000) have shown that down regulation of 9-O-acetyl GD3 by stable transfection of O-acetylesterase cDNA and antisense vector against GD3-synthase gene expression have effects on cell proliferation and differentiation. The diminished expression of 9-O-acetyl GD3 in these cells promoted cellular differentiation characterized by the formation of elongated processes in parallel with a decreased cellular proliferation (Birkle et al., 1999, 2000). These findings together with the results of this paper suggest that the expression of 9-O-acetyl GD3 is correlated with an undifferentiated cellular state: maintenance of undifferentiated melanoma cells and migrating neuronal precursors (undifferentiated neurons). Several lines of evidence support the idea that gangliosides control cell adhesion through modulation of integrin receptor function (Cheresh et al., 1986, Cheresh, 1987; Burns et al., 1988; Stallcup, 1988; Kojima and Hakamori, 1991). Recently, a novel inhibition mechanism for RGD-dependent cell adhesion to fibronectin was described (Probstmeier and Pesheva, 1999). This effect is mediated by the interaction of tenascin-C with membrane-associated disialogangliosides. This interaction at the cellular surface modulates the 1 integrin receptor and affects protein kinase C (PKC) related signaling pathways, most likely by the induction of ganglioside turnover (Probstmeier and Pesheva, 1999). The same group has shown that a different class of tenascin, tenascin-R, interferes with oligodendrocyte adhesion through the same mechanism (Probstmeier et al., 1999). Together with our observation that immunoblockage of 9-O-acetyl GD3 (a disialoganglioside) inhibits neuronal migration along glial fibers and that the developing cerebellum expresses both tenascin-C (Bartsch et al., 1992) and  1 integrin (Stallcup 1988, Stallcup et al., 1989, Husmann and Sievers, 1985), it is possible to postulate a mechanism for action of this ganglioside in neuronal migration. It has been proposed that integrins are involved in neuron-glial recognition during glial-guided neuronal migration (Anton et al., 1999). It is then possible to suggest that the ganglioside 9-O-acetyl GD3 is acting as a modulating molecule in the recognition between these two cells regulating the neuronal migration program in the developing cerebellum and possibly in other glial-guided neuronal migration systems. In the developing cerebellum the apposing surfaces of the migrating neuron and the radial glia form a
496 specialized migration junction, the interstitial junction. This junction consists of a widening of the intercellular space containing filamentous material that spans the cleft and membranes of each cell, contiguous to cytoskeletal elements (Gregory et al., 1988). This close apposition suggests that membrane components of the cell surface may mediate migration. A number of neuronal and glial receptors systems have been implicated in the directed migration of neurons along radial glial (see Rakic et al., 1994, Pearlman et al., 1998, Hatten, 1999, for review). Our observation that only glial cells in contact with migrating neurons express 9-O-acetyl GD3 (cf. also Mendez-Otero and Constantine-Paton, 1990) leads to the suggestion that this ganglioside might be associated with the functional state of the glial cell and that it may act directly or indirectly as a membrane modulator for gliophylic migration. The particular expression of the acetylated form of GD3 only on glial cells supporting migration can be explained by local acetylation of the ganglioside GD3 present in the glial cells or by transport of the 9-O-acetyl GD3 from the neuron to the apposing glial cell by lipid-binding proteins (Feng et al., 1994). Quantitative inhibition results showed that the inhibition of neuronal migration by Jones mAb was extremely specific in a dose-dependent manner. In similar experiments, immunoblockage with A2B5 mAb, an antibody especific for c-series gangliosides, showed comparable values to the control group. A2B5 mAb was also used as a control for a possible unspecific effect of an IgM, since Jones mAb comes from the same immunoglobulin class. Moreover, the effect of Jones mAb appears to be specific on the migrating neurons since the immunoblockage of 9-O-acetyl GD3 had no effect on radial glial fibers extension from the explant. The arrest of granular cells by Jones mAb in the microexplant cultures was confirmed using postnatal cerebellar slice cultures (Fig. 5). In these cultures the original cerebellar cytoarchitecture is maintained and the immunoblockage effect on the postmitotic granular cell migration could be evaluated in an enviroment very similar to the in vivo situation. In these experiments, it was also possible to observe that Jones mAb did not interfere with the incorporation of BrdU by proliferating cells since the number of labeled cells in the presence of Jones mAb is similar to that found in the control cultures. In conclusion, the ganglioside 9-O-acetyl GD3 could provide a new cell signaling mechanism in glial-guided neuronal migration in the developing postnatal cerebellum. Moreover, it is important to note that granule cells migrate by extension of a leading, neurite-like process
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(Rakic, 1971) and that 9-O-acetyl GD3 has been implicated in both neuronal migration and neurite outgrowth (Mendez-Otero and Friedman, 1996; Araujo et al., 1997). The potential roles for this ganglioside in identical mechanisms for neuronal migration and neurite outgrowth suggest an emerging framework in which glycolipids are involved in cell movement in general.
EXPERIMENTAL METHODS Animals. Lister rats were obtained from our breeding colony. All experiments were carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals and the experimental protocols were approved by the Committee for the Use of Experimental Animals of our institution. Light microscope immunohistochemistry. Immunostaining of cerebellar sections was performed by the immunogold method as described previously (MendezOtero et al., 1988) or by immunofluorescence. Briefly, rats of 4 –7 postnatal (P) days of age were perfused through the ascending aorta with 4% paraformaldehyde in 0.1 M phosphate buffer at, pH 7.4. The cerebella were then dissected, fixed by immersion for 4 – 8 h and then transferred to a 30% phosphate-buffered sucrose for 12 h. Sagittal sections (15 m) were cut using a cryostat at ⫺20°C and were mounted on coated slides. Sections were preincubated for 15 min with 5% normal goat serum (NGS) and then incubated overnight at 4°C with Jones mAb (either purified IgM, 4 g/ml in 5% NGS; or ascites, 1:100; Sigma, St. Louis, MO). The slides were then rinsed three times in PBS and incubated with the immunogold staining reagent (goat IgG directed against mouse IgM linked to 5 nm colloidal gold particles, 1:40 dilution; Amersham, Sa˜o Paulo, Brasil) and intensified with the silver-intensification kit (Amersham) or incubated with a Cy3-conjugated goat antimouse IgG (1:1000, Jackson Lab. Inc., West Grove, PA). Electron microscope immunocytochemistry. Indirect immunoelectron microscopy was performed on parasagittal vibratome sections by a preembedding PAP procedure. Shortly, 7-day-old rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and were perfused through the left ventricle with 4 ml of heparin (1000 U/ml in 0.15 M NaCl), and 20 ml of paraformaldehyde (4%) in 0.1 M phosphate buffer (PB), pH 7.4, followed by buffered 4% paraformaldehyde and 0.5% glutaraldehyde. The brains were removed and postfixed for 30 min in buffered 4% paraformaldehyde and vibratome sections were made through the cerebel-
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lum at a thickness of 50 m. The vermis and intermediate region were sectioned sagittally, whereas sections through the lateral cerebellum were in an oblique plane, thus, providing transversely cut folia. Tissue sections were then rinsed in phosphate-buffered saline (PBS), preincubated for 30 min in 5% NGS and incubated overnight with Jones mAb (purified IgM) in 2% NGS. Sections were then incubated with a link (unlabeled) goat anti-mouse IgM (Dako, Carpinteria, CA) in 2% NGS for 2 h and, finally, reacted with mouse PAP (Dako, Carpinteria, CA) in PBS. After several washes with PBS, HRP histochemistry was performed with 0.0002 % H 2O 2 and 0.05% diaminobenzidine in 0.1 M phosphate buffer, pH 7.6. After several washes, the sections were postfixed with buffered 2% glutaraldehyde followed by 1% osmium tetroxide in phosphate buffer. Experimental and control sections were dehydrated with increasing ethanol concentrations and embedded in Spurrs’ resin between Teflon-coated slides and coverslips and polymerized. Small pieces of these blocks were cut from the embedded sections, glued onto the tops of blank blocks or reembedded in BEEM capsules. After preliminary trimming, 1-m sections were cut and examined under the light microscope to identify sections of interest. After that, ultrathin sections were made and short ribbons collected onto parallel bar grids or Formvar-coated slot grids. Alternate ribbons were contrasted with uranyl acetate and lead citrate or left without contrast for assessment in a Zeiss EM-900 electron microscope. Microexplant cultures. Cerebella taken from 4- to 6-day-old rats were dissected, cleaned for meninges and major blood vessels, and cut into small pieces with a blade under sterile conditions. The pieces were plated onto glass coverslips coated with poly-l-lysine (MW ⫽ 389.000; 20 g/ml; Sigma, St. Louis, MO) and laminin (40 g/ml; Gibco Brl; Gainthersburg, MD). Four to six similar sized explants of about 600 m in diameter were placed on each coverslip. About 1 h after plating the media was changed in order to eliminate the floating explants and 300 l of fresh media was added to each well. At this point the antibodies Jones mAb (1:100; Sigma, St. Louis, MO) and A2B5 mAb (1:1000; donation from Dr. Paola Bovolenta; Cajal Institute, MadridSpain), which recognizes a group of c-series gangliosides (Eisenbarth et al., 1979), were added to the culture mixed with the media. Cultures were maintained in a 95% air/5% CO 2 incubator at 37°C and the explants were grown in DMEM/F12 containing 10% fetal calf serum for 72 h after which they were fixed with 4% paraformaldehyde in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2 (pH 6.9; PHEM buffer) for
497 30 min. For immunocytochemistry, cultures were labeled either with Jones mAb (1:100) or A2B5 mAb (1: 1000) or with antibodies against GFAP (1:100, polyclonal; Sigma) or MAP2 (1:100, monoclonal; Sigma). Primary antibodies were incubated for 18 –24 h at 4°C. The binding of these antibodies was visualized with Cy3-conjugated goat anti-mouse IgG (1:1000; Jackson Lab., Inc.; West Grove, PA), and fluorescein isothiocyanate-coupled goat anti-rabbit IgG (1:100; Sigma). After 2 h incubation at room temperature, the cultures were washed three times in PBS. In the last wash the fluorescent dye 4⬘-6-diamidino-2-phenylindole (DAPI, Sigma) was added in order to visualize the nuclei. Stained cultures were mounted with VectaShield (Vector Labs., Burlingame, CA) and viewed with an inverted or with a confocal Zeiss microscope. Quantitative analysis. The extent of cell migration was estimated by measuring the distance between the border of the explant and the wavefront of cells labeled with DAPI at eight sites located at 45° angles to each other. A mean was obtained for each explant. These measures were made using a 20⫻ objective lens in an Axioplan Zeiss microscope connected to a computer equipped with Morpho 3.2 software (UFRJ, 1988 –1995) capable of encoding x and y coordinates. A minimum of five explants was analyzed for each treatment, and the effect of different treatments on neuronal migration was evaluated for significant differences using the Student’s t test. Slice cultures. Cerebella were taken from P4 –7 rats and dissected out in sterile conditions. Slices were cut at 350 m, using a tissue chopper (MacIlwain, U.S.A.), and plated on millicell inserts (Sigma, St. Louis, MO). To distinguish between proliferating and postmitotic cells we used 5-bromo-2⬘-deoxyuridine (BrdU; Sigma), which is incorporated by cells synthesizing DNA (Gratzner, 1982). A 10 g/ml solution of BrdU was added to the culture medium for 15 min. In some slices Jones mAb was added at a 1:20 concentration simultaneously with BrdU. The slices were then carefully washed and new culture medium was added with or without antibodies. Jones mAb was added at 1:20 concentration and A2B5 mAb (Boehringer-Mannhein Co., Germany) was used at a 1:100 concentration. Cultures were maintained in a 95% air/5% CO 2 incubator at 37°C and the slices were grown in DMEM/F12 containing 10% fetal calf serum for 72 h, after which they were fixed with 4% paraformaldehyde in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2 (pH 6.9; PHEM buffer) for 30 min. Following antigen retrieval in a microwave oven (Dover and Patel, 1994), cultures were washed with PBS and incubated overnight at 4°C
498 with a mouse monoclonal antibody against BrdU (RPN 202, Amersham, Little Chalfont, UK), according to the manufacturer’s instructions and developed with a CY3conjugated goat anti-mouse IgG (1:1000; Jackson Lab). The slices were analyzed and documented using a Zeiss confocal microscope.
ACKNOWLEDGMENTS The authors thank Dr. Cecı´lia Hedin-Pereira for crucial comments during the elaboration of this work, Dr. Elizabeth Debski for comments on the manuscript, and Felipe Marins for technical assistance. This work was supported by PRONEX, FINEP, CNPq, and FAPERJ grants to R.M-O. and L.A.C. and TWAS to R.M-O.
REFERENCES Altman, J. (1972a). Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145: 353–398. Altman, J. (1972b). Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Comp. Neurol. 145: 399 – 464. Altman, J. (1972c). Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145: 465–514. Anton, E. S., Kreidberg, J. A., and Rakic, P. (1999). Distinct functions of ␣ 3 and ␣ v integrin receptors in neuronal migration and laminar organization of the cerebral cortex. Neuron 22: 277–289. Antonicek, H., Persohn, E., and Schachner, M. (1987). Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration. J. Cell Biol. 104: 1587–1595. Araujo, H., Menezes, M., and Mendez-Otero, R (1997). Blockage of 9-O-acetyl gangliosides induces microtubule depolymerization in growth cones and neurites. Eur. J. Cell Biol. 72: 202–213. Bartsch, S., Bartsch, U., Do¨rries, U., Faissner, A., Weller, A., Ekblom, P., and Schachner, M. (1992). Expression of tenascin in the developing and adult cerebellar cortex. J. Neurosci. 12: 736 –749. Birkle´, S., Ren, S., Slominski, A., Zeng, G., Gao, L., and Yu, R. K. (1999). Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis. J. Neurochem. 72: 954 –961. Birkle´, S., Gao, L., Zeng, G., and Yu, R. K. (2000). Down regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells: Effects on cellular growth, melanogenesis, and dendricity. J. Neurochem. 74: 547–554. Bonafede, D. M., Macala, L. J., Constantine-Paton, M., and Yu, R. K. (1989). Isolation and characterization of ganglioside 9-O-acetylGD3 from bovine buttermilk. Lipids 24: 680 – 684. Burns, G. F., Lucas, C. M., Krissansen, G. W., Werkmeister, J. A., Scanlon, D. B., Simpson, R. J., and Vadas, M. A. (1988). Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells. J. Cell Biol. 107: 1225–1230.
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Cameron, R. S., and Rakic, P. (1994). Polypeptides that comprise the plasmalemmal microdomain between migrating neuronal and glial cells. J. Neurosci. 14: 3139 –3355. Caviness, V. S., Jr., and Rakic, P. (1978). Mechanisms of cortical development: A view from mutations in mice. Annu. Rev. Neurosci. 1: 297–326. Cheresh, D. A. (1987). Ganglioside involvement in tumor cell-substratum interactions. In Development and Recognition of the Transformed Cell (M. I. Greene and T. Hamaoka, Eds.) Plenum Press, New York, NY. Cheresh, D. A., Pierbacher M. D., Herzig, M. A., and, Mujoo, J. (1986). Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. J. Cell Biol. 102: 688 – 696. Cheresh, D. A., Varki, A. P., Varki, N. W., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984). A monoclonal antibody recognizes an O-acetylated sialic acid in a human melanoma-associated ganglioside. J. Biol. Chem. 259: 7453–7459. Constantine-Paton, M., Blum, A. S., Mendez-Otero, R., and Barnstable, C. J. (1986). A cell surface molecule distributed in a dorsoventral gradiente in the perinatal rat retina. Nature (London) 324: 459 – 462. Dover, R., and Patel, K. (1994). Improved methodology for detecting bromodeoxyuridine in cultured cells and tissues sections by immunocytochemistry. Histochemistry 102: 383–387. Edmondson, J. C., and Hatten, M. E. (1987). Glial-guided neuronal migration in vitro: A high resolution time-lapse videomicroscopic study. J. Neurosci. 7: 1928 –1934. Eisenbarth, G. S., Walsh, F. S., and Nirenberg, M. (1979). Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. USA 76: 4913– 4917. Feng, L., Hatten, M. E., and Heintz, N. (1994). Brain lipid-binding protein (BLBP): A novel signaling system in the developing mammalian CNS. Neuron 12: 895–908. Fishell, G., and Hatten, M. E. (1991). Astrotactin provides a receptor system for CNS neuronal migration. Development 113: 755–765. Gao, W.-Q., and Hatten, M. E. (1993). Neuronal differentiation rescued by implantation of weaver granule cell precursos into wildtype cerebellar cortex. Science 260: 367–369. Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218: 474 – 478. Gregory, W. A., Edmondson, J. C., Hatten, M. E., and Mason, C. A. (1988). Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro. J. Neurosci. 8: 1728 –1738. Hakomori, S. (1990). Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J. Biol. Chem. 265: 18713–18716. Hatten, M. E. (1990). Riding the glial monorail: A common mechanism for glial-guided neuronal migration in different regions of the developing brain. Trends. Neurosci. 13: 179 –187. Hatten, M. E. (1999). Central nervous system neuronal migration. Annu. Rev. Neurosci. 22: 511–539. Hatten, M. E., Liem, R. K. H., and Mason, C. A. (1984). Defects in specific associations between astroglia and neurons occur in microcultures of weaver mouse cerebellar cells. J. Neurosci. 4: 1163–1172. Hatten, M. E., Liem, R. K. H., and Mason, C. A. (1986). Weaver mouse cerebellar granule cells fail to migrate on wild-type astroglial process in vitro. J. Neurosci. 9: 2676 –2683. Hockberger, P. E., Tseng, H.-Y., and Connor, J. A. (1987). Immunocytochemical and eletrophysiological differentiation of rat cerebellar granule cells in explant cultures. J. Neurosci. 7: 1370 –1383.
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Husmann K., Faissner, A., and Schachner, M. (1992). Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats. J. Cell Biol. 116: 145–186. Husmann, B., and Sievers, J. (1985). Cerebellar external granule cells are attached to the basal lamina from the onset of migration up to the end of their proliferative activity. J. Comp. Neurol. 241: 50 – 62. Kojima, N., and Hakomori, S. (1991). Synergistic effect of two cell recognition systems: Glycosphingolipid-glycosphingolipid and integrin receptor interaction with pericellular matrix protein. Glycobiology 1: 623– 630. Komuro, H., and Rakic, P. (1993). Modulation of neuronal migration by NMDA receptors. Science 260: 95–97. Komuro, H., and Rakic, P. (1995). Dynamics of granule cell migration: A confocal microscopic study in acute cerebellar slice preparations. J. Neurosci. 15: 1110 –1120. Ledeen, R. (1985). Gangliosides of neurons. Trends Neurosci. 8: 169 – 174. Lloyd, K. O., and Furukawa, K. (1998). Biosynthesis and functions of gangliosides: Recent advances. Glycoconj. J. 7: 627– 636. Mendez-Otero, R., and Cavalcante, L. A. (1996). Expression of 9-Oacetylated gangliosides is correlated with tangential cell migration in the rat brain. Neurosci. Lett. 204: 97–100. Mendez-Otero, R., and Constantine-Paton, M. (1990). Granule cell induction of 9-O-acetyl gangliosides on cerebellar glia in microcultures. Dev. Biol. 138: 400 – 409. Mendez-Otero, R., and Friedman, J. E. (1996). Role of acetylated gangliosides on neurite extension. Eur. J. Cell Biol. 71: 192–198. Mendez-Otero, R., and Ramon-Cueto, A. (1994). Expression of 9-Oacetylated gangliosides during development of the rat olfatory system. Neuroreport 5: 1755–1759. Mendez-Otero, R., Schlosshauer, B., Barnstable, C. J., and Constantine-Paton, M. (1988). A developmentally regulated antigen associated with neural cell and process migration. J. Neurosci. 8: 564 –579. Miale, I. L., and Sidman, R. L. (1961). An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Neurol. 4: 277–296. O’Shea, K. S., Rheinheimer, J. S., and Dixit, V. M. (1990). Deposition and role of thrombospondin in the histogenesis of cerebellar cortex. J. Cell Biol. 110: 1275–1283. Pearlman, A. L., Faust, P. L., Hatten, M. E., and Brunstrom, J. E. (1998). New directions for neuronal migration. Curr. Opin. Neurobiol. 8: 45–54. Probstmeier, R., Michels, M., Franz, T., Chan, B. M. C., and Pesheva,
499 P. (1999). Tenascin-R interferes with integrin-dependent oligodendrocyte cell adhesion by a ganglioside-mediated signalling mechanism. Eur. J. Neurosci. 11: 2472–2588. Probstmeier, R., and Pesheva, P. (1999). Tenascin-C inhibits 1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism. Glycobiology 9: 101–114. Rakic, P. (1971). Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electromicroscopic study in Macacus rhesus. J. Comp. Neurol. 141: 283–312. Rakic, P. (1976). Synaptic specificity in the cerebellar cortex: Study of anomalous circuits induced by single gene mutation in mice. Cold Spring Harbor Symp. Quant. Biol. 40: 333–346. Rakic, P. (1981). Neuronal-glial interactions during brain development. Trends Neurosci. 4: 184 –187. Rakic, P. (1990). Principles of cell migration. Experientia 46: 882– 891. Rakic, P., Cameron, R. S., and Komuro, H. (1994). Recognition, adhesion, transmembrane signaling and cell motility in guided neuronal migration. Curr. Opin. Neurobiol. 4: 63– 69. Rakic, P., and Sidman, R. L. (1973a). Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J. Comp. Neurol. 152: 103–132. Rakic, P., and Sidman, R. L. (1973b). Weaver mouse mutant cerebellum: defective neuronal migration secondary to abnormality of Bergmann glia. Proc. Natl. Acad. Sci. USA 70: 240 –244. Ramo´n y Cajal, S. (1911). Histologie du Systeme Nerveux de’l Homme et des Vertebre´s, Maloine, Paris. [Reprinted by Consejo Superior de Investigaciones Cientificas, Madrid, 1972] Stallcup, W. B. (1988). Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin. Exp. Cell Res. 177: 90 –102. Stallcup, W. B., Pytela, R., and Ruoslahti, E. (1989). A neuroectodermassociated ganglioside participates in fibronectin receptor mediated adhesion of germinal cells to fibronectin. Dev. Biol. 132: 212–229. Varki, A., Hooshmand, F., Diaz, S. Varki, N. M., and Hedrick, S. M. (1991). Developmental abnormalities in transgenic mice expressing a sialic acid-specific 9-O-acetylesterase. Cell 65: 65–74. Yu, R. K. (1994). Development regulation of ganglioside metabolism. Prog. Brain Res. 101: 31– 44. Wichterle, H., Garcı´a-Verdugo, J. M., and Alvarez-Buylla, A. (1997). Direct evidence for homotypic, glia-independent neuronal migration. Neuron 18: 779 –791. Received July 19, 2000 Revised November 29, 2000 Accepted November 30, 2000
Molecular and Cellular Neuroscience 17, 488 – 499 (2001) doi:10.1006/mcne.2000.0943, available online at http://www.idealibrary.com on
Expression and Function of Ganglioside 9-O-Acetyl GD3 in Postmitotic Granule Cell Development Marcelo F. Santiago, Marcia Berredo-Pinho, Marcos R. Costa, Mario Gandra, Leny A. Cavalcante, and Rosalia Mendez-Otero Instituto de Biofisica Carlos Chagas Filho, Universidade Federal do Rio de Janeiro, RJ 21941-590, Rio de Janeiro, Brazil
We have shown previously that the Jones monoclonal antibody (Jones mAb) recognizes 9-O-acetyl GD3 expressed during periods of neuronal migration and neurite outgrowth in the developing rat nervous system. In the present study we investigated the expression of this ganglioside in the developing cerebellum and correlated this expression with granule cell migration. Electron microscopic immunocytochemistry revealed that around the peak of cerebellar neuronal migration (7-day-old rat), 9-Oacetyl GD3 was localized at the contact sites between migrating granule cells and radial glia in the external granular layer and prospective molecular layer. In addition, using microexplant and slice cultures of the postnatal rat cerebellum, we tested whether the ganglioside detected by our antibody contribute to the regulation of neuronal migration in the cerebellar cortex. We have shown that the Jones mAb blocks the migration of neurons in a dosedependent manner. These findings suggest strongly that 9-O-acetyl GD3 is involved in granule cell migration in the developing cerebellum.
INTRODUCTION During development of the central nervous system, postmitotic neurons undergo a marked migration toward their final positions. This active displacement of immature neurons is essential for the establishment of normal cytoarchitecture and synaptic connectivity in the vertebrate brain (Caviness and Rakic, 1978; Hatten, 1990; Rakic, 1990). In the cerebellar cortex, for example, granule cell precursors migrate from the external granular cell layer where they are generated, to reach their final positions in the internal granular layer (Ramo´n y Cajal, 1911; Miale and Sidman, 1961). Several studies have supported the hypothesis that
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functional interactions between postmitotic granule cells and Bergmann glial fibers are essential for this migration (Rakic and Sidman, 1973a, b; Rakic, 1976, 1981; Hatten et al., 1984, 1986; Gao and Hatten, 1993). Although the molecular mechanisms influencing granule cell migration are not yet fully understood, it has become clear from various in vitro studies that a number of molecular and cellular mechanisms, including cell– cell recognition, cell– cell adhesion, cell motility, intracellular Ca 2⫹ dynamics, and activity of NMDA receptors are involved in the control of the migration program (Antonicek et al., 1987; Edmondson and Hatten, 1987; Fishell and Hatten, 1991; Komuro and Rakic, 1993; Cameron and Rakic, 1994; Rakic et al., 1994; Komuro and Rakic, 1995). The possibility that cell surface carbohydrates and/or lipids mediate cellular interactions within the vertebrate nervous system has been considered on numerous occasions although most of the studies in cell migration have focused on protein–protein interactions. Gangliosides (sialic acid-containing glycosphingolipids) constitute a major group of cell surface carbohydrate-containing molecules. They have been implicated in numerous cellular functions in the developing and adult mammalian nervous system, since they are enriched in the outer surface of neural membranes (Ledeen, 1985; Hakomori, 1990; Yu, 1994). In previous works we observed that the ganglioside 9-O-acetyl GD3 recognized by the Jones monoclonal antibody (Jones mAb—Constantine-Paton et al., 1986; Bonafede et al., 1989) is developmentally regulated and expressed in regions of cell migration and neurite outgrowth (Mendez-Otero et al., 1988; Mendez-Otero and Ramon-Cueto, 1994; Men1044-7431/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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FIG. 1. Heterogeneous patterns of immunoreactivity to 9-O-acetyl GD3 in P7 rat cerebellum. (A) Low power photomicrograph, (B) confocal image, and (C) electromicrograph. Observe the heterogeneous binding of Jones mAb on different folia and the apparently compact labeling of one caudal folium (A). In the confocal image (B) notice the labeling of cell bodies and processes in the external granular cell layer and presumptive molecular layer. In the electronmicrograph (C), notice the selective labeling of few granular-Bergmann membranes. (A) Silverenhanced colloidal gold labeling of the secondary antibody. (B) Cy3-labeling of the secondary antibody. (C) PAP procedure. Calibration bars: (A) 375 m, (B) 25 m, and (C) 3 m.
dez-Otero and Cavalcante, 1996). We have also observed that cultured neurons from rat cerebella when in contact with glial cells that support cell migration (radial glial cells), express the Jones-reactive ganglioside, as do the glial cells. On the other hand, glial cells with a stellate morphology, even when in contact with granule neurons do not express this antigen (Mendez-Otero and Constantine-Paton, 1990). In the present study, we have chosen the rat cerebellum to further elucidate the functional role of ganglioside 9-O-acetyl GD3 in neuronal migration. We chose this system because it has been thoroughly characterized histologically and correlation with neurogenesis, migration and differentiation are easy to perform (Altman, 1972a, 1972b, 1972c). Furthermore, migration of neuronal precursors occurs postnatally and is therefore more available to experimental analysis. Initially, the localization of 9-O-acetyl gangliosides in explants of rat cerebellum was determined using cellular markers. We also report that the antibody against 9-O-acetyl GD3
inhibits migration of granule neuron precursors along glial fibers in a dose-dependent manner in this model system. This is the first time in which a specific ganglioside has been implicated as a signaling molecule in neuronal migration.
RESULTS Expression of 9-O-Acetyl GD3 in Postnatal Cerebellum The ganglioside recognized by the Jones mAb was expressed in a stacked pattern in the external granule cell layer (EGL) during the first postnatal week (Fig. 1A). The intensity of expression of this antigen differed in the various folia of the cerebellum, which may reflect different maturational stages of these structures. Granular cell precursors leaving the EGL with a typical migratory morphology express 9-O-
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FIG. 2. Pattern of expression of gangliosides in cerebellar explants. Phase contrast (A and C) and fluorescence photomicrographs of cerebellar explants labeled with Jones (B) or A2B5 (D) antibodies. Observe labeling of cell bodies and neurites (insets) with either antibody. Calibration bar, 20 m.
acetyl GD3 (Fig. 1B). At the EM level, Jones immunoreactivity in the presumptive molecular layer was detected mainly at sites of contact between migrating granule cells and Bergmann glial fibers (arrows - Fig. 1C). Labeling of granular cells and/or adjoining Bergmann fibers is heterogeneous with many unlabeled sets of granule–Bergmann elements in the close vicinity of labeled cells.
Expression of 9-O-Acetyl GD3 in Cerebellar Microexplants In cerebellar microexplants, several cells were observed migrating out of the explant 24 h after plating. Based on their size (8 –10 m) and nuclear morphol-
ogy, these cells are most likely granule cells which constitute the majority of the neurons in cerebella explant cultures (Hockberger et al., 1987). Figures 2A and 2C show the typical morphology of explants with granule cells migrating in a radial orientation from the core (bottom-left in all pictures) after 72 h in vitro. These cells when stained with Jones mAb show immunoreactivity for 9-O-acetyl GD3 on their cell bodies and neurites (Fig. 2B). Migrating cells also bind other antibodies against gangliosides. For instance, immunoreaction with the A2B5 monoclonal antibody (A2B5 mAb) also labels cell bodies and neurites in these explants (Fig. 2D). Both antibodies stain the cell body very strongly and delineate the membrane of the neurites (insets in Figs. 2B and 2D).
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Characterization of the Jones-Positive Cells on Cerebellar Microexplants To further characterize the cell types present in these cultures we double labeled them with antibodies against MAP-2 to identify neurons and GFAP to identify glia (Figs. 3A–3C). In Fig. 3A we observe a large population of radial glial fibers labeled with GFAP (green). Closely associated with these glial cells are a great number of granular neurons labeled with MAP-2 (red). These neurons seem to migrate out of the explant closely apposed to the radial glial fibers (arrows, Fig. 3B) in a fashion very similar to that shown in vivo by electron microscopy (see also Fig. 1C) and in vitro with video-enhanced differential contrast microscopy (Rakic, 1971; Hatten, 1990). Besides this glial-guided neuronal migration, we could also observe neurons labeled with MAP-2 (red) migrating without any apparent association with glial fibers (Fig. 3C). These cells probably migrate associated with the neurites of other neurons similarly to the chain migration that occurs in the rostral migratory stream of the developing and adult telencephalon (Wichterle et al., 1997; Mendez-Otero and Cavalcante, 1996). In order to determine which cell types expressed Jones antigen in these cultures we performed double label experiments. Figure 3D shows a double staining for MAP-2 (green) and 9-O-acetyl GD3 (red). The colocalization of the neuronal marker with the 9-O-acetyl GD3 (yellow) shows that these migrating cells are neurons (arrows). In our model system, the expression of Jones antigen is not restricted to granule neurons. Double labeling of GFAP (green) and Jones mAb (red) reveals that radial glial cells also express 9-O-acetyl GD3 (yellow, Fig. 3E). Nevertheless, the expression of this antigen is restricted to glial cells that contact migrating granule neurons and is not observed on glial cells that are not associated with neurons (outline in Fig. 3E). These results confirm our previous suggestion from cerebellar dissociated microcultures that there exists an inducible effect from the apposition of these two cells (Mendez-Otero and Constantine-Paton, 1990).
Jones mAb Inhibits the Migration of Cerebellar Neurons in Microexplant Cultures The influence of Jones mAb on neuronal migration in microexplant cultures prepared from P4 –P7 rat cerebellum was tested. In this in vitro system, migration of granule cells occurs over a period of 3 days. The effects on neuronal migration were therefore assessed after 3
491 days following the addition of Jones mAb and A2B5 mAb at the first day. Similar-sized explants (600 –700 m in diameter) were analyzed for each experimental group. Neuronal cells were labeled with the nucleophilic dye DAPI and then measurements were made in eight sites located at 45° angles to each other at the periphery of each explant. The distance reached by the wavefront of cells was measured from the border of the explant. Using this methodology we found a 66% decrease in the distance from the explant reached by granule cells in the presence of Jones mAb (214 ⫾ 34 m, n ⫽ 13; Figs. 4C and 4D) as compared to those of control cultures in which no antibody was added (638 ⫾ 64 m, n ⫽ 18; Figs. 4A and 4D). When the same test was performed in the presence of A2B5 mAb, granule cell migration was not modified (614 ⫾ 28 m; n ⫽ 21; Figs. 4B and 4D) when compared with control cultures. This experiment showed that only Jones mAb specifically interferes with migration of granule cells (Fig. 4D). Using different dilutions of Jones mAb we verified that the immunoblockade effect occurs in a dose-dependent manner and that it has a plateau of activity around the 1:100 dilution (Table 1). Since granule neurons and radial glial cells express the Jones antigen, the decrease in migration distance could be due to interference of this monoclonal antibody on the extension of the glial cells and/or on the migration of the granule neurons. To answer this question, Jones blocked explants were double labeled with GFAP (green) and MAP-2 (red). Figure 3F shows a phase contrast image of an immunoblocked explant with a small number of cells migrating out of it (arrowheads) and some fibers that may be neurites and/or glial fibers (arrows). A similar explant was double labeled for GFAP and MAP-2 (Fig. 3G), and it showed that even when the explant grows in the presence of the Jones mAb, glial cells are able to extend their process. The staining for MAP-2 reveals that some neurons migrate a short distance from the border of the explant in this condition. These results suggest a specific effect of Jones mAb on the migration of granule cells along glial fibers. Posmitotic Granular Cell Migration Is Arrested by Jones mAb in Cerebellum Slice Cultures To investigate whether the immunoblockage obtained with the explant cultures could be also extended to organotypic slice cultures we labeled proliferating cells with a 15-min pulse of 5-Bromo-2⬘-deoxyuridine (BrdU) and mapped their migratory movement throughout the laminar structure of the cerebellar slice
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FIG. 3. Characterization of 9-O-acetyl GD3 immunoreactive cells in cerebellar explants. (A–C) Cerebellar explants, double-labeled with GFAP (green) and MAP-2 (red), showing the presence of radial glial fibers and their close association with neurons. In (B), neurons (arrows) closely associated with glial fibers are shown and neurons that migrate outside the region of glial fibers are illustrated in (C). (D) Explant double-labeled for 9-O-acetyl GD3 (red) and MAP-2 (green). Notice that all MAP-2-positive cells (arrows) are also labeled for 9-O-acetyl GD3 (yellow). (E) Explants double-labeled for GFAP (green) and 9-O-acetyl GD3 (red). Some of the glial cells are double-labeled (yellow) and others are only stained for GFAP (outline). Most of the neurons (red) are positive for 9-O-acetyl GD3. (F and G) Explants blocked with Jones mAb showing that radial glia cells are present in these explants but neurons do not migrate very far out of the explant. Calibration bar, 20 m.
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FIG. 4. Immunoblocking of cerebellar explants with different antibodies. (A) Phase contrast photomicrograph of an explant in the absence of antibodies. (B) Similar explant in the presence of A2B5 mAb or (C) Jones mAb. Observe the blockage of cell migration out of the explant in the presence of Jones mAb (C and D). (D) Quantitative analysis of several explants in the presence or absence of different ganglioside-reactive antibodies. *P ⬍ 0.01. Calibration bar, 40 m.
cultures. In the control group, the slices were incubated with fresh medium after the BrdU pulse (Fig. 5B). In two other sets of cultures Jones mAb was added at the same time as BrdU (Figs. 5D and 5E). After 15 min this
medium was removed and changed for fresh culture medium without Jones mAb (Fig. 5D) or with Jones mAb (Fig. 5E). In another set of slices A2B5 mAb was used instead of Jones mAb and maintained throughout
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TABLE 1 Dose-Dependent Effect of Jones mAb in Neuronal Migration Jones mAb concentration
Migrated distance (m) Percentage
Control
1:800
1:400
1:200
1:100
1:50
638 ⫾ 64 (n⫽18) 100
512 ⫾ 34 (n⫽7) 80
270 ⫾ 27 (n⫽5) 42
225 ⫾ 32 (n⫽5) 35
214 ⫾ 34 (n⫽13) 33.5
216 ⫾ 24 (n⫽6) 33.8
Note. The quantitative analysis of neuronal migration was performed as described under Experimental Methods. The number of explants analyzed is shown in parentheses. The mean ⫾ SEM of the migrated distance after each treatment is shown in the first line. The percentage in relation to the control is shown in the second line.
culture period (Fig. 5C). After 72 h in vitro the cultures were fixed and an antibody against BrdU was used to trace the destination of the postmitotic cells in the different conditions. In the control experiments (Fig. 5B) the BrdU-labeled cells migrated well throughout the cerebellar layers. Comparing with a similar slice stained with DAPI (Fig. 5A), we could observe BrdU-positive cells in all layers, including the internal granular layer (Fig. 5B). Similar results were obtained with A2B5 mAb. The pattern of BrdU staining was quite similar to the
control group with positive cells throughout cerebellar layers (Fig. 5C). The presence of Jones mAb for 15 min in the culture medium did not arrest cells in the external granule cell layer and BrdU positive cells were observed migrating throughout all layers in a pattern similar to the control cultures (Fig. 5D). In addition, this experiment also suggests that Jones mAb did not interfere with cell proliferation since the number of BrdUpositive cells is similar to that found in the control cultures (Figs. 5B–5D). However, the presence of Jones
FIG. 5. Immunoblocking of 9-O-acetyl-GD3 arrests cells in the external granular cell layer. Cerebellar slices were pulse labeled with BrdU and maintained in vitro for 72 h in the presence or absence of antibodies. (A) DAPI labeling to show the cerebellar layers. (B–E) Confocal immunofluorescence micrographs of slices immunolabeled for BrdU. In (B) observe the migration of cells from the external granular cell layer, where they had incorporated BrdU, to the molecular and internal granular cell layer. (C) A similar pattern is observed in the presence of A2B5 mAb during the period in culture. In (D) the slices received Jones mAb for 15 min at the same time they were pulse labeled with BrdU. Notice that the number of BrdU-positive cells is similar to (B) and (C). (E) In the presence of Jones mAb during the 72-h period, cells were arrested in the external granular cell layer and in the superficial portion of the molecular layer. EGL, external granular cell layer; ML, molecular layer; PCL, Purkinje cell layer; IGL, Internal granular cell layer. Calibration bar, 25 m.
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mAb during the culture period arrests BrdU-positive cells in the external granular cell layer and in the superficial portion of the molecular layer (Fig. 5E).
DISCUSSION During development of the central nervous system, widespread cell migration takes place ubiquitously. Most of the neuronal precursors migrate from the proliferative zones along processes of radial glia. Several molecules have been implicated in the directed migration of neurons along astroglial substrates (for review see Pearlman et al., 1998; Hatten, 1999). The developing cerebellar cortex has been used as a model for studies of glial-guided migration since the postmitotic granule cells migrate to the presumptive granule cell layer apposed to Bergman fibers. This system has been widely described and several molecules have been involved in its neuronal migration program (see Hatten, 1999, for review). Among these, several components of the extracellular matrix (ECM) have been proposed to influence granule cell migration (O’Shea et al., 1990; Husmann et al., 1992). In this work, we have shown that the ganglioside 9-O-acetyl GD3 is expressed in the developing cerebellum and that an antibody against this ganglioside perturbs the migration of granule cell on cerebellar explants and slices. The results obtained from the functional studies are compatible with the developmental profile of this ganglioside in the cerebellar cortex and further support our view that this molecule is involved in cell migration (Mendez-Otero et al., 1988; MendezOtero and Ramon-Cueto, 1994; Mendez-Otero and Cavalcante, 1996). We specifically tested the role of this ganglioside in gliophylic radial migration of granule cells but the expression of this molecule is also associated with Purkinje cell migration that takes place at embryonic ages and with the tangential migration of precursors from the rhombic lip to form the external granule cell layer (Mendez-Otero et al., 1988). The differential distribution of 9-O-acetyl GD3 in the developing nervous system is a strong indication that it plays specific functions in this tissue. Cleavage of 9-Oacetyl groups by transgenic expression of influenza C virus hemaglutinin caused abnormalities in the development of mice retina (Varki et al., 1991). However, the specific functions of 9-O-acetyl GD3 are not known. Association of this ganglioside with cell migration has been well characterized in cancer research where it was found that tumors arising from neural crest-derived tissues express high levels of 9-O-acetyl GD3. These
495 gangliosides are concentrated in adhesion plaques and are involved in cell adhesion (Cheresh et al., 1984, see also Lloyd and Furukawa, 1998, for review). Using the melanoma cell line abc-1, Birkle and colleagues (1999, 2000) have shown that down regulation of 9-O-acetyl GD3 by stable transfection of O-acetylesterase cDNA and antisense vector against GD3-synthase gene expression have effects on cell proliferation and differentiation. The diminished expression of 9-O-acetyl GD3 in these cells promoted cellular differentiation characterized by the formation of elongated processes in parallel with a decreased cellular proliferation (Birkle et al., 1999, 2000). These findings together with the results of this paper suggest that the expression of 9-O-acetyl GD3 is correlated with an undifferentiated cellular state: maintenance of undifferentiated melanoma cells and migrating neuronal precursors (undifferentiated neurons). Several lines of evidence support the idea that gangliosides control cell adhesion through modulation of integrin receptor function (Cheresh et al., 1986, Cheresh, 1987; Burns et al., 1988; Stallcup, 1988; Kojima and Hakamori, 1991). Recently, a novel inhibition mechanism for RGD-dependent cell adhesion to fibronectin was described (Probstmeier and Pesheva, 1999). This effect is mediated by the interaction of tenascin-C with membrane-associated disialogangliosides. This interaction at the cellular surface modulates the 1 integrin receptor and affects protein kinase C (PKC) related signaling pathways, most likely by the induction of ganglioside turnover (Probstmeier and Pesheva, 1999). The same group has shown that a different class of tenascin, tenascin-R, interferes with oligodendrocyte adhesion through the same mechanism (Probstmeier et al., 1999). Together with our observation that immunoblockage of 9-O-acetyl GD3 (a disialoganglioside) inhibits neuronal migration along glial fibers and that the developing cerebellum expresses both tenascin-C (Bartsch et al., 1992) and  1 integrin (Stallcup 1988, Stallcup et al., 1989, Husmann and Sievers, 1985), it is possible to postulate a mechanism for action of this ganglioside in neuronal migration. It has been proposed that integrins are involved in neuron-glial recognition during glial-guided neuronal migration (Anton et al., 1999). It is then possible to suggest that the ganglioside 9-O-acetyl GD3 is acting as a modulating molecule in the recognition between these two cells regulating the neuronal migration program in the developing cerebellum and possibly in other glial-guided neuronal migration systems. In the developing cerebellum the apposing surfaces of the migrating neuron and the radial glia form a
496 specialized migration junction, the interstitial junction. This junction consists of a widening of the intercellular space containing filamentous material that spans the cleft and membranes of each cell, contiguous to cytoskeletal elements (Gregory et al., 1988). This close apposition suggests that membrane components of the cell surface may mediate migration. A number of neuronal and glial receptors systems have been implicated in the directed migration of neurons along radial glial (see Rakic et al., 1994, Pearlman et al., 1998, Hatten, 1999, for review). Our observation that only glial cells in contact with migrating neurons express 9-O-acetyl GD3 (cf. also Mendez-Otero and Constantine-Paton, 1990) leads to the suggestion that this ganglioside might be associated with the functional state of the glial cell and that it may act directly or indirectly as a membrane modulator for gliophylic migration. The particular expression of the acetylated form of GD3 only on glial cells supporting migration can be explained by local acetylation of the ganglioside GD3 present in the glial cells or by transport of the 9-O-acetyl GD3 from the neuron to the apposing glial cell by lipid-binding proteins (Feng et al., 1994). Quantitative inhibition results showed that the inhibition of neuronal migration by Jones mAb was extremely specific in a dose-dependent manner. In similar experiments, immunoblockage with A2B5 mAb, an antibody especific for c-series gangliosides, showed comparable values to the control group. A2B5 mAb was also used as a control for a possible unspecific effect of an IgM, since Jones mAb comes from the same immunoglobulin class. Moreover, the effect of Jones mAb appears to be specific on the migrating neurons since the immunoblockage of 9-O-acetyl GD3 had no effect on radial glial fibers extension from the explant. The arrest of granular cells by Jones mAb in the microexplant cultures was confirmed using postnatal cerebellar slice cultures (Fig. 5). In these cultures the original cerebellar cytoarchitecture is maintained and the immunoblockage effect on the postmitotic granular cell migration could be evaluated in an enviroment very similar to the in vivo situation. In these experiments, it was also possible to observe that Jones mAb did not interfere with the incorporation of BrdU by proliferating cells since the number of labeled cells in the presence of Jones mAb is similar to that found in the control cultures. In conclusion, the ganglioside 9-O-acetyl GD3 could provide a new cell signaling mechanism in glial-guided neuronal migration in the developing postnatal cerebellum. Moreover, it is important to note that granule cells migrate by extension of a leading, neurite-like process
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(Rakic, 1971) and that 9-O-acetyl GD3 has been implicated in both neuronal migration and neurite outgrowth (Mendez-Otero and Friedman, 1996; Araujo et al., 1997). The potential roles for this ganglioside in identical mechanisms for neuronal migration and neurite outgrowth suggest an emerging framework in which glycolipids are involved in cell movement in general.
EXPERIMENTAL METHODS Animals. Lister rats were obtained from our breeding colony. All experiments were carried out in accordance with the National Institute of Health guide for the care and use of laboratory animals and the experimental protocols were approved by the Committee for the Use of Experimental Animals of our institution. Light microscope immunohistochemistry. Immunostaining of cerebellar sections was performed by the immunogold method as described previously (MendezOtero et al., 1988) or by immunofluorescence. Briefly, rats of 4 –7 postnatal (P) days of age were perfused through the ascending aorta with 4% paraformaldehyde in 0.1 M phosphate buffer at, pH 7.4. The cerebella were then dissected, fixed by immersion for 4 – 8 h and then transferred to a 30% phosphate-buffered sucrose for 12 h. Sagittal sections (15 m) were cut using a cryostat at ⫺20°C and were mounted on coated slides. Sections were preincubated for 15 min with 5% normal goat serum (NGS) and then incubated overnight at 4°C with Jones mAb (either purified IgM, 4 g/ml in 5% NGS; or ascites, 1:100; Sigma, St. Louis, MO). The slides were then rinsed three times in PBS and incubated with the immunogold staining reagent (goat IgG directed against mouse IgM linked to 5 nm colloidal gold particles, 1:40 dilution; Amersham, Sa˜o Paulo, Brasil) and intensified with the silver-intensification kit (Amersham) or incubated with a Cy3-conjugated goat antimouse IgG (1:1000, Jackson Lab. Inc., West Grove, PA). Electron microscope immunocytochemistry. Indirect immunoelectron microscopy was performed on parasagittal vibratome sections by a preembedding PAP procedure. Shortly, 7-day-old rats were anesthetized with sodium pentobarbital (100 mg/kg, i.p.) and were perfused through the left ventricle with 4 ml of heparin (1000 U/ml in 0.15 M NaCl), and 20 ml of paraformaldehyde (4%) in 0.1 M phosphate buffer (PB), pH 7.4, followed by buffered 4% paraformaldehyde and 0.5% glutaraldehyde. The brains were removed and postfixed for 30 min in buffered 4% paraformaldehyde and vibratome sections were made through the cerebel-
9-O-Acetyl GD3 in Neuronal Migration
lum at a thickness of 50 m. The vermis and intermediate region were sectioned sagittally, whereas sections through the lateral cerebellum were in an oblique plane, thus, providing transversely cut folia. Tissue sections were then rinsed in phosphate-buffered saline (PBS), preincubated for 30 min in 5% NGS and incubated overnight with Jones mAb (purified IgM) in 2% NGS. Sections were then incubated with a link (unlabeled) goat anti-mouse IgM (Dako, Carpinteria, CA) in 2% NGS for 2 h and, finally, reacted with mouse PAP (Dako, Carpinteria, CA) in PBS. After several washes with PBS, HRP histochemistry was performed with 0.0002 % H 2O 2 and 0.05% diaminobenzidine in 0.1 M phosphate buffer, pH 7.6. After several washes, the sections were postfixed with buffered 2% glutaraldehyde followed by 1% osmium tetroxide in phosphate buffer. Experimental and control sections were dehydrated with increasing ethanol concentrations and embedded in Spurrs’ resin between Teflon-coated slides and coverslips and polymerized. Small pieces of these blocks were cut from the embedded sections, glued onto the tops of blank blocks or reembedded in BEEM capsules. After preliminary trimming, 1-m sections were cut and examined under the light microscope to identify sections of interest. After that, ultrathin sections were made and short ribbons collected onto parallel bar grids or Formvar-coated slot grids. Alternate ribbons were contrasted with uranyl acetate and lead citrate or left without contrast for assessment in a Zeiss EM-900 electron microscope. Microexplant cultures. Cerebella taken from 4- to 6-day-old rats were dissected, cleaned for meninges and major blood vessels, and cut into small pieces with a blade under sterile conditions. The pieces were plated onto glass coverslips coated with poly-l-lysine (MW ⫽ 389.000; 20 g/ml; Sigma, St. Louis, MO) and laminin (40 g/ml; Gibco Brl; Gainthersburg, MD). Four to six similar sized explants of about 600 m in diameter were placed on each coverslip. About 1 h after plating the media was changed in order to eliminate the floating explants and 300 l of fresh media was added to each well. At this point the antibodies Jones mAb (1:100; Sigma, St. Louis, MO) and A2B5 mAb (1:1000; donation from Dr. Paola Bovolenta; Cajal Institute, MadridSpain), which recognizes a group of c-series gangliosides (Eisenbarth et al., 1979), were added to the culture mixed with the media. Cultures were maintained in a 95% air/5% CO 2 incubator at 37°C and the explants were grown in DMEM/F12 containing 10% fetal calf serum for 72 h after which they were fixed with 4% paraformaldehyde in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2 (pH 6.9; PHEM buffer) for
497 30 min. For immunocytochemistry, cultures were labeled either with Jones mAb (1:100) or A2B5 mAb (1: 1000) or with antibodies against GFAP (1:100, polyclonal; Sigma) or MAP2 (1:100, monoclonal; Sigma). Primary antibodies were incubated for 18 –24 h at 4°C. The binding of these antibodies was visualized with Cy3-conjugated goat anti-mouse IgG (1:1000; Jackson Lab., Inc.; West Grove, PA), and fluorescein isothiocyanate-coupled goat anti-rabbit IgG (1:100; Sigma). After 2 h incubation at room temperature, the cultures were washed three times in PBS. In the last wash the fluorescent dye 4⬘-6-diamidino-2-phenylindole (DAPI, Sigma) was added in order to visualize the nuclei. Stained cultures were mounted with VectaShield (Vector Labs., Burlingame, CA) and viewed with an inverted or with a confocal Zeiss microscope. Quantitative analysis. The extent of cell migration was estimated by measuring the distance between the border of the explant and the wavefront of cells labeled with DAPI at eight sites located at 45° angles to each other. A mean was obtained for each explant. These measures were made using a 20⫻ objective lens in an Axioplan Zeiss microscope connected to a computer equipped with Morpho 3.2 software (UFRJ, 1988 –1995) capable of encoding x and y coordinates. A minimum of five explants was analyzed for each treatment, and the effect of different treatments on neuronal migration was evaluated for significant differences using the Student’s t test. Slice cultures. Cerebella were taken from P4 –7 rats and dissected out in sterile conditions. Slices were cut at 350 m, using a tissue chopper (MacIlwain, U.S.A.), and plated on millicell inserts (Sigma, St. Louis, MO). To distinguish between proliferating and postmitotic cells we used 5-bromo-2⬘-deoxyuridine (BrdU; Sigma), which is incorporated by cells synthesizing DNA (Gratzner, 1982). A 10 g/ml solution of BrdU was added to the culture medium for 15 min. In some slices Jones mAb was added at a 1:20 concentration simultaneously with BrdU. The slices were then carefully washed and new culture medium was added with or without antibodies. Jones mAb was added at 1:20 concentration and A2B5 mAb (Boehringer-Mannhein Co., Germany) was used at a 1:100 concentration. Cultures were maintained in a 95% air/5% CO 2 incubator at 37°C and the slices were grown in DMEM/F12 containing 10% fetal calf serum for 72 h, after which they were fixed with 4% paraformaldehyde in 60 mM Pipes, 25 mM Hepes, 10 mM EGTA, and 2 mM MgCl 2 (pH 6.9; PHEM buffer) for 30 min. Following antigen retrieval in a microwave oven (Dover and Patel, 1994), cultures were washed with PBS and incubated overnight at 4°C
498 with a mouse monoclonal antibody against BrdU (RPN 202, Amersham, Little Chalfont, UK), according to the manufacturer’s instructions and developed with a CY3conjugated goat anti-mouse IgG (1:1000; Jackson Lab). The slices were analyzed and documented using a Zeiss confocal microscope.
ACKNOWLEDGMENTS The authors thank Dr. Cecı´lia Hedin-Pereira for crucial comments during the elaboration of this work, Dr. Elizabeth Debski for comments on the manuscript, and Felipe Marins for technical assistance. This work was supported by PRONEX, FINEP, CNPq, and FAPERJ grants to R.M-O. and L.A.C. and TWAS to R.M-O.
REFERENCES Altman, J. (1972a). Postnatal development of the cerebellar cortex in the rat. I. The external germinal layer and the transitional molecular layer. J. Comp. Neurol. 145: 353–398. Altman, J. (1972b). Postnatal development of the cerebellar cortex in the rat. II. Phases in the maturation of Purkinje cells and of the molecular layer. J. Comp. Neurol. 145: 399 – 464. Altman, J. (1972c). Postnatal development of the cerebellar cortex in the rat. III. Maturation of the components of the granular layer. J. Comp. Neurol. 145: 465–514. Anton, E. S., Kreidberg, J. A., and Rakic, P. (1999). Distinct functions of ␣ 3 and ␣ v integrin receptors in neuronal migration and laminar organization of the cerebral cortex. Neuron 22: 277–289. Antonicek, H., Persohn, E., and Schachner, M. (1987). Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration. J. Cell Biol. 104: 1587–1595. Araujo, H., Menezes, M., and Mendez-Otero, R (1997). Blockage of 9-O-acetyl gangliosides induces microtubule depolymerization in growth cones and neurites. Eur. J. Cell Biol. 72: 202–213. Bartsch, S., Bartsch, U., Do¨rries, U., Faissner, A., Weller, A., Ekblom, P., and Schachner, M. (1992). Expression of tenascin in the developing and adult cerebellar cortex. J. Neurosci. 12: 736 –749. Birkle´, S., Ren, S., Slominski, A., Zeng, G., Gao, L., and Yu, R. K. (1999). Down-regulation of the expression of O-acetyl-GD3 by the O-acetylesterase cDNA in hamster melanoma cells: effects on cellular proliferation, differentiation, and melanogenesis. J. Neurochem. 72: 954 –961. Birkle´, S., Gao, L., Zeng, G., and Yu, R. K. (2000). Down regulation of GD3 ganglioside and its O-acetylated derivative by stable transfection with antisense vector against GD3-synthase gene expression in hamster melanoma cells: Effects on cellular growth, melanogenesis, and dendricity. J. Neurochem. 74: 547–554. Bonafede, D. M., Macala, L. J., Constantine-Paton, M., and Yu, R. K. (1989). Isolation and characterization of ganglioside 9-O-acetylGD3 from bovine buttermilk. Lipids 24: 680 – 684. Burns, G. F., Lucas, C. M., Krissansen, G. W., Werkmeister, J. A., Scanlon, D. B., Simpson, R. J., and Vadas, M. A. (1988). Synergism between membrane gangliosides and Arg-Gly-Asp-directed glycoprotein receptors in attachment to matrix proteins by melanoma cells. J. Cell Biol. 107: 1225–1230.
Santiago et al.
Cameron, R. S., and Rakic, P. (1994). Polypeptides that comprise the plasmalemmal microdomain between migrating neuronal and glial cells. J. Neurosci. 14: 3139 –3355. Caviness, V. S., Jr., and Rakic, P. (1978). Mechanisms of cortical development: A view from mutations in mice. Annu. Rev. Neurosci. 1: 297–326. Cheresh, D. A. (1987). Ganglioside involvement in tumor cell-substratum interactions. In Development and Recognition of the Transformed Cell (M. I. Greene and T. Hamaoka, Eds.) Plenum Press, New York, NY. Cheresh, D. A., Pierbacher M. D., Herzig, M. A., and, Mujoo, J. (1986). Disialogangliosides GD2 and GD3 are involved in the attachment of human melanoma and neuroblastoma cells to extracellular matrix proteins. J. Cell Biol. 102: 688 – 696. Cheresh, D. A., Varki, A. P., Varki, N. W., Stallcup, W. B., Levine, J., and Reisfeld, R. A. (1984). A monoclonal antibody recognizes an O-acetylated sialic acid in a human melanoma-associated ganglioside. J. Biol. Chem. 259: 7453–7459. Constantine-Paton, M., Blum, A. S., Mendez-Otero, R., and Barnstable, C. J. (1986). A cell surface molecule distributed in a dorsoventral gradiente in the perinatal rat retina. Nature (London) 324: 459 – 462. Dover, R., and Patel, K. (1994). Improved methodology for detecting bromodeoxyuridine in cultured cells and tissues sections by immunocytochemistry. Histochemistry 102: 383–387. Edmondson, J. C., and Hatten, M. E. (1987). Glial-guided neuronal migration in vitro: A high resolution time-lapse videomicroscopic study. J. Neurosci. 7: 1928 –1934. Eisenbarth, G. S., Walsh, F. S., and Nirenberg, M. (1979). Monoclonal antibody to a plasma membrane antigen of neurons. Proc. Natl. Acad. Sci. USA 76: 4913– 4917. Feng, L., Hatten, M. E., and Heintz, N. (1994). Brain lipid-binding protein (BLBP): A novel signaling system in the developing mammalian CNS. Neuron 12: 895–908. Fishell, G., and Hatten, M. E. (1991). Astrotactin provides a receptor system for CNS neuronal migration. Development 113: 755–765. Gao, W.-Q., and Hatten, M. E. (1993). Neuronal differentiation rescued by implantation of weaver granule cell precursos into wildtype cerebellar cortex. Science 260: 367–369. Gratzner, H. G. (1982). Monoclonal antibody to 5-bromo and 5-iododeoxyuridine: A new reagent for detection of DNA replication. Science 218: 474 – 478. Gregory, W. A., Edmondson, J. C., Hatten, M. E., and Mason, C. A. (1988). Cytology and neuron-glial apposition of migrating cerebellar granule cells in vitro. J. Neurosci. 8: 1728 –1738. Hakomori, S. (1990). Bifunctional role of glycosphingolipids. Modulators for transmembrane signaling and mediators for cellular interactions. J. Biol. Chem. 265: 18713–18716. Hatten, M. E. (1990). Riding the glial monorail: A common mechanism for glial-guided neuronal migration in different regions of the developing brain. Trends. Neurosci. 13: 179 –187. Hatten, M. E. (1999). Central nervous system neuronal migration. Annu. Rev. Neurosci. 22: 511–539. Hatten, M. E., Liem, R. K. H., and Mason, C. A. (1984). Defects in specific associations between astroglia and neurons occur in microcultures of weaver mouse cerebellar cells. J. Neurosci. 4: 1163–1172. Hatten, M. E., Liem, R. K. H., and Mason, C. A. (1986). Weaver mouse cerebellar granule cells fail to migrate on wild-type astroglial process in vitro. J. Neurosci. 9: 2676 –2683. Hockberger, P. E., Tseng, H.-Y., and Connor, J. A. (1987). Immunocytochemical and eletrophysiological differentiation of rat cerebellar granule cells in explant cultures. J. Neurosci. 7: 1370 –1383.
9-O-Acetyl GD3 in Neuronal Migration
Husmann K., Faissner, A., and Schachner, M. (1992). Tenascin promotes cerebellar granule cell migration and neurite outgrowth by different domains in the fibronectin type III repeats. J. Cell Biol. 116: 145–186. Husmann, B., and Sievers, J. (1985). Cerebellar external granule cells are attached to the basal lamina from the onset of migration up to the end of their proliferative activity. J. Comp. Neurol. 241: 50 – 62. Kojima, N., and Hakomori, S. (1991). Synergistic effect of two cell recognition systems: Glycosphingolipid-glycosphingolipid and integrin receptor interaction with pericellular matrix protein. Glycobiology 1: 623– 630. Komuro, H., and Rakic, P. (1993). Modulation of neuronal migration by NMDA receptors. Science 260: 95–97. Komuro, H., and Rakic, P. (1995). Dynamics of granule cell migration: A confocal microscopic study in acute cerebellar slice preparations. J. Neurosci. 15: 1110 –1120. Ledeen, R. (1985). Gangliosides of neurons. Trends Neurosci. 8: 169 – 174. Lloyd, K. O., and Furukawa, K. (1998). Biosynthesis and functions of gangliosides: Recent advances. Glycoconj. J. 7: 627– 636. Mendez-Otero, R., and Cavalcante, L. A. (1996). Expression of 9-Oacetylated gangliosides is correlated with tangential cell migration in the rat brain. Neurosci. Lett. 204: 97–100. Mendez-Otero, R., and Constantine-Paton, M. (1990). Granule cell induction of 9-O-acetyl gangliosides on cerebellar glia in microcultures. Dev. Biol. 138: 400 – 409. Mendez-Otero, R., and Friedman, J. E. (1996). Role of acetylated gangliosides on neurite extension. Eur. J. Cell Biol. 71: 192–198. Mendez-Otero, R., and Ramon-Cueto, A. (1994). Expression of 9-Oacetylated gangliosides during development of the rat olfatory system. Neuroreport 5: 1755–1759. Mendez-Otero, R., Schlosshauer, B., Barnstable, C. J., and Constantine-Paton, M. (1988). A developmentally regulated antigen associated with neural cell and process migration. J. Neurosci. 8: 564 –579. Miale, I. L., and Sidman, R. L. (1961). An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp. Neurol. 4: 277–296. O’Shea, K. S., Rheinheimer, J. S., and Dixit, V. M. (1990). Deposition and role of thrombospondin in the histogenesis of cerebellar cortex. J. Cell Biol. 110: 1275–1283. Pearlman, A. L., Faust, P. L., Hatten, M. E., and Brunstrom, J. E. (1998). New directions for neuronal migration. Curr. Opin. Neurobiol. 8: 45–54. Probstmeier, R., Michels, M., Franz, T., Chan, B. M. C., and Pesheva,
499 P. (1999). Tenascin-R interferes with integrin-dependent oligodendrocyte cell adhesion by a ganglioside-mediated signalling mechanism. Eur. J. Neurosci. 11: 2472–2588. Probstmeier, R., and Pesheva, P. (1999). Tenascin-C inhibits 1 integrin-dependent cell adhesion and neurite outgrowth on fibronectin by a disialoganglioside-mediated signaling mechanism. Glycobiology 9: 101–114. Rakic, P. (1971). Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A Golgi and electromicroscopic study in Macacus rhesus. J. Comp. Neurol. 141: 283–312. Rakic, P. (1976). Synaptic specificity in the cerebellar cortex: Study of anomalous circuits induced by single gene mutation in mice. Cold Spring Harbor Symp. Quant. Biol. 40: 333–346. Rakic, P. (1981). Neuronal-glial interactions during brain development. Trends Neurosci. 4: 184 –187. Rakic, P. (1990). Principles of cell migration. Experientia 46: 882– 891. Rakic, P., Cameron, R. S., and Komuro, H. (1994). Recognition, adhesion, transmembrane signaling and cell motility in guided neuronal migration. Curr. Opin. Neurobiol. 4: 63– 69. Rakic, P., and Sidman, R. L. (1973a). Sequence of developmental abnormalities leading to granule cell deficit in cerebellar cortex of weaver mutant mice. J. Comp. Neurol. 152: 103–132. Rakic, P., and Sidman, R. L. (1973b). Weaver mouse mutant cerebellum: defective neuronal migration secondary to abnormality of Bergmann glia. Proc. Natl. Acad. Sci. USA 70: 240 –244. Ramo´n y Cajal, S. (1911). Histologie du Systeme Nerveux de’l Homme et des Vertebre´s, Maloine, Paris. [Reprinted by Consejo Superior de Investigaciones Cientificas, Madrid, 1972] Stallcup, W. B. (1988). Involvement of gangliosides and glycoprotein fibronectin receptors in cellular adhesion to fibronectin. Exp. Cell Res. 177: 90 –102. Stallcup, W. B., Pytela, R., and Ruoslahti, E. (1989). A neuroectodermassociated ganglioside participates in fibronectin receptor mediated adhesion of germinal cells to fibronectin. Dev. Biol. 132: 212–229. Varki, A., Hooshmand, F., Diaz, S. Varki, N. M., and Hedrick, S. M. (1991). Developmental abnormalities in transgenic mice expressing a sialic acid-specific 9-O-acetylesterase. Cell 65: 65–74. Yu, R. K. (1994). Development regulation of ganglioside metabolism. Prog. Brain Res. 101: 31– 44. Wichterle, H., Garcı´a-Verdugo, J. M., and Alvarez-Buylla, A. (1997). Direct evidence for homotypic, glia-independent neuronal migration. Neuron 18: 779 –791. Received July 19, 2000 Revised November 29, 2000 Accepted November 30, 2000