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Cells positive for the O4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes
Developmental Brain Research, 46 (1989) 115-122
115
Elsevier BRD50877
Cells positive for the 0 4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes Jacqueline Trotter and Melitta Schachner Department of Neurobiology, University of Heidelberg, Heidelberg (F.R. G. ) (Accepted 18 October 1988)
Key words: Oligodendrocyte; Astrocyte; Differentiation; Antigen 04; Cell sorting
Cells expressing the surface antigen 04 were isolated as pure populations from cultures of murine brain or cerebellum using fluorescence activated cell sorting. When these O4-positive cells were further cultured in the presence of fetal calf serum (FCS) many ceils expressed both 04 and the astrocyte marker glial fibrillary acidic protein (GFAP) after 4 days of culture. Cells not exposed to FCS expressed 04, but never GFAP. GFAP-positive cells in the presence of fetal calf serum very rarely expressed the myelin associated glycoprotein (MAG) or O1, both of which are expressed on more differentiated oligodendrocytes, and never expressed O10 that is characteristic of even more mature oligodendrocytes. These results show that glial cells expressing 04, but not MAG, O1, O10 or GFAP are bipotential precursor cells able to differentiate into astrocytes or oligodendrocytes depending on the culture conditions and suggest that bipotentiality of glial precursor cells is retained up to a later developmental stage than that of the O2A progenitor cell.
INTRODUCTION The differentiation pathways for neural cells and the stages at which different cell lineages diverge, such as those of glia and neurones, are poorly defined. Most progress has been made in studying differentiation of the two main glial cell types in the central nervous system, the oligodendrocytes and astrocytes, in a relatively simple area, the optic nerve, where only glial cell bodies are present 7,14,26. Here it was shown that a subclass of astrocytes (type 2) and oligodendrocytes appear to share a c o m m o n precursor cell which could be recognised by the A2B5 antibody 6. This precursor cell was shown to differentiate into glial fibrillary acidic protein (GFAP)-positive astrocytes in the presence of fetal calf serum or into galactocerebroside-positive oligodendrocytes in serum-free medium 25. Whether these observations hold true for other brain regions and other species has, however, not been studied in detail. Furthermore, it is not known up to which time point along a
differentiation pathway cells retain a developmental choice. The present study was undertaken to analyse more accurately the developmental stage at which precursor cells from different brain regions are able to still differentiate into oligodendrocytes and astro-
cytes. In general, it is not possible to study cell lineage relationships in heterogeneous cell cultures and thus homogeneous populations of neural cells of a particular developmental stage, defined by their expression of surface markers, would be useful for the precise assessment of their differentiation potential. Murine oligodendrocytes have been identified in cell culture by their expression of several cell surface components, designated as O antigens and recognised by monoclonal antibodies 1°,16,17,22. The antibodies 0 4 , O1 and O10 define increasingly more mature differentiation stages of oligodendrocytes, 0 4 being expressed on the least differentiated cells which then additionally acquire expression of O1 and O 1 0 1 0 ' 2 3 . In this paper we show that O4-positive cells from mu-
Correspondence: J. Trotter, Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 6900 Heidelberg,
F.R.G.
0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
116 rine cerebellum or whole brain isolated by fluorescence-activated cell sorting (FACS) exhibit a developmental plasticity in that they are able to mature both along the oligodendrocyte lineage into O1- and O10-positive oligodendrocytes or along the astrocyte lineage into GFAP-positive astrocytes.
ated spontaneously during the first days of culture. Cells were harvested after 4-5 days in culture for cerebellum or 7-9 days for whole brain using 0.05% trypsin and 0.02% EDTA and immunostained for analysis or for fluorescence activated cell sorting (see below).
MATERIALS AND METHODS
Fluorescence activated cell sorting and analysis Cells were harvested from the Petri dishes, immediately added to a solution of soybean trypsin inhibitor (Serva, Heidelberg) at a final concentration of 1 mg/ml and collected by centrifugation at 1200 g for 10 min at 4 °C. After resuspension in culture medium and filtration through a Nylon gauze (130 pm pore size, Eckert, Waldkirch, F.R.G.) cells were again collected by centrifugation and resuspended at approximately 7 × 106 cells/ml in culture medium containing 10 mM HEPES as buffer (HEPES culture medium). Primary antibodies were then added for 20 rain on ice. The cell suspension was underlaid with about 1 ml of fetal calf serum (FCS) and centrifuged to pellet the cells. Cells were resuspended in 0.5 ml HEPES culture medium and again washed over a cushion of FCS. Cell pellets were finally resuspended at 7 x 106 cells/ml in HEPES culture medium and the second antibody incubated with the cells for 20 min on ice. The washing procedures were repeated and cells resuspended for analysis or sorting in the fluorescence-activated cell sorter at 5 × 10 6 cells/ml. At the last step propidium iodide (3 ~M) to label dead cells and DNase (10 ~g/ml, Serva, Heidelberg, F.R.G.) to prevent cell clumping induced by DNA released from dead cells were added to the cell suspension. Dead cells were distinguished in a distinct channel on the cell sorter and excluded from the analysis or sort. Negative controls consisted of cells stained with second antibody alone. These controls were used to determine the sorting windows and to eliminate weakly stained macrophages. For analysis or cell sorting an Ortho system 50-H equipped with a Diagnostic System 2150 (Ortho Instruments, Westwood, U.S.A.) was used. For analysis, 20,000-40,000 events were examined per experimental sample. Forward light scatter was plotted against fluorescein (FITC) fluorescence. The area outside the background fluorescence was determined and 70% of this area, which included the brightest fluorescent cells was taken as the antigen-positive
Antibodies Monoclonal antibodies 04, O1 and O1010'17'22 were used as dialysed ammonium sulphate fractions of ascites fluids or serum-free hybridoma supernatants. Rabbit antiserum to human glial fibrillary acidic protein (GFAP) 2 was a kind gift of Dr. L. Eng, Stanford University, U.S.A. Rabbit antiserum and a monoclonal antibody (513) to the myelin-associated glycoprotein (MAG) recognising extracellularly localised epitopes on MAG 13 were also used. To confirm the absence of neurones rabbit antiserum to L115 was used. Fluorescein- or rhodamine-conjugated second antibodies to mouse or rabbit IgG were obtained from Dakopatts (Hamburg, F.R.G.) or Cappel (Paesel, Frankfurt, F.R.G.). For double-immunofluorescence labelling with 513, 04, O1 and O10 antibodies cells were stained by goat anti-mouse IgG (fluorescein-conjugated; FITC). Antibodies to GFAP or MAG were visualised by goat anti-rabbit IgG (tetramethyl-rhodamine-conjugated, TRITC) second antibodies. For sorting, anti-mouse IgG (fluoresceincoupled) antibodies from Dakopatts were used. Cell culture Monolayer cultures of cerebellar cells from 6- to 8day-old or whole brains from 0- to 2-day-old NMRI mice were prepared and plated onto poly-L-lysinecoated Petri dishes (10 crn in diameter). Cells prepared from 12 cerebella (yield approximately 8 × 107 cells) or 12 whole brains (yield approximately 1 × 108 cells) were distributed into 4 Petri dishes in Basal medium Eagle's (BME) containing 10% horse serum, penicillin and streptomycin (culture medium) as described 1s,27. After 3-4 days the neurones in the cerebellar cultures were immunocytolysed by incubation with M5 antibody in the presence of guinea pig complement 9. In cultures from whole brain neurones did not survive the preparation procedure and degener-
117 fraction for sorting. The negative fraction consisted of the cells within the background fluorescence. For cell sorting, the cell suspension was stirred continuously at very slow speed with a small magnetic flea in a small plastic vial in an ice-cooled chamber to prevent cell clumping. The outlet tube from the cell suspension consisted of a small glass funnel over which a Nylon filter (47 ~m pore size, Eckert, Waldkirch, F.R.G.) was stretched. Ceils were sorted at a flow rate of 200-500 cells/s using a nozzle diameter of 76 /~m and phosphate-buffered saline, pH 7.2, as sheath fluid. The sorted positive and negative fractions were collected on ice in 100% FCS. Samples from each fraction were monitored continuously for purity in a fluorescence microscope. After sorting, cells were collected by centrifugation, resuspended in a small volume of culture medium containing 10% FCS and plated at 10-20 x 103 cells/coverslip (7 mm in diameter). They were left overnight at 37 °C in a 5% CO 2 incubator. Coverslips were then flooded with serumfree medium (Raft's modification of Bottenstein and Sato's serum-free medium containing Dulbecco's modified Eagle's medium, 0.2% (w/v) sodium bicarbonate, 0.01 mg/ml insulin, 0.1 mg/ml transferrin, 2 mM glutamine, 200 nM progesterone, 100/~M putrescine, 220 nM sodium selenite, 400 nM triiodothyronine, 500 nM thyroxine, and 0.025 mg/ml gentamycin sulphate). After four to five days in culture coverslips were stained by indirect immunofluorescence TM. When cells are sorted on the basis of 0 4 positivity the losses both during the sorting and after plating are considerable suggesting that this method is not suitable for the bulk isolation of oligodendrocytes in quantity for biochemical analysis. This loss may be due to several reasons: oligodendrocytes are fairly large, process bearing cells and the shear forces encountered during the sorting are unfavourable for the survival of such cells. Thus, the cells that survive best are probably the less differentiated O4-positive cells that have fewer processes. When we sorted with A2B5 antibodies 6 and obtained glial precursor cells, oligodendrocytes and type 2 astrocytes from neurone-free cultures of neonatal or embryonic brain, losses were much lower probably because these cells are more robust, since they lack elaborate processes. Also, these cells may divide further in culture. Abney et al.1 separated A2B5-positive cells from embryonic
rats by cell sorting with high yield. In addition, St. John et al. 24 showed that cells could be sorted from embryonic rat or mouse CNS with good viability and yield but very few viable cells could be recovered from late embryonic or early postnatal tissue. Thus cell sorting can be successfully used to separate populations of neural cells provided that they lack extensive processes. Furthermore, the purity of sorted cells depends on their proliferation relative to contaminating cells. Although the O4-positive sorted fraction is very pure (>99% of all cells express O4), a single contaminating astrocyte divides rapidly in culture and such cells thus represent an appreciable contamination if the oligodendrocytes are cultured for longer periods before analysis by staining. RESULTS Prior to fluorescence activated cell sorting the percentage of 0 4 antigen-positive cells was determined in 7- to 9-day-old cultures of 0- to 2-day-old brain or 4-day-old cultures of 6- to 8-day-old cerebella after elimination of neurones by immunocytolysis. Approximately 8% of all cells from total brain expressed 0 4 (Table I). The percentage of O4-positive cells from cerebellum after removal of neurones was higher: up to 25% of all cells were 0 4 antigen-positive and 17% expressed in addition the O1 antigen (Table I). When the 0 4 antigen-positive cells were isolated by FACS using monoclonal antibodies against 0 4 as surface marker, cell losses were very high (approximately 50% of the input cells) and viability of sorted cells was rather low, so that only 5-10% of all cells recovered in the sorted fractions survived in culture one day after plating. However, TABLE I Percentage of cells that express 04 and 01 antigens in cultures of neonatal murine brain or cerebellum from 6- to 8-day-old mice after 4 days in vitro
The results represent the mean of 3 experiments. Twenty to forty thousand events were analysed per sample on the cell sorter. DIV, days in vitro; P6-P8, postnatal day 6-8. Culture
% 04-positive
% O l-positive
neonatal brain (7-9 DIV) P6-P8 cerebella* (4 DIV)
8 __+.4 25 +_4
4+ 1 17 + 2
* Neurones were killed with M5 antibody plus complementafter 3 days in culture.
118
Fig. 1. Indirect immunofluorescence of the 0 4 antigen-positive cell fraction isolated by fluorescence activated cell sorting from early postnatal mouse brain after maintenance in culture for 4 days using 0 4 (a) and O10 (c) antibodies, b, d are the corresponding phase contrast micrographs of fluorescence images a, c, respectively. Bar = 20/~m.
Fig. 2. Double immunofluorescence of 0 4 antigen-positive fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using 0 4 (a,d) and polyclonal GFAP (b,e) antibodies, c, f, are the corresponding phase contrast micrographs to fluorescence images a, b, d, e, respectively. Bar = 10~tm (a-c); and 20~tm (d-f).
119 the sorted O4-positive cells were very pure ( > 9 9 % ) at the time of plating as revealed by fluorescence microscopy. Prior to sorting none of the 0 4 antigenpositive cells expressed G F A P (data not shown). Most of the cells in the positively selected fraction had a morphology typical of differentiated oligodendrocytes with many interconnected processes extended in a halo around the cell body (Fig. 1). Four days after plating, in addition to expressing 0 4 antigens (Fig. la,b), many of these cells expressed the oligodendrocyte markers O1 (Fig. 4a,c), O10 (Figs. lc,d and 4d,f) and myelin associated glycoprotein ( M A G ) (not shown). W h e n cells in the O4-positive fraction were stained after 4 days for G F A P , approximately 1 0 - 2 0 % of all 0 4 antigen-positive cells also expressed G F A P as seen by double-immunolabelling (Fig. 2a-f). These G F A P - and O4-positive cells had several processes. Such double-immunolabelled cells were also rarely seen ( < 1 % of all cells) in the negatively selected cell fraction (Fig. 3a-c). Often, G F A P immunoreactivity was seen confined to the cell body and larger processes whereas 0 4 immunoreactivity extended into the fine cellular processes (Fig. 2d-f). The percentage of cells positive for both 0 4 and G F A P varied greatly between experiments. However, the percentage of double-immunolabelled cells in the O4-positive sorted fraction was usually between 10 and 20% and in one sort as many as 60% of all O4-positive cells expressed G F A P (Table II).
TABLE II The percentage of 04-, 01-, 010- and MAG-positive cells that are double-labelled with antibodies to G FA P
Cells were isolated by cell sorting using the 04 antibody from P6-P8 cerebella after 4 days in vitro and elimination of neurones. The O4-positive fraction was cultured for 4 days in medium and the cells were stained with antibodies to one of the 'O' markers, or MAG and also with antibodies against GFAP as described in Materials and Methods. The results shown are the percentage of each 'O' stained group or MAG-positive cells that express GFAP. For each experiment several hundred cells were counted and the results given indicate the range over 5 different experiments. Antibody
% of cells co-expressing GFAP
04 O1 MAG O10
10-60% <1% <1% 0
Fig. 3. Double immunofluorescence of cells in the O4-negative fraction isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using 04 (a) and polyclonal GFAP (b) antibodies, c is the phase contrast micrograph to fluorescence images a, b. Bar = 10~m.
O4-positive cells cultured in the absence of FCS were never seen to co-express G F A P . When cultures were double-labelled with antibodies to O1 and G F A P a double-labelled cell was only very seldom seen (Fig. 4 a - c , Table II). G F A P was never detectable in O10-positive cells (Fig. 4 d - f ,
120
Fig. 4. Double immunofluorescence of 04 antigen-positive fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using O1 (a), O10 (d) and polyclonal GFAP (b,e) antibodies, c, f represent the corresponding phase contrast micrographs to fluorescence images a, b and d, e, respectively. Bar ~ 10~tm(a-c); and 20pm (d-f).
Table II). Oligodendrocytes expressing M A G rarely also expressed G F A P (Fig. 5a-c, Table II). DISCUSSION In this study we show that O4-positive cells are able to differentiate into GFAP-positive astrocytes and that this differentiation does not depend on the presence of neurones. O4-positive cells have previously been shown to be the precursor cells to O1positive oligodendrocytes in culture z3 and therefore belong to the oligodendroeytic lineage. However, we could show in this study that O4-positive cells not only give rise to oligodendrocytes, but can also differentiate into astrocytes when cultured in the presence, but not in the absence of FCS. FCS thus induces GFAP expression in O4-positive cells rather than selects for the survival of cells expressing the two markers, since O4-positive cells were never seen to express GFAP prior to sorting. These features are characteristic of progenitor :ells which have been observed in the rat optic nerve to give rise to both oligodendrocytes and type 2 astrocytes 7'14'2°'25'26. These so-called O2A progenitor cells express the A2B5 antigen and tetanus toxin receptors, both of which later cease to be expressed on more differentiated oligodendrocytes 5'19'27. A partial
overlap in expression of 04, A2B5 and tetanus toxin receptors has been seen 5A9'27. The fact that the more differentiated 0 4 antigen-positive glial precursors are able to differentiate both into astrocytes and oligodendrocytes thus suggests that developmentally more advanced glial precursors retain bipotentiality and differentiate according to the culture conditions, as has been observed previously for the A2B5 and tetanus toxin receptor-positive O2A progenitor cells. Our study has also shown that cells expressing markers characteristic of the more differentiated oligodendrocytes, i.e., O1, MAG and O10 (in that order of differentiation) cease to show this bipotentiality, because they have extremely rarely, or in the case of the O10 antigen, never been observed to express GFAP. Thus, with the expression of O1 the developmental plasticity and ability to differentiate into GFAP-positive astrocytes is lost. It is not clear, however, whether the expression of GFAP in these O4positive cells commits them irreversibly to the astrocyte lineage or if they can still differentiate along the oligodendrocyte lineage, after loss of GFAP. Bipotential O4-positive cells have been reported for the rat optic nerve 21, but not described in experimental detail. Also, Levi et al. 12have recently shown that in heterogeneous cultures of rat cerebellum containing neurones and astrocytes some cells express
121
t~
both 0 4 antigen and GFAP in the presence of FCS and have hypothesised that these cells may also develop from a bipotential A2B5-positive precursor 11. The O4-positive population in heterogeneous cultures of rat optic nerve has previously been shown to represent a distinct differentiation stage which is slowly proliferating and dependent on the presence of insulin and/or neuronal factors for further specialisation along the oligodendrocyte lineage 5. An important observation of our study is that the differentiation into O1- and O10-positive oligodendrocytes or GFAP-positive astrocytes from O4-positive precursor cells occurs in the absence of neurones. In addition, it is likely that it does not depend on the influence of astrocytes either, since the proportion of astrocytes in the sorted O4-positive fraction after 4 days is very small (less than 10% of all cells). Since previous experiments on the differentiation potential of O4-positive cells were always carried out in the presence of other cell types, our study is the first to show this independence of differentiation. It is likely that O4-positive precursor cells are induced to become astrocytes in vivo by factors that are mimicked in culture by FCS. The cellular source of this factor in vivo and the regulation of its production and effects still remain to be clarified. A 25,000 Da protein can be isolated from rat optic nerve which induces the differentiation of O2A progenitor cells in vitro along the astrocyte lineage 8. It remains to be clarified if the protein isolated from the optic nerve also regulates commitment to the astrocyte lineage in other brain regions and which signals regulate the constitutive differentiation along the oligodendrocyte lineage in vivo 3'4.
ACKNOWLEDGEMENTS
Fig. 5. Double immunofluorescenceof 04 antigen-negative fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using monoclonal 513 MAG (a) and polyclonal GFAP (b) antibodies, c represents the phase contrast micrograph to fluorescence images a andb. Bar = 10/~m.
The authors are grateful to I. Biinzli-Ehret and D. Kendel for excellent technical assistance, to K. Hexel and M. St6hr for skillful operation of the cell sorter, and to the Gemeinntitzige Hertie-Stiftung and Bundesministerium fiir Forschung und Technologie for support.
122 REFERENCES 1 Abney, E.R., Williams, B.P. and Raft, M.C., Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence activated cell sorting and cell culture, Dev. Biol., 100 (1983) 166-171. 2 Bignami, A., Eng, L.F., Dahl, D. and Uyeda, C.T., Localisation of the g!ial fibrillary acidic protein in astrocytes by immunofluorescence, Brain Res., 43 (1972) 429-435. 3 Choi, B.H. and Kim, R.C., Expression of the glial fibrillary acidic protein in immature oligodendroglia, Science, 233 (1984) 407-408. 4 Choi, B.H., Kim, R.C. and Lapham, L.W., Do radial glia give rise to both astroglial and oligodendroglial cells?, Dev. Brain Res., 8 (1983) 119-130. 5 Dubois-Dalq, M., Characterisation of a slowly proliferative cell along the oligodendrocyte differentiation pathway, EMBOJ., 6 (1987) 2587-2595. 6 Eisenbarth, G.S., Walsh, F. and Nirenberg, M., Monoclonal antibody to a plasma membrane antigen of neurones, Proc. Natl. Acad. Sci. U.S.A., 76 (1979) 4913-4917. 7 ffrench-Constant, C. and Raft, M.C., Proliferating bipotential glial cells in adult optic nerve, Nature (Lond.), 319 (1986) 499-502. 8 Hughes, S.M. and Raff, M.C., An inducer protein may control the timing of fate switch in a bipotential glial progenitor cell in rat optic nerve, Development, 101 (1987) 157-167. 9 Keilhauer, G., Faissner, A. and Schachner, M., Differential inhibition of neurone-neurone, neurone-astrocyte and astrocyte-astrocyte adhesion by L1, L2 and N-CAM antibodies, Nature (Lond.), 316 (1985) 728-730. 10 Kuhlmann-Krieg, S., Sommer, I. and Schachner, M., Ultrastructural features of cultured oligodendrocytes expressing stage specific cell surface antigens, Dev. Brain Res., 39 (1988) 281-293. 11 Levi, G., Gallo, V. and Ciotti, M.T., Bipotential precursors of putative fibrous astrocytes and oligodendrocytes in rat cerebellar cultures express distinct features and 'neurone-like' gamma-aminobutyric acid transport, Proc. Natl. Acad. Sci. U.S.A., 83 (1986) 1504-1508. 12 Levi, G., Aloisi, F. and Wilkin, G.P., Differentiation ofcerebellar bipotential glial precursors into oligodendrocytes in primary culture: developmental profile of surface antigens and mitotic activity, J. Neurosci. Res., 18 (1987) 407-417. 13 Poltorak, M., Sadoul, R., Keilhauer, G., Landa, C., Fahrig, T. and Schachner, M., Myelin associated glycoprotein, a member of the L2/HNK-1 family of neural cell adhesion molecules is involved in neurone-oligodendrocyte and oligodendrocyte-oligodendrocyte interaction, J. Cell Biol., 105 (1987) 1893-1895.
14 Raft, M.C., Miller, R.H. and Noble, M., A glial progenitor cell that develops into an astrocyte or an oligodendrocyte depending on the culture medium, Nature (Lond.), 303 (1983) 390-396. 15 Rathjen, F.G. and Schachner, M., Immunocytological and biochemical characterization of a new neuronal cell surface component (L1 antigen) which is involved in cell adhesion, EMBOJ., 3 (1984) 1-10. 16 Schachner, M., Cell-type specific antigens in the mammalian central nervous system, J. Neurochem., 39 (1982) 1-8. 17 Schachner, M., Kim, S.K. and Zehnle, R., Developmental expression in central and peripheral nervous system of oligodendrocyte cell surface antigens (O antigens) recognized by monoclonal antibodies, Dev. BioL, 83 (1981) 328-338. 18 Schnitzer, J. and Schachner, M., Expression of Thy-1, H-2 and NS-1 cell surface antigens and tetanus toxin receptors in early postnatal and adult mouse cerebellum, J. Neuroimmunol., 1 (1981) 429-456. 19 Schnitzer, J. and Schachner, M., Cell type specificity of a neuronal cell surface antigen recognised by the monoclonal antibody A2B5, Cell Tissue Res., 224 (1982) 625-636. 20 Small, R.K., Riddle, P. and Noble, M., Evidence for migration of oligodendrocyte type 2 astrocyte progenitor cells into the developing rat optic nerve, Nature (Lond.), 328 (1987) 155-157. 21 Sommer, I. and Noble, M., Plasticity and commitment in oligodendrocyte development, Soc. Neurosci. A bstr. , (1986) 427.13. 22 Sommer, I. and Schachner, M., Monoclonal antibodies (O1 to 04) to oligodendrocyte cell surfaces: an immunological study in the central nervous system, Dev. Biol., 83 (1981) 311-327. 23 Sommer, I. and Schachner, M., Cells that are 04 antigenpositive and O1 antigen-negative differentiate into O1 antigen-positive oligodendrocytes, Neurosci. Lett., 29 (1982) 183-188. 24 St. John, P.A., Kell, W.M., Mazzetta, J.S., Lange, G.D. and Barker, J.L., Analysis and isolation of embryonic mammalian neurones by fluorescence activated cell sorting, J. Neurosci., 6 (1986) 1492-1512. 25 Temple, S. and Raft, M.C., Differentiation of a bipotential glial progenitor cell in single cell microculture, Nature (Lond.), 313 (1985) 223-225. 26 Temple, S. and Raft, M.C., Clonal analysis of oligodendrocyte development in culture: evidence for a developmental clock that counts cell divisions, Cell 44 (1986) 773-779. 27 Trotter, J., Bitter-Suermann, D. and Schachner, M., Differentiation-regulated loss of the polysialylated embryonic form and expression of the different polypeptides of the neural cell adhesion molecule (N-CAM) by cultured oligodendrocytes and myelin, J. Neurosci. Res., in press.
115
Elsevier BRD50877
Cells positive for the 0 4 surface antigen isolated by cell sorting are able to differentiate into astrocytes or oligodendrocytes Jacqueline Trotter and Melitta Schachner Department of Neurobiology, University of Heidelberg, Heidelberg (F.R. G. ) (Accepted 18 October 1988)
Key words: Oligodendrocyte; Astrocyte; Differentiation; Antigen 04; Cell sorting
Cells expressing the surface antigen 04 were isolated as pure populations from cultures of murine brain or cerebellum using fluorescence activated cell sorting. When these O4-positive cells were further cultured in the presence of fetal calf serum (FCS) many ceils expressed both 04 and the astrocyte marker glial fibrillary acidic protein (GFAP) after 4 days of culture. Cells not exposed to FCS expressed 04, but never GFAP. GFAP-positive cells in the presence of fetal calf serum very rarely expressed the myelin associated glycoprotein (MAG) or O1, both of which are expressed on more differentiated oligodendrocytes, and never expressed O10 that is characteristic of even more mature oligodendrocytes. These results show that glial cells expressing 04, but not MAG, O1, O10 or GFAP are bipotential precursor cells able to differentiate into astrocytes or oligodendrocytes depending on the culture conditions and suggest that bipotentiality of glial precursor cells is retained up to a later developmental stage than that of the O2A progenitor cell.
INTRODUCTION The differentiation pathways for neural cells and the stages at which different cell lineages diverge, such as those of glia and neurones, are poorly defined. Most progress has been made in studying differentiation of the two main glial cell types in the central nervous system, the oligodendrocytes and astrocytes, in a relatively simple area, the optic nerve, where only glial cell bodies are present 7,14,26. Here it was shown that a subclass of astrocytes (type 2) and oligodendrocytes appear to share a c o m m o n precursor cell which could be recognised by the A2B5 antibody 6. This precursor cell was shown to differentiate into glial fibrillary acidic protein (GFAP)-positive astrocytes in the presence of fetal calf serum or into galactocerebroside-positive oligodendrocytes in serum-free medium 25. Whether these observations hold true for other brain regions and other species has, however, not been studied in detail. Furthermore, it is not known up to which time point along a
differentiation pathway cells retain a developmental choice. The present study was undertaken to analyse more accurately the developmental stage at which precursor cells from different brain regions are able to still differentiate into oligodendrocytes and astro-
cytes. In general, it is not possible to study cell lineage relationships in heterogeneous cell cultures and thus homogeneous populations of neural cells of a particular developmental stage, defined by their expression of surface markers, would be useful for the precise assessment of their differentiation potential. Murine oligodendrocytes have been identified in cell culture by their expression of several cell surface components, designated as O antigens and recognised by monoclonal antibodies 1°,16,17,22. The antibodies 0 4 , O1 and O10 define increasingly more mature differentiation stages of oligodendrocytes, 0 4 being expressed on the least differentiated cells which then additionally acquire expression of O1 and O 1 0 1 0 ' 2 3 . In this paper we show that O4-positive cells from mu-
Correspondence: J. Trotter, Department of Neurobiology, University of Heidelberg, Im Neuenheimer Feld 364, 6900 Heidelberg,
F.R.G.
0165-3806/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division)
116 rine cerebellum or whole brain isolated by fluorescence-activated cell sorting (FACS) exhibit a developmental plasticity in that they are able to mature both along the oligodendrocyte lineage into O1- and O10-positive oligodendrocytes or along the astrocyte lineage into GFAP-positive astrocytes.
ated spontaneously during the first days of culture. Cells were harvested after 4-5 days in culture for cerebellum or 7-9 days for whole brain using 0.05% trypsin and 0.02% EDTA and immunostained for analysis or for fluorescence activated cell sorting (see below).
MATERIALS AND METHODS
Fluorescence activated cell sorting and analysis Cells were harvested from the Petri dishes, immediately added to a solution of soybean trypsin inhibitor (Serva, Heidelberg) at a final concentration of 1 mg/ml and collected by centrifugation at 1200 g for 10 min at 4 °C. After resuspension in culture medium and filtration through a Nylon gauze (130 pm pore size, Eckert, Waldkirch, F.R.G.) cells were again collected by centrifugation and resuspended at approximately 7 × 106 cells/ml in culture medium containing 10 mM HEPES as buffer (HEPES culture medium). Primary antibodies were then added for 20 rain on ice. The cell suspension was underlaid with about 1 ml of fetal calf serum (FCS) and centrifuged to pellet the cells. Cells were resuspended in 0.5 ml HEPES culture medium and again washed over a cushion of FCS. Cell pellets were finally resuspended at 7 x 106 cells/ml in HEPES culture medium and the second antibody incubated with the cells for 20 min on ice. The washing procedures were repeated and cells resuspended for analysis or sorting in the fluorescence-activated cell sorter at 5 × 10 6 cells/ml. At the last step propidium iodide (3 ~M) to label dead cells and DNase (10 ~g/ml, Serva, Heidelberg, F.R.G.) to prevent cell clumping induced by DNA released from dead cells were added to the cell suspension. Dead cells were distinguished in a distinct channel on the cell sorter and excluded from the analysis or sort. Negative controls consisted of cells stained with second antibody alone. These controls were used to determine the sorting windows and to eliminate weakly stained macrophages. For analysis or cell sorting an Ortho system 50-H equipped with a Diagnostic System 2150 (Ortho Instruments, Westwood, U.S.A.) was used. For analysis, 20,000-40,000 events were examined per experimental sample. Forward light scatter was plotted against fluorescein (FITC) fluorescence. The area outside the background fluorescence was determined and 70% of this area, which included the brightest fluorescent cells was taken as the antigen-positive
Antibodies Monoclonal antibodies 04, O1 and O1010'17'22 were used as dialysed ammonium sulphate fractions of ascites fluids or serum-free hybridoma supernatants. Rabbit antiserum to human glial fibrillary acidic protein (GFAP) 2 was a kind gift of Dr. L. Eng, Stanford University, U.S.A. Rabbit antiserum and a monoclonal antibody (513) to the myelin-associated glycoprotein (MAG) recognising extracellularly localised epitopes on MAG 13 were also used. To confirm the absence of neurones rabbit antiserum to L115 was used. Fluorescein- or rhodamine-conjugated second antibodies to mouse or rabbit IgG were obtained from Dakopatts (Hamburg, F.R.G.) or Cappel (Paesel, Frankfurt, F.R.G.). For double-immunofluorescence labelling with 513, 04, O1 and O10 antibodies cells were stained by goat anti-mouse IgG (fluorescein-conjugated; FITC). Antibodies to GFAP or MAG were visualised by goat anti-rabbit IgG (tetramethyl-rhodamine-conjugated, TRITC) second antibodies. For sorting, anti-mouse IgG (fluoresceincoupled) antibodies from Dakopatts were used. Cell culture Monolayer cultures of cerebellar cells from 6- to 8day-old or whole brains from 0- to 2-day-old NMRI mice were prepared and plated onto poly-L-lysinecoated Petri dishes (10 crn in diameter). Cells prepared from 12 cerebella (yield approximately 8 × 107 cells) or 12 whole brains (yield approximately 1 × 108 cells) were distributed into 4 Petri dishes in Basal medium Eagle's (BME) containing 10% horse serum, penicillin and streptomycin (culture medium) as described 1s,27. After 3-4 days the neurones in the cerebellar cultures were immunocytolysed by incubation with M5 antibody in the presence of guinea pig complement 9. In cultures from whole brain neurones did not survive the preparation procedure and degener-
117 fraction for sorting. The negative fraction consisted of the cells within the background fluorescence. For cell sorting, the cell suspension was stirred continuously at very slow speed with a small magnetic flea in a small plastic vial in an ice-cooled chamber to prevent cell clumping. The outlet tube from the cell suspension consisted of a small glass funnel over which a Nylon filter (47 ~m pore size, Eckert, Waldkirch, F.R.G.) was stretched. Ceils were sorted at a flow rate of 200-500 cells/s using a nozzle diameter of 76 /~m and phosphate-buffered saline, pH 7.2, as sheath fluid. The sorted positive and negative fractions were collected on ice in 100% FCS. Samples from each fraction were monitored continuously for purity in a fluorescence microscope. After sorting, cells were collected by centrifugation, resuspended in a small volume of culture medium containing 10% FCS and plated at 10-20 x 103 cells/coverslip (7 mm in diameter). They were left overnight at 37 °C in a 5% CO 2 incubator. Coverslips were then flooded with serumfree medium (Raft's modification of Bottenstein and Sato's serum-free medium containing Dulbecco's modified Eagle's medium, 0.2% (w/v) sodium bicarbonate, 0.01 mg/ml insulin, 0.1 mg/ml transferrin, 2 mM glutamine, 200 nM progesterone, 100/~M putrescine, 220 nM sodium selenite, 400 nM triiodothyronine, 500 nM thyroxine, and 0.025 mg/ml gentamycin sulphate). After four to five days in culture coverslips were stained by indirect immunofluorescence TM. When cells are sorted on the basis of 0 4 positivity the losses both during the sorting and after plating are considerable suggesting that this method is not suitable for the bulk isolation of oligodendrocytes in quantity for biochemical analysis. This loss may be due to several reasons: oligodendrocytes are fairly large, process bearing cells and the shear forces encountered during the sorting are unfavourable for the survival of such cells. Thus, the cells that survive best are probably the less differentiated O4-positive cells that have fewer processes. When we sorted with A2B5 antibodies 6 and obtained glial precursor cells, oligodendrocytes and type 2 astrocytes from neurone-free cultures of neonatal or embryonic brain, losses were much lower probably because these cells are more robust, since they lack elaborate processes. Also, these cells may divide further in culture. Abney et al.1 separated A2B5-positive cells from embryonic
rats by cell sorting with high yield. In addition, St. John et al. 24 showed that cells could be sorted from embryonic rat or mouse CNS with good viability and yield but very few viable cells could be recovered from late embryonic or early postnatal tissue. Thus cell sorting can be successfully used to separate populations of neural cells provided that they lack extensive processes. Furthermore, the purity of sorted cells depends on their proliferation relative to contaminating cells. Although the O4-positive sorted fraction is very pure (>99% of all cells express O4), a single contaminating astrocyte divides rapidly in culture and such cells thus represent an appreciable contamination if the oligodendrocytes are cultured for longer periods before analysis by staining. RESULTS Prior to fluorescence activated cell sorting the percentage of 0 4 antigen-positive cells was determined in 7- to 9-day-old cultures of 0- to 2-day-old brain or 4-day-old cultures of 6- to 8-day-old cerebella after elimination of neurones by immunocytolysis. Approximately 8% of all cells from total brain expressed 0 4 (Table I). The percentage of O4-positive cells from cerebellum after removal of neurones was higher: up to 25% of all cells were 0 4 antigen-positive and 17% expressed in addition the O1 antigen (Table I). When the 0 4 antigen-positive cells were isolated by FACS using monoclonal antibodies against 0 4 as surface marker, cell losses were very high (approximately 50% of the input cells) and viability of sorted cells was rather low, so that only 5-10% of all cells recovered in the sorted fractions survived in culture one day after plating. However, TABLE I Percentage of cells that express 04 and 01 antigens in cultures of neonatal murine brain or cerebellum from 6- to 8-day-old mice after 4 days in vitro
The results represent the mean of 3 experiments. Twenty to forty thousand events were analysed per sample on the cell sorter. DIV, days in vitro; P6-P8, postnatal day 6-8. Culture
% 04-positive
% O l-positive
neonatal brain (7-9 DIV) P6-P8 cerebella* (4 DIV)
8 __+.4 25 +_4
4+ 1 17 + 2
* Neurones were killed with M5 antibody plus complementafter 3 days in culture.
118
Fig. 1. Indirect immunofluorescence of the 0 4 antigen-positive cell fraction isolated by fluorescence activated cell sorting from early postnatal mouse brain after maintenance in culture for 4 days using 0 4 (a) and O10 (c) antibodies, b, d are the corresponding phase contrast micrographs of fluorescence images a, c, respectively. Bar = 20/~m.
Fig. 2. Double immunofluorescence of 0 4 antigen-positive fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using 0 4 (a,d) and polyclonal GFAP (b,e) antibodies, c, f, are the corresponding phase contrast micrographs to fluorescence images a, b, d, e, respectively. Bar = 10~tm (a-c); and 20~tm (d-f).
119 the sorted O4-positive cells were very pure ( > 9 9 % ) at the time of plating as revealed by fluorescence microscopy. Prior to sorting none of the 0 4 antigenpositive cells expressed G F A P (data not shown). Most of the cells in the positively selected fraction had a morphology typical of differentiated oligodendrocytes with many interconnected processes extended in a halo around the cell body (Fig. 1). Four days after plating, in addition to expressing 0 4 antigens (Fig. la,b), many of these cells expressed the oligodendrocyte markers O1 (Fig. 4a,c), O10 (Figs. lc,d and 4d,f) and myelin associated glycoprotein ( M A G ) (not shown). W h e n cells in the O4-positive fraction were stained after 4 days for G F A P , approximately 1 0 - 2 0 % of all 0 4 antigen-positive cells also expressed G F A P as seen by double-immunolabelling (Fig. 2a-f). These G F A P - and O4-positive cells had several processes. Such double-immunolabelled cells were also rarely seen ( < 1 % of all cells) in the negatively selected cell fraction (Fig. 3a-c). Often, G F A P immunoreactivity was seen confined to the cell body and larger processes whereas 0 4 immunoreactivity extended into the fine cellular processes (Fig. 2d-f). The percentage of cells positive for both 0 4 and G F A P varied greatly between experiments. However, the percentage of double-immunolabelled cells in the O4-positive sorted fraction was usually between 10 and 20% and in one sort as many as 60% of all O4-positive cells expressed G F A P (Table II).
TABLE II The percentage of 04-, 01-, 010- and MAG-positive cells that are double-labelled with antibodies to G FA P
Cells were isolated by cell sorting using the 04 antibody from P6-P8 cerebella after 4 days in vitro and elimination of neurones. The O4-positive fraction was cultured for 4 days in medium and the cells were stained with antibodies to one of the 'O' markers, or MAG and also with antibodies against GFAP as described in Materials and Methods. The results shown are the percentage of each 'O' stained group or MAG-positive cells that express GFAP. For each experiment several hundred cells were counted and the results given indicate the range over 5 different experiments. Antibody
% of cells co-expressing GFAP
04 O1 MAG O10
10-60% <1% <1% 0
Fig. 3. Double immunofluorescence of cells in the O4-negative fraction isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using 04 (a) and polyclonal GFAP (b) antibodies, c is the phase contrast micrograph to fluorescence images a, b. Bar = 10~m.
O4-positive cells cultured in the absence of FCS were never seen to co-express G F A P . When cultures were double-labelled with antibodies to O1 and G F A P a double-labelled cell was only very seldom seen (Fig. 4 a - c , Table II). G F A P was never detectable in O10-positive cells (Fig. 4 d - f ,
120
Fig. 4. Double immunofluorescence of 04 antigen-positive fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using O1 (a), O10 (d) and polyclonal GFAP (b,e) antibodies, c, f represent the corresponding phase contrast micrographs to fluorescence images a, b and d, e, respectively. Bar ~ 10~tm(a-c); and 20pm (d-f).
Table II). Oligodendrocytes expressing M A G rarely also expressed G F A P (Fig. 5a-c, Table II). DISCUSSION In this study we show that O4-positive cells are able to differentiate into GFAP-positive astrocytes and that this differentiation does not depend on the presence of neurones. O4-positive cells have previously been shown to be the precursor cells to O1positive oligodendrocytes in culture z3 and therefore belong to the oligodendroeytic lineage. However, we could show in this study that O4-positive cells not only give rise to oligodendrocytes, but can also differentiate into astrocytes when cultured in the presence, but not in the absence of FCS. FCS thus induces GFAP expression in O4-positive cells rather than selects for the survival of cells expressing the two markers, since O4-positive cells were never seen to express GFAP prior to sorting. These features are characteristic of progenitor :ells which have been observed in the rat optic nerve to give rise to both oligodendrocytes and type 2 astrocytes 7'14'2°'25'26. These so-called O2A progenitor cells express the A2B5 antigen and tetanus toxin receptors, both of which later cease to be expressed on more differentiated oligodendrocytes 5'19'27. A partial
overlap in expression of 04, A2B5 and tetanus toxin receptors has been seen 5A9'27. The fact that the more differentiated 0 4 antigen-positive glial precursors are able to differentiate both into astrocytes and oligodendrocytes thus suggests that developmentally more advanced glial precursors retain bipotentiality and differentiate according to the culture conditions, as has been observed previously for the A2B5 and tetanus toxin receptor-positive O2A progenitor cells. Our study has also shown that cells expressing markers characteristic of the more differentiated oligodendrocytes, i.e., O1, MAG and O10 (in that order of differentiation) cease to show this bipotentiality, because they have extremely rarely, or in the case of the O10 antigen, never been observed to express GFAP. Thus, with the expression of O1 the developmental plasticity and ability to differentiate into GFAP-positive astrocytes is lost. It is not clear, however, whether the expression of GFAP in these O4positive cells commits them irreversibly to the astrocyte lineage or if they can still differentiate along the oligodendrocyte lineage, after loss of GFAP. Bipotential O4-positive cells have been reported for the rat optic nerve 21, but not described in experimental detail. Also, Levi et al. 12have recently shown that in heterogeneous cultures of rat cerebellum containing neurones and astrocytes some cells express
121
t~
both 0 4 antigen and GFAP in the presence of FCS and have hypothesised that these cells may also develop from a bipotential A2B5-positive precursor 11. The O4-positive population in heterogeneous cultures of rat optic nerve has previously been shown to represent a distinct differentiation stage which is slowly proliferating and dependent on the presence of insulin and/or neuronal factors for further specialisation along the oligodendrocyte lineage 5. An important observation of our study is that the differentiation into O1- and O10-positive oligodendrocytes or GFAP-positive astrocytes from O4-positive precursor cells occurs in the absence of neurones. In addition, it is likely that it does not depend on the influence of astrocytes either, since the proportion of astrocytes in the sorted O4-positive fraction after 4 days is very small (less than 10% of all cells). Since previous experiments on the differentiation potential of O4-positive cells were always carried out in the presence of other cell types, our study is the first to show this independence of differentiation. It is likely that O4-positive precursor cells are induced to become astrocytes in vivo by factors that are mimicked in culture by FCS. The cellular source of this factor in vivo and the regulation of its production and effects still remain to be clarified. A 25,000 Da protein can be isolated from rat optic nerve which induces the differentiation of O2A progenitor cells in vitro along the astrocyte lineage 8. It remains to be clarified if the protein isolated from the optic nerve also regulates commitment to the astrocyte lineage in other brain regions and which signals regulate the constitutive differentiation along the oligodendrocyte lineage in vivo 3'4.
ACKNOWLEDGEMENTS
Fig. 5. Double immunofluorescenceof 04 antigen-negative fractions isolated by FACS from early postnatal mouse cerebellum after maintenance in culture for 4 days using monoclonal 513 MAG (a) and polyclonal GFAP (b) antibodies, c represents the phase contrast micrograph to fluorescence images a andb. Bar = 10/~m.
The authors are grateful to I. Biinzli-Ehret and D. Kendel for excellent technical assistance, to K. Hexel and M. St6hr for skillful operation of the cell sorter, and to the Gemeinntitzige Hertie-Stiftung and Bundesministerium fiir Forschung und Technologie for support.
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