332 amine (Sigma), sealed with nail polish and visualized on an axiophot fluorescence microscope (Zeiss) with appropriate filters. Controls were done by omitting the primary antibody. Immunocytochemistry. Coverslips were washed in PBS once and overlayed with methanol at -20 °C for 5 min. Slides were reconstituted in PBS and processed for fluorescence microscopy in the same way as the cryostat sections. Histogenesis of cell cultures. Within the first 24 h, cells attach to the culture substratum and spread. Small round, phase-bright cells start to aggregate on top of cells with a flattened morphology. Aggregates become larger (approx. 150 ~m in diameter) and are interconnected by fasciculated fibers formed by radial glia-like cells (see below). After a period of 6 days in vitro (DIV), the typical morphology is observed (see Fig. 1). Nearly all phase-bright cells are reaggregated on top of the glial sheets. After 12 DIV, aggregates start to detach. This morphology is characteristic of tectum cultures, but not

of those made from other brain regions (medulla oblongata and telencephalon). The course of histogenesis of the CNS cultures is basically the same for all developmental stages of tadpoles (stages 24-40). Identification of radial glia in vivo. GFAP is a marker protein of astrocytes 2. The antibody XC9D8 raised against Xenopus GFAP 21 exclusively stains radial glia in the developing brain of Discoglossus. The somata of the cells at the ventricular surface as well as glial endfeet contacting the pial surface are stained (see Fig. 2). Occasionally, stained processes can be traced through the entire thickness of the brain. These are specific features of radial glial cells 17. Together with the observed staining of cytoskeletal filaments in culture (see Fig. 3), these findings confirm the specificity of the antibody for GFAP in Discoglossus pictus. Identification of cell types in culture. Cells in culture are identified by their morphology as seen in phasecontrast microscopy. Glial cells are additionally characterized by indirect GFAP immunofluorescence. Cells

Fig. 1. q~ypicalmorphologyof tectum culture taken from stage-36 Discoglossus tadpoles after 7 DIV. Note the homogenous size of aggregates. Arrows indicate migrating cells which moved at least one cell diameter during one hour. Bar = 100/zm.

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Fig. 2. Section through the optic tectum of a Discoglossustadpole (stage 36) Stained with anti-GFAP. Somata at the ventricular surface (open arrow) as well as glial endfeet contacting the pial surface are stained (arrows). Bar = 50/~m. Inset: higher magnificationof ghal endfeet staining in a section through the optic tectum of a postmetamorphic frog. Orientation: pial surface up, ventricle down. Bar = 12.5/~m.

with a neuron-like morphology appear as small round and phase-bright cells with thin processes originating directly from the soma and occasionally ending in a growth cone. Migrating neuron-like cells are identified by their elliptic form and their close association to glial fibers. Cells labeled with the GFAP antibody show various morphologies. One cell type is polygonally shaped with only short, if any, protrusions. The other type, called 'radial gila-like', has very long and straight processes (up to 1 ram). These processes fasciculate, interconnect aggregates and facilitate migration of neuron-like cells along their extension (see Fig. 3A-D). Other fiat cells in the culture are assumed to be either undifferentiated astroblasts or contaminating fibroblasts. Migration along radial glial fibers. Migration of neuron-like cells along glial fibers is frequently seen in tectum cultures (see Fig. 1). Proof that cells actively migrate along radial glial fibers is given by time-lapse photography (see Fig. 4). This mode of migration is

observed as early as 24 h in vitro. It peaks after 6 DIV and decreases thereafter. Even though there are nearly as many radial gila-like cells in medulla cultures (see Fig. 3) as in tectum cultures, migrating cells are rare in cultures of medulla and telencephalon. In our cell culture system, cells of the frog optic tectum interact in a manner similar to cells of mammalian laminated CNS regions in vitro 9. Neuron-like cells of the frog optic tectum recognize glial cells and migrate along glial processes in cell culture. The constitution of laminae in the vertebrate brain seems to require extensive radial migration. In vitro, cells taken from the optic rectum exhibit a higher degree of migration than those taken from medulla and telencephalon. This corresponds with the degree of cell migration in the respective brain regions in vivo, with the optic tectum showing the highest degree of lamination 11. Our results point to an involvement of radial migration in the constitution of laminae in the frog optic rectum. In other systems, neuronal migration along glial

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Fig. 3. A: immunofluorescence image of a GFAP-positive glial strand with an apposed GFAP-negative cell in a tectum culture of Discoglossus tadpoles (stage 36) after 8 DIV. B: corresponding phase-contrast image to A. Bar = 10/~m. C: radial glia-like cells, stained for GFAP, are connectinl~ two ap_~relzates in a medulla culture of stage-37 tadpoles after 8 DIV. D: corresponding phase-contrast image to C. Bar = 50 #m.

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Fig. 4. Time-lapse study of two migrating cells in a tectum culture of stage-37 Discoglossus tadpoles, 6 DIV. One cell is migrating 'up' and the other one is more slowly migrating 'down'. Cells come into direct contact, but prefer to adhere to the glial strand rather than to each other. Temporal order is from left to right (0 h, 1 h, 1 h 40 min, 2 h, 3 h, 3 h 40 rain.) Bar -- 20/zm.

1 Antonicek, H., Persohn, E. and Schachner, M., Biochemical and functional characterization of a novel neuron-glia adhesion molecule that is involved in neuronal migration, J. Cell BIOL, 104 (1987) 1587-1595. 2 Bignami, A., Eng, L.F., Dahl, D. and Uyeda, C.T., Localization of the glial fibrillary acidic protein in astrocytes by immunofluoreseence, Brain Research, 43 (1972) 429-435. 3 Chuong, C.M., Crossin, K.L. and Edelman, G.M., Sequential expression and differential function of multiple adhesion molecules during the formation of cerebellar cortical layers, J. Cell Biol., 104 (1987) 331-342. 4 Edmondson, J.C., Liem, R.K.H., Kuster, J.E. and Hatten, M.E., Astrotactin: a novel neuronal cell surface antigen that mediates neuron-astroglial interactions in cerebellar microcultures, J. Cell BIOL, 106 (1988) 505-517. 5 Engel, A.K. and Miiller, C.M., Postnatal development of vimentin-immunoreactive radial glial cells in the primary visual cortex of the eat, J. Neurocytol., 18 (1989) 437-450. 6 Gianonatti, C., Bodega, G. and Bardasano, J.L., Neuroglia of the optic tectum in the Bufo bufo (amphibian anura), first trials, J. Hirnforsch., 28 (1987) 139-143. 7 Gosner, R.L. and Rossman, D.A., Eggs and larval development of the tree frogs Hyla crucifer and Hyla ocularis, Herpetologica, 16 (1960) 225-232. 8 Gray, G.E., Leber, S.M. and Sanes, J.S., Migratory patterns of clonally related cells in the developing central nervous system, Experientia, 46 (1990) 929-940. 9 Hatten, M.E., Riding the glial monorail: a common mechanism for gliai-guided neuronal migration in different regions of the developing mammalian brain, Trends Neurosci., 13 (1990) 179-184. 10 Hatten, M.E. and Liem, R.K.H., Astroglial cells provide a template for the positioning of developing cerebellar neurons in vitro, J. Cell Biol., 90 (1981) 622-630. 11 Kemali, M. and Braitenberg, V., Atlas of the frog's brain, Springer, Berlin, 1969. 12 Krfiger, C.G., Becker, T. and Ernst, M., Characterization of amphibian CNS cells in primary cell culture. In N. Eisner and G.

processes has been attributed to highly specific interactions of cell adhesion molecules mediating cell interaction or to specific molecules of the extracellular matrix. Antibodies to NgCAM, cytotactin3 and A M O G 1 can block granule cell migration in cerebellum slices. Astrotactin 4 and laminin 14 are likewise thought to be involved in neuron-glia interaction during CNS development. Laminin is expressed along radial glia in the developing mouse brain as well as on radial glial cells in the optic tectum of the frog Rana pipiens 15. The in vitro assay we have established will be used to elucidate the role of specific molecules in neuron-glia interaction during frog brain histogenesis.

We thank Dr. B.G. Szaro for the gift of the antibody XC9D8 and Drs. C. Naujoks-Manteuffel, G. Roth, A. Schmidt and D.B. Wake for helpful comments on the manuscript. Supported by the DFG.

Roth (Eds.), Brain-Perception-Cogniton: Proceedings of the 18th Grttingen Neurobiology Conference, Thieme, Stuttgart, 1990, p.

467. 13 Levitt, P. and Rakic, P., Immunoperoxidase localization of glial fibrillary acidic protein in radial glial ceils and astrocytes of the developing rhesus monkey brain, J. Comp. Neurol., 193 (1980) 815-840. 14 Liesi, P., Do neurons in the vertebrate CNS migrate on laminin?, EMBO J., 4 (1985) 1163-1170. 15 Liesi, P., Laminin-immunoreactive glia distinguishes regenerative adult CNS systems from non-regenerative ones, EMBO J., 4 (1985) 2505-2511. 16 Naujoks-Manteuffel, C. and Roth, G., Astroglial cells in a salamander brain (Salamandra salamandra) as compared to mammals: a glial fibrillary acidic protein immunohistoehemistry study, Brain Research, 487 (1989) 397-401. 17 Paul, E., Ober die Typen tier Ependymzellen und ihre regionale Verteilung bei Rana temporaria, Z. Zeilforsch., 80 (1967) 461-487. 18 Rakic, P., Mode of cell migration to the superficial layers of the fetal monkey neocortex, Z Comp. NeuroL, 145 (1972) 61-84. 19 Rakic, P., Neuron-glia relationship during granule cell migration in developing cerebellar cortex. A golgi and electronmicroseopic study in Macacus rhesus, J. Comp. Neurol., 141 (1971) 283-312. 20 Sidman, R.L. and Rakic, P., Neuronal migration, with special reference to developing human brain: a review, Brain Research, 62 (1973) 1-35. 21 Szaro, B.G. and Gainer, H., Immunocytochemical identification of non-neuronal intermediate filament proteins in the developing Xenopus laevis nervous system, Dev. Brain Res., 43 (1988) 207-224. 22 Szrkely, G. and l_Azdr, G., Cellular and synaptic architecture of the optic tectum. In R. Llinds, and W. Precht, (Eds.), Frog Neurobiology: A Handbook, Springer, Heidelberg, 1976, pp. 407-434. 23 Trenkner, E. and Sidman, R.L., Histogenesis of mouse cerebellum in microwell cultures, J. Cell Biol., 75 (1977) 915-940.

Cell migration along glial fibers in dissociated cell culture of the frog optic tectum.

Migration of neurons along radial glial fibers is associated with the development of laminated regions in the mammalian brain. We examined cell intera...
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