Summary
Reaggregate cultures were obtained from single-cell suspensions of fetal and early postnatal cerebellum, and fetal telencephalon and mesencephalon from C57BL/6J and NMRI mice and maintained in suspension under constant rotation as described previously (Seeds 1971). The percentage of dead cells in the aggregates as measured by the uptake of the fluorescent dye propidium iodide was always less than 5% of all cells. During the initial phase of reaggregation up to 20 h in vitro (hiv) several immunocytochemically defined cell types had a random distribution within the aggregate. Astrocytes were identified by indirect immunofluorescence by the use of the markers glial fibrillary acidic protein (GFAP), C1 and M1 antigens; neurons by NS-4 antigen and tetanus-toxin receptors; fibroblasts or fibroblast-like cells by fibronectin and laminin; and oligodendrocytes by myelin basic protein (MBP). Choleratoxin receptors and M 2 antigen served to distinguish the more mature from the less mature neurons. In reaggregates of early postnatal cerebellar cells neurons had started to redistribute after 40 hiv, forming an outer region containing more immature neurons and a core with more mature neurons. After 5 days in vitro (div) immature neurons were no longer detectable. From 3–8 div M1-and GFAP-positive astrocytic processes in the outer region showed a tendency for radial orientation. At later stages the processes appeared more randomly distributed and formed a dense glial network. Few oligodendrocytes and fibronectin-positive cells were present in the reaggregates. When reaggregates were prepared from 15 day-old embryonic cerebella, formation of radially oriented astrocytic processes and redistribution of neurons proceeded more slowly, but in a similar pattern as described for early postnatal cerebellum. GFAP was detectable at earlier ages than in situ. In reaggregates of 15 to 17 day old embryonic telencephalic anlage or midbrain, radially oriented astrocytic processes were not detectable. Similar to cerebellar reaggregates, accumulation of neurons in the inner region was observed.
Similar content being viewed by others
References
Biber A, Englert D, Dommasch D, Hempel K (1981) Myelin basic protein in cerebrospinal fluid of patients with multiple sclerosis and other neurological diseases. J Neurol 225:231–236
Bignami A, Eng LF, Dahl D, Uyeda CT (1972) Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res 43:429–435
Bignami A, Dahl D, Rueger DC (1980a) Glial fibrillary acidic protein (GFA) in normal neural cells and in pathological conditions. Advances in Cellular Neurobiology 1:285–310
Bignami A, Kozak LP, Dahl D (1980b) Molecular markers for the analysis of neural differentiation in culture. Tissue culture in neurobiology, Giacobini et al. (ed). Raven Press, New York, 63–73
Caviness VS Jr, Sidman RL (1973) Time of origin of corresponding cell classes in the cerebral cortex of normal and reeler mutant mice: an autoradiographic analysis. J Comp Neurol 148:141–152
Dimpfel W, Neale GH, Habermann E (1975) 125I-labelled tetanus toxin as a neuronal marker in tissue cultures derived from embryonic CNS. Naunyn-Schmiedeberg's Arch Pharmacol 290:329–333
Eng LF, Vanderhaegen GG, Bignami A, Gerstl B (1971) An acidic protein isolated from fibrous astrocytes. Brain Res 28:351–354
Garber BB (1977) Cell aggregation and recognition in the selfassembly of brain tissues. Cell tissue and organ culture in neurobiology, Federoff and Hertz (eds). Academic Press, New York, 515–537
Garber BB, Moscona AA (1972a) Reconstruction of brain tissue from cell suspensions. I. Aggregation patterns of cells dissociated from different regions of the developing brain. Dev Biol 27:217–234
Garber BB, Moscona AA (1972b) Reconstruction of brain tissue from cell suspensions. II. Specific enhancement of aggregation of embryonic cerebral cells by supernatant from homologous cell cultures. Dev Biol 27:235–271
Goridis C, Martin J, Schachner M (1978) Characterization of an antiserum to synaptic glomeruli from rat cerebellum. Brain Res Bull 3:45–52
Hicks SP, D'Amato CJ (1968) Cell migrations to the isocortex in the rat. Anat Rec 160:619–634
Honegger P, Richelson E (1976) Biochemical differentiation of mechanically dissociated mammalian brain in aggregating cell culture. Brain Res 109:335–354
Honegger P, Richelson E (1979) Neurotransmitter synthesis, storage and release by aggregating cell cultures of rat brain. Brain Res 162:89–101
Kozak LP (1977) The transition from embryonic to adult isozyme expression in reaggregating cell cultures of mouse brain. Dev Biol 55:160–169
Kozak LP (1978) Increased synthesis of L-glycerol-3-phosphate dehydrogenase during in vitro differentiation of reaggregating cerebellar cells. Dev Biol 66:539–600
Kozak LP, Eppig JJ, Dahl D, Bignami A (1977) Ultrastructural and immunohistological characterization of a cell culture model for the study of neuronal-glial interactions. Dev Biol 59:206–227
Kozak LP, Eppig JJ, Dahl D, Bignami A (1978a) Enhanced neuronal expression in reaggregating cells of mouse cerebellum cultured in the presence of poly-L-lysine. Dev Biol 64:252–264
Kozak LP, Dahl D, Bignami A (1978b) GFA-protein in reaggregating and monolayer cultures of fetal mouse cerebral hemispheres. Brain Res 150:631–637
Lagenaur C, Schachner M (1981) Monoclonal antibody (M2) to glial and neuronal cell surfaces. J Surpamol Struct Cell Biochem 15:335–346
Lagenaur C, Sommer I, Schachner M (1980) Subclass of astroglia in mouse cerebellum recognized by monoclonal antibody. Dev Biol 79:367–378
Lagenaur C, Masters C, Schachner M (1982) Changes in expression of glial antigens M1 and C1 after cerebellar injury. J Neuroscience, in press
Levitt P, Moore RY, Garber BB (1976) Selective cell association of catecholamine neurons in brain aggregates in vitro. Brain Res 111:311–320
Matthieu JM, Honegger P, Trapp BD, Cohen SR, Webster HdeF (1978) Myelination in rat brain aggregating cell cultures. Neuroscience 3:565–572
Matthieu JM, Honegger P, Favrod P, Poduslo JF, Costantino-Cessarini E, Krstic R (1980) Myelination and demyelination in aggregating cultures of rat brain cells. In: Giacobini et al. (ed) Tissue culture in neurobiology. Raven Press, New York, 441–459
Moscona AA (1961) Rotation mediated histogenetic aggregation of dissociated cells: a quantifiable approach to cell interactions in vitro. Exp Cell Res 22:455–475
Ramirez G, Seeds NW (1977) Temporal changes in embryonic nerve cell recognition: correlate with cholinergic development in aggregate cultures. Dev Biol 60:153–162
Rohde H, Wick G, Timpl R (1979) Immunochemical characterization of the basement membrane glycoprotein laminin. Eur J Biochem 102:195–201
Schachner M, Wortham KA, Carter LD, Chaffee JK (1975) NS-4, a cell surface antigen of developing and adult mouse brain and sperm. Dev Biol 44:313–325
Schachner M, Schoonmaker G, Hynes RO (1978) Cellular and subcellular localization of LETS-protein in the nervous system. Brain Res 158:149–158
Schnitzer J, Schachner M (1981) Expression of Thy-1, H-2 and NS-4 cell surface antigens and tetanus toxin receptors in early postnatal and adult mouse cerebellum. J Neuroimmunol 1:429–456
Seeds NW (1971) Biochemical differentiation in reaggregating brain cell culture. Proc Natl Acad Sci (USA) 68:1858–1861
Seeds NW (1973) Differentiation of aggregating brain cell cultures. In: Sato (ed) Tissue culture of the nervous system. Plenum Press, New York, 35–53
Seeds NW (1975) Expression of differentiated activities in reaggregated brain cell cultures. J Biol Chem 250:5455–5458
Seeds NW, Haffke SC (1978) Cell junction and ultrastructural development of reaggregated mouse brain cultures. Dev Neurosci 1:69–79
Seeds NW, Vatter AE (1971) Synaptogenesis in reaggregating brain cell culture. Proc Natl Acad Sci (USA) 68:3219–3222
Seeds NW, Ramirez G, Marks MJ (1977) Aggregate cultures: A model for studies of brain development. In: Acton and Lynn (eds) Cell culture and its applications. Academic Press, New York, 23–37
Seeds NW, Haffke SC, Krystosek A (1980) Cell migration and recognition in cerebellar reaggregate cultures. In: Giacobini et al. (ed) Tissue culture in neurobiology. Raven Press, New York
Sheffield JB (1982) Sorting behavior among cells from the 14 day embryonic chick neural retina. Dev Biol 89:41–47
Sommer I, Schachner M (1981) Monoclonal antibodies (01 to 04) to oligodendrocyte cell surfaces: an immunological study in the central nervous system. Dev Biol 83:311–327
Sommer I, Lagenaur C, Schachner M (1981) Recognition of Bergmann glial and ependymal cells in the mouse nervous system by monoclonal antibody. J Cell Biol 90:448–458
Spiegel J, Garber B (1981a) Sorting out of coaggregated brain cell types mediated by specific cell recognition factors in vitro. Dev Biol 85:1–15
Spiegel J, Garber B (1981b) Sorting out of coaggregated brain cell types mediated by specific cell recognition factors in vitro. Dev Biol 85:16–25
Sternberger NH, Hoyama Y, Kies MW, Webster HdeF (1978) Myelin basic protein demonstrated immunocytochemically in oligodendroglia prior to myelin sheath formation. Proc Natl Acad Sci 75:2521–2524
Theiler K (1972) The house mouse. Springer Verlag, Berlin Heidelberg New York
Trapp BD, Honegger P, Richelson E, Webster HdeF (1979) Morphological differentiation of mechanically dissociated fetal rat brain in aggregating cell cultures. Brain Res 160:117–130
Willinger M, Schachner M (1980) GM1-ganglioside as a marker for neuronal differentiation in mouse cerebellum. Dev Biol 74:101–117
Wood BT, Thompson SH, Goldstein G (1965) Fluorescent antibody staining. J Immunol 95:225–229
Zwerner RK, Acton RT, Seeds NW (1977) The developmental appearance of Thy-1 in mouse reaggregating brain cell cultures. Dev Biol 60:331–335
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lindner, J., Schachner, M. Immunocytochemical localization of cell type-specific markers in reaggregating cell cultures of mouse cerebellum. Cell Tissue Res. 227, 677–690 (1982). https://doi.org/10.1007/BF00204797
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00204797