A quantitative immunohistochemical study of macroglial cell development in the rat optic nerve: In vivo evidence for two distinct astrocyte lineages

DEVELOPMENTAL

BIOLOGY 1 I I, 35-41 (19%)

A Quantitative lmmunohistochemical Study of Macroglial Cell Development in the Rat Optic Nerve: ln Viva Evidence for Two Distinct Astrocyte Lineages ROBERT H. MILLER,' SAM DAVTD,~ RAMILLA PATEL,~ Medical

Research Council Neurm~mmunolcgy

Project, Department

ERIKA

of Zoology,

R. ABNEY,

University

Received May 16, 1984; accepted in revised .fm

March

AND MARTIN

C. RAFF

College Londma, London WCIE 6BT, England 7, 1985

We have shown previously that three antibodies-anti-galactocerebroside (GC), anti-glial fibrillary acidic protein (GFAP), and the A2B5 monoclonal antibody-can be used to help distinguish three classes of glial cells in the rat optic nerve: oligodendrocytes are GC+,GFAP-, almost all type-l astrocytcs are AZB5-,GFAP’, and almost all type-2 astrocytes are A2B5+,GFAPt. In the present study we have used these antibodies to examine the timing and sequence of the development of the three types of glial cells in viva. We show that type-l astrocytes first appear at embryonic Day 16 (E16), oligodendrocytes at birth (E21), and type-2 astrocytes between postnatal Days ‘7 and 10 (P7-10). Moreover, we demonstrate quantitatively that astrocytes in the optic nerve develop in two waves, with more than 95% of type-l astrocytes developing before P15 and more than 95% of type-2 astrocytes developing after P15. Finally, we provide indirect evidence that type-2 astrocytes do not develop from type-l astrocytes in viva, supporting previous direct evidence that the two types of astrocytes develop from two serologically distinct precursor cells in vitro. 0 1985 Academic Press, Inc.

INTRODUCTION

opment, they were hampered by the problem of distinguishing between immature cell types and failed to answer the critical question concerning the lineage relationship between the two types of glial cells: Do astrocytes and oligodendrocytes develop from separate precursor cells, or do they have a common progenitor cell? Since it is unlikely that morphological and autoradiographical studies of intact optic nerve will ever, on their own, be able to provide a definitive answer to the cell lineage question, we took a different approach. We studied gliogenesis in cultures of perinatal rat optic nerve, using antibodies to identify and manipulate the different types of glial cells and their precursors. In this way, we made two surprising observations. The first was that optic nerve cultures contain two serologically distinct types of astrocytes: whereas both types contain glial filaments composed of glial fibrillary acidic protein (GFAP) (Bignami et al, 1972), one (which we call a type-2 astrocyte) binds tetanus toxin and the A2B5 monoclonal antibody (Eisenbarth et ah, 1979), while the other (type-l astrocyte) usually does not (Raff et aZ., 1983a). The second was that the two types of astrocytes develop in vitro from two different precursor cells. Type-Z astrocytes develop from bipotential A2B5+,GFAP- progenitor cell, which differentiates into a type-2 astrocyte if cultured in 10% fetal calf serum (FCS) and into an oligodendrocyte if cultured in serumfree media (Raff et al., 198313). (The oligodendrocytes in these studies were identified using anti-galactocere-

The optic nerve develops from the optic stalk, which is an extension of the neural tube. It is an attractive part of the central nervous system (CNS) for studying gliogenesis since the neuroepithelial cells that form the optic stalk develop only into astrocytes and oligodendrocytes and not into neurons, allowing developing glial cells to be studied without the complication of having to distinguish thern from developing nerve cells. Glial cell development has been particularly well studied in the rat optic nerve, where light and electron microscopic experiments have shown that astrocytes begin to develop several (days before birth, while oligodendrocytes begin to develop several days after birth (Vaughn, 1969; Kuwabara., 1974; Skoff et al., 19’76a,b). Autoradiographic experiments following injection of r3H]thymidine have suggested that most of the astrocytes in the nerve have developed by the end of the first postnatal week, while most of the oligodendrocytes develop during the second and third postnatal weeks (Skoff et aZ., 1976a,b; Valat et ah, 1983). Although these studies have made important contributions to our understanding of astrocyte and oligodendrocyte devel’ Present address: Department of Anatomy, Case Western Reserve School of Medicine, Cleveland, Ohio 44106. ‘Present address: Division of Neurology, The Montreal General Hospital, Montreal H3G lA4, Canada. 3 Present address: Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Mass. 02139. 35

0012-1606/85 $3.00 Copyright 0 1985 by Academic Press. Inc. All rights of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

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(Difco) in Tris-buffered saline, pH 7.7, with collagenase (Worthington) added to a final concentration of 0.02% (w/v). After a second 20-min incubation in the same solution, the cells were incubated for 20 min at 37°C in 1 ml of trypsin solution and 1 ml of 0.02% (w/v) EDTA in Tris-buffered saline with collagenase. The cells were then dissociated in DNAase (Sigma, 0.04 mg/ml) and trypsin inhibitor (Sigma, 0.05 mg/ml) in Ca’+- and Mga+-free MEM by pipetting 15 times in a 2-ml graduated pipet, followed by syringing 3 times through a 25-gauge hypodermic needle on a 2-ml syringe. The resulting cell suspension was passed through a fine nylon mesh to remove debris and washed in 10 ml of MEM-Hepes containing 10% FCS and 0.2% sodium azide. The cells were counted in a hemacytometer in the presence of trypan blue. Frozen sections. Semithin frozen sections (Tokuyasu, 1973) of Pl, P7, P15, P21, and adult (>3 months) S-D rat optic nerves were prepared as previously described (Miller and Raff, 1984) after the length of the nerves was measured from just behind the eyeball to the chiasm. Briefly, the optic nerves were fixed by immersion (in Pl rats) or perfusion (in older rats) in 3% paraformaldehyde and 0.5% glutaraldehyde, cut into small pieces, and immersed in 1 M sucrose for 1 hr. The nerves were then mounted on the stub of a freezing microtome and frozen by immersion in a slush of Freon 22, which was cooled in a bath of liquid nitrogen. Semithin sections (0.25-0.5 pm) were cut using a Sorval MT2B microtome with an FTS cryoattachment on a 25-mm knife at -60 to -70°C. In some cases, optic nerves were fixed as above, dehydrated through graded alcohols, and embedded in Epon 812. Transverse, semithin sections were cut and MATERIALS AND METHODS stained with 1% toluidine blue. Immunojhn-escence labeling. All of the antibodies Cell suspensions. Pregnant Sprague-Dawley (S-D) rats were obtained from the breeding colony of the used in these studies have been previously described. Imperial Cancer Research Fund, Mill Hill. To time the Two monoclonal antibodies, A2B5 (Eisenbarth et al., pregnancies, female rats were caged with males over- 1979) and anti-GC (Ranscht, 1982), were used as ascites night and then removed: this was taken as Day 0 of fluid, diluted 1:500, except for staining semithin frozen the pregnancy. Embryos were taken from El7 to E20 sections, when A2B5 supernatant fluid was used, diluted and their ages were checked by measuring crown- 1:4. Rabbit anti-GFAP serum (Pruss, 1979) was diluted rump length (Angulo Y Gonzales, 1932) and by exam- 1:lOOO.The binding of the monoclonal antibodies was detected with a rhodamine-conjugated goat anti-mouse ining the morphology of the paws (Long and Burlingame, 1938). When there was a discrepancy between immunoglobulin (G anti-MIg-Rd, Cappel, diluted the dating by time and that by measurement and l:lOO), while the binding of the anti-GFAP serum was detected with a fluorescein-conjugated sheep anti-rabbit morphology, the latter was taken as correct. Optic nerves from E15, E16, E20, PO (the day of Ig (G anti-Rig-Fl, Wellcome, diluted l:lOO), which had birth, usually on E21), P2, P7, and PlO rats were been absorbed with mouse Ig coupled to Sepharose 4B. Cells in suspension were labeled with anti-GC antidissected from just behind the eye to the chiasm, cut body or double labeled with A2B5 and anti-GFAP into small pieces with iridectomy scissors, and incubated for 20 min at 37°C in 1 ml of minimal Eagle’s antibodies as previously described (Raff et al., 1983a). medium (MEM) with 0.02 M Hepes buffer (MEM- Semithin frozen sections were double labeled with Hepes) and an equal volume of 0.25% (w/v) trypsin A2B5 and anti-GFAP antibodies as previously described broside (GC) antibodies (Raff et al., 1978; Ranscht et al, 1982).) Type-l astrocytes, on the other hand, develop from a different precursor cell, which can be distinguished serologically from the oligodendrocyte-type-2 astrocyte (O-2A) progenitor cell as early as embryonic Day 17 (E17) in the rat optic nerve (Raff et al., 1984a). Although these experiments in culture suggested that the optic nerve contains two distinct types of astrocytes that develop as two separate cell lineages, it can be dangerous to extrapolate from findings in vitro to the situation in vivo. For this reason, we used A2B5 and anti-GFAP antibodies to study astrocytes in semithin frozen sections (Tokuyasu, 1973) of rat optic nerve. We found that the great majority of astrocytes in the interior of the adult nerve were A2B5+, while the astrocytes that formed the glial limiting membrane at the periphery of the nerve were A2B5- (Miller and Raff, 1984), suggesting that A2B5- (type-l) and A2B5+ (type-2) astrocytes are bona fide astrocyte subpopulations, which are largely segregated in the adult optic nerve. Moreover, in sections of developing nerve, type1 astrocytes were already present at birth, while type2 astrocytes only appeared during the second week of life (Miller and Raff, 1984). While the latter findings were consistent with the hypothesis that the two types of astrocytes arise from separate cell lineages, they could not exclude the possibility that type-l astrocytes converted to type-2 astrocytes by acquiring A2B5 antigen during and after the second postnatal week. The present quantitative study makes this possibility highly unlikely, thereby providing the first in vivo evidence for the two-lineage hypothesis.

MILLER ET AL.

(Miller and Raff, 1984). Cells and frozen sections were examined in a Zeiss Universal incidence fluorescence microscope and photographed using Tri-X or Ektachrome color slide film rated at 400 ASA as previously described (Raff et al., 19’79,1983a). No significant staining was observed when normal ascites fluid or normal rabbit serum was used in place of the monoclonal antibodies or anti-GFAP serum, respectively. Autoradiography. S-D rats were injected ip with 5 &i/g of [3H]thymidine (27 Ci/mmole, Amersham) on P14, P17, P20, P23, and P26. On P30, the rats were perfused and semithin frozen sections were cut and labeled with anti-GFAP antibodies, or l-pm plastic sections were cut, as described above. The sections (on glass slides) were coated with Ilford K5 or Kodak NTBB emulsion, stored at 4°C for 4-8 weeks in the dark, developed with Ilford Super Contrast FF, and examined and stored as previously described (Raff et al., 1983a). The plastic sections were stained with 1% toluidine blue for 5 min after they were developed. Attempts to double-label the semithin frozen sections with A2B5 and anti-GF.AP antibodies and to examine them by autoradiograph[y failed as the A2B5 staining did not withstand the processing. RESULTS

Cell Suspension

Cells were dissociated1 from E15, E16, E20, PO, P2, P7, and PlO optic nerves. The total number of cells was counted and the proportions of the different types of glial cells were determined by immunofluorescence: oligodendrocytes were i’dentified as GC+ cells, type-l astrocytes as A2B5-,GFAP+ cells, and type-2 astrocytes as A2B5+,GFAP+ cells; putative O-2A progenitor cells were identified as A2B5+,GFAP- cells, although not all cells with this antigenic phenotype are O-2A progenitor

Astrocyte

37

Lineages

cells; for example, from P2 onward, some are GC+ oligodendrocytes (Abney et al., 1983). As can be seen in Table 1, A2B5+,GFAP- cells were present at the youngest age examined (E15). Type-l astrocytes first appeared at E16, oligodendrocytes at PO, and type-2 astrocytes between P7 and PlO. Type-l astrocytes and A2B5+,GFAP- cells increased dramatically between El6 and PO, while a similar increase in oligodendrocytes occurred between PO and PlO. There were two worrying aspects of the cell suspension data that can be seen in Table 1. First, the number of type-l astrocytes decreased more than twofold between P2 and PlO, raising the possibility that many of these cells were not being released into suspension by our dissociation procedure in the older nerves. Second, only about 60-70% of the total cells could be identified as astrocytes, oligodendrocytes, or putative O-2A progenitor cells by antibody labeling of suspensions of PO and P2 optic nerves, and this percentage fell to about 40% in P7 and PlO nerves. While we did not attempt to identify the other cells, it seems likely that they were mainly a mixture of leptomeningeal, endothelial, and microglial cells. To circumvent these problems, and to examine the development of type-2 astrocytes in older animals, we studied semithin frozen sections of optic nerve. Unfortunately, anti-GC antibody did not label individual oligodendrocytes in such sections, so that we could not identify these cells directly. Semithin

Frozen Sections

Semithin frozen sections of Pl, P7, P15, P21, and adult (>3 months) optic nerves were double labeled with A2B5 and anti-GFAP antibodies. The total number of glial cells per cross section of nerve was counted under phase microscopy, while the numbers of type-l (A2B5-,GFAP+) and type-2 (A2B5+,GFAP+) astrocytes

TABLE 1 GLIAL CELLS IN SUSPENSIONSOF DEVELOPOING OPTIC NERVE”

Age

Total cells obtained per nerve

El5 El6 E20 PO P2 P7 PlO

1,748 3,255 8,765 11,756 19,756 24,555 30,540

f L + f f f 5

385 540 847 1171 1342 2768 3722

Type1 Oligodendrocytes (GC’) ND ND 0 47 + 16 242+93 1473 f 695 4275 f 992

astrocytes (A2B5,GFAP+)

35 5762 6874 7902 3438 2277

0 f f + + f k

8 922 828 1121 972 658

Type2 astrocytes (A2B5+,GFAP+) 0 0 0 0 0
A2B5+,GFAPcells 206 323 863 1598 3163 5654 6054

f f k + f + f

67 182 216 460 897 984 1131

a The numbers of specific cells were determined by multiplying the percentage of each cell type (assessed by immunofluorescence) by the total number of cells per nerve. Results are expressed as means f SD of at least three separate experiments. The numbers representing the time of first appearance of a cell type are underlined.

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DEVELOPMENTALBIOLOGY

VOLUME111,1985

and of A2B5+,GFAPp cells were counted by fluorescence microscopy (Fig. 1). The relative numbers of these cells at different ages were estimated by multiplying the cell number per cross section by the length of the nerve (from eye to chiasm). In some nerves, the total number of glial cells was counted in semithin plastic sections stained with toluidine blue; the numbers were always within 10% of those obtained in semithin frozen sections. Type-l (A2B5-) astrocytes were seen at all ages examined, while type-2 (A2B5+) astrocytes were seen at >P15 but not at P7 (Table 2). At Pl and P’7, many of the astrocytes (all of which were type-l) were located at the periphery of the nerve, where they and their processes formed the glial limiting membrane. As reported previously (Miller and Raff, 1984), in the adult nerve almost all of the GFAP+ cells in the interior of the nerve were A2B5+ type-2 astrocytes, while those forming the glial limiting membrane at the periphery of the nerve were A2B5- type-l astrocytes. As can be seen in Table 2, the fall in astrocyte numbers seen in cell suspensions after P2 was not seen in sections, suggesting that the majority of astrocytes in older nerves were not released into suspension by our dissociation procedure. In fact, the total number of astrocytes remained relatively constant between Pl and P7 and then increased more than fivefold by adulthood. Most of this late increase in astrocyte numbers was due to the accumulation of type-2 astrocytes, which accounted for more than 65% of the astrocytes in the adult nerve (Table 2). As indicated in Table 2, although the number of A2B5+,GFAPcells decreased dramatically after P21, a small number of such cells were still seen in the adult nerve. Although we were unable to look at oligodendrocytes directly, it is likely that they constituted most of the GFAP- cells from P15 onward (Vaughn, 1969). The numbers of such cells increased greatly between P’7 and P21 and then remained relatively constant (Table 2).

FIG. 1. Semithin transverse frozen section of P21 optic nerve labeled with A2B5 and anti-GFAP antibodies. The A2B5 antibody was detected with G anti-MIg-Rd (C), the anti-GFAP antibodies with G anti-Rig-Fl (B), and the section was viewed by phase contrast in (A). Four cell nuclei (arrows) are readily seen: an A2B5-,GFAP+ type-l astrocyte (ASl) at the pial surface of the nerve, an A2B5+,GFAP+ type-2 astrocyte (AS2) in the interior of the nerve, an A2B5+,GFAPm cell (P), which is probably an O-2A progenitor cell, and an A2B5-,GFAPcell (OL), which is probably an oligodendrocyte. A second A2B5+,GFAPf type-2 astrocyte is seen at the top left corner of the section, although its nucleus is difficult to make out in phase contrast (A). Scale bar = 10 pm.

MILLER ET AL.

39

Astrocyte Lixeages TABLE

2

GLIAL CELLSIN SEMITHIN FROZENSECTIONS OF DEVELOPING OPTIC NERVE= Type 1 astrocytes (A2B5-,GFAP+)

Type 2 astrocytes (A2B5+,GFAP+)

Total length of nerve (mm)

Total glial cells per section

Pl

3 f 0.5

120 + 10 (360)

63 +- 2 (189)

0

ND

P7

4 f 1.2

170 * 20

54 f 2

0

53 f 12

(680)

(216)

338 f 11 (2298)

62 k 7

5 f 1.5 (34)

77 f 12

376 f 14 (3045)

53 iz 2 (429)

46 f 11

(372)

38 2 12 (307)

331 * 22 (3630)

39 k 4 (429)

‘78 i 10

2 f 1.4

(858)

(22)

Age

P15 P21 Adult

6.8 +- 0.3 8.1 f 0.15 11 + 0.4

(421)

A2B5+,GFAPm cells

(212) (523)

‘Sections were cut approximately midway between the eye cup and the optic chiasm; counts on sections cut just behind the eye or just anterior to the chiasm gave similar results. Only cell nuclei within the boundaries of the glial limiting membrane were counted, excluding endothelial cells. The sections were labeled with A2B5 and anti-GFAP antibodies as described in text. Results are expressed as means + SD of counts taken from five comlplete transverse sections of three different optic nerves. The numbers in parentheses were obtained by multiplying the number of each cell type by the length of the nerve and thus give an estimate of the relative numbers of each cell type in the whole nerve

Autoradiography Rats were injected with [3H]thymidine every 3 days from P14 to P26 and killeld at P30. Following perfusion fixation, their optic nerve13 were removed and semithin frozen sections labeled with anti-GFAP antibodies, as well as toluidine blue stained plastic sections, were studied by autoradiography. While 50-70% of the astrocytes in the interior of the nerve were found to have incorporated [3H]thymidine, less than 5% of the astrocytes at the periphery of the nerve had done so (Figs. 2 and 3). Approximately 25% of the oligodendrocytes had also incorporated [3H]thymidine (Fig. 2). DISCUSSION

Using antigenic markers to define them, we have established the sequence and timing of the development of the three major classes of macroglial cells in the rat optic nerve. Type-l astrocytes (A2B5-,GFAP’) first appeared at E16, oligodendrocytes (GC+) at birth (PO), and type-2 astrocytes (A2135+,GFAP+) between P7 and PlO. Cells that were A2B5’-,GFAP- were found as early as El5 and, in small numbers, even in adult optic nerves. Many of the cells with this antigenic phenotype in E17, Pl, and P7 optic nerve have been previously shown to be glial progenitor cells that can develop in vitro into either oligodendrocytes or type-2 astrocytes, depending on the culture conditions (Raff et al, 1983b, 1984a). We did not attempt in this study to show that

this is the case for the A2B5+,GFAPcells in El5 or adult optic nerve. To obtain quantitative data on glial cell development, we determined the number of each cell type in suspensions of optic nerves prepared from rats of different ages. While this approach probably gave a reasonably accurate picture of development in perinatal animals, it gave misleading results in older animals. For example, we found far fewer astrocytes in cell suspensions prepared from P7 and PlO rats than from P2 rats, suggesting that most of the astrocytes in the older nerves were not being released into suspension by our dissociation procedure. This suspicion was confirmed in quantitative immunofluorescence studies on semithin frozen sections. The latter studies showed that astrocyte development occurred in two waves in the optic nerve. The number of type-l astrocytes reached adult levels by P15, while the great majority of type-2 astrocytes, which made up more than 65% of the astrocytes in the adult nerve, developed after P15. Could the second wave of astrocyte development reflect a conversion of type-l to type-2 astrocytes? If this were the case, one would expect the number of type-l astrocytes to fall as the number of type-2 astrocytes increases, unless type-l astrocytes proliferate or are continually produced from GFAPprecursor cells during the period of type-2 astrocyte development. In fact, the number of type-l astrocytes remained remarkably stable after P15, while the num-

40

DEVELOPMENTAL BIOLOGY

FIG. 2. Autoradiographs of l-nrn plastic transverse sections from P14 to P25 and killed at P30; the optic nerves were fixed, blue as described in text, and viewed with phase contrast. Note nerve are radiolabeled in (A). In (B), an astrocyte in the interior The focus is on the silver grains rather than on the cells. Scale

VOLUME 111. 1985

of P30 optic nerve. The rat was injected with [3H]thymidine every 3 days embedded, cut, processed for autoradiography, and stained with toluidine that two astrocytes (AS) and an oligodendrocyte (OL) in the interior of the of the nerve is radiolabeled while the astrocyte at the pial surface is not. bar = 15 grn.

ber of type-2 astrocytes increased by at least 25-fold. Moreover, while 50-70% of type-2 astrocytes incorporated [3H]thymidine when it was given every 3 days between P14 and P26, less than 5% of the type-l astrocytes did so, suggesting that cells of the type-l astrocyte lineage proliferated very little after P15. These results strongly suggest that type-2 astrocytes do not arise from type-l astrocytes but instead develop as a separate cell lineage. This supports our previous observations that type-l and type-2 astrocytes develop

in culture from serologically distinct precursor cells that separate as early as El7 in the rat optic nerve (Raff et ab, 1984a). The finding that the number of A2B5+,GFAPcells fell reciprocally as the number of type-2 astrocytes increased from P15 to adulthood is consistent with the in vitro experiments that demonstrated that the latter cells develop from the former (Raff et ab, 1983a,b). Our results and conclusions are quite different from those of previous workers, who studied gliogenesis in

FIG. 3. Autoradiographs of semithin frozen sections of P30 optic nerve stained with anti-GFAP serum. The rat and sections were cut, stained for immunofluorescence, and processed for autoradiography as described in text, contrast (A and C) and fluorescence (B and D) optics. Note that the GFAP+ astrocyte in the interior of the nerve radiolabeled, while the two GFAP+ astrocytes at the pial surface (arrowed in C and D) are not. A meningeal cell focus in (A) and (C) is on the silver grains rather than on the cells. Scale bar = 10 pm.

was injected as in Fig. 2, and viewed with phase(arrowed in A and B) is is radiolabeled in C. The

MILLER ET AL.

the rat optic nerve and concluded that the majority of oligodendrocytes differentiate after most astrocytes have already developed (Vaughn, 1969; Skoff et al., 1976a,b; Valat et al., 1983). We attribute these differences largely to the fact that, in previous studies, morphological criteria were used to identify astrocytes, oligodendrocytes, and their precursor cells, while we have used antigenic markers. Thus, until antibodies were used to distinguish them, type-l and type-2 astrocytes were not recognized as distinct classes of glial cells because they have very similar morphologies in the adult optic nerve. Diistinguishing between different types of glial cells by mo’rphological appearances alone is even more difficult in developing tissues, where many of the cells have not yet acquired their mature morphological phenotype. Flor example, we have recently found that O-2A progenitor cells in developing optic nerve have vimentin filaments (Raff et al, 1984b), which could be mistaken for glial filaments in immature astrocytes. Two different views of glial cell lineages emerged from previous morphological and autoradiographical studies of gliogenesis in the normal (Skoff et ah, 1976a,b) and transected (Privat et ah, 1981) optic nerve. One suggested that oligodendrocytes and astrocytes develop from separate precursor cells, which can be distinguished in the neonatal nerve (Skoff et al., 1976a,b), while the other suggested that the two types of glial cells develop from a common glioblast (Privat et ak, 1981). Our present and previous experiments suggest that both of theise views are partly right and partly wrong: the type-l astrocyte lineage and oligodendrocyte-type-2 astrocyte lineage have already diverged in the perinatal optic nerve (Raff et al., 1984a), and oligodendrocytes and type-2 (but not type-l) astrocytes develop from a common progenitor cell, at least in culture (Raff et ab, 198313). We thank Dr. Marshal Nirenberg for the A2B5 clone. SD. was supported by a Canadian MRC Centennial Fellowship and R.H.M. by a fellowship from the National Fund for Research into Crippling Diseases. REFERENCES ABNEY, E., WILLIAMS, B., and RAFF, M. C. (1983). Tracing the development of oligodendrocytes from precursor cells using monoclonal antibodies, fluorescence activated cell sorting and cell culture. Dev. BioL 100, 166-1’71. ANGULO Y GONZALES, A. W. (1932). The prenatal growth of the albino rat. Anat. Rec. 52, 117.-138.

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BIGNAMI, A., ENG, L. F., DAHL, D., and UYEDA, C. T. (1972). Localization of the glial fibrillary acidic protein in astrocytes by immunofluorescence. Brain Res 43, 429-435. 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. KUWABARA, T. (1974). Development of the optic nerve of the rat. Invest. OphthalmoL 13, 732-745. LONG, J. A., and BURLINGAME, P. L. (1938). The development of the external form of the rat with observations on the origin of the extra embryonic coelom and foetal membranes. Univ. Calij PubL ZooL 43, 143-184. MILLER, R. H., and RAFF, M. C. (1984). Fibrous and protoplasmic astrocytes are biochemically and developmentally distinct. J. Neurosci. 4, 585-592. PRIVAT, A., VALAT, J., and FULCRAND, J. (1981). Proliferation of neuroglial cell lines in the degenerating optic nerve of young rats. A radioautographic study. J. NeuropathoL Exp. NezwoL 40, 46-60. PRUSS, R. (1979). Thy-l antigen on astrocytes in long-term cultures of rat central nervous system. Nature (Lmzdon) 280, 688-690. RAFF, M. C., MIRSKY, R., FIELDS, K. L., LISAK, R. P., DORFMAN, S. H., SILBERBERG, D. H., GREGSON, N. A., LIEBOWITZ, S., and KENNEDY, M. (1978). Galactocerebroside: A specific cell surface antigenic marker for oligodendrocytes in culture. Nature (Loxdon) 274, 813-816. RAFF, M. C., FIELDS, K. L., HAKOMORI, S., MIRSKI, R., PRUSS, R. M., and WINTER, J. (1979). Cell-type-specific markers for distinguishing and studying neurons and the major classes of glial cells in culture. Brain Res. 174, 283-308. RAFF, M. C., ABNEY, E. A., COHEN, J., LINDSAY, R., and NOBLE, M. (1983a). Two types of astrocytes in cultures of developing rat white matter: Differences in morphology, surface gangliosides and growth characteristics. J. Neurosci. 3, 1289-1300. RAFF, M. C., MILLER, R. H., and NOBLE, M. (1983b). A glial progenitor cell that develops in vitro into an astrocyte or an oligodendrocyte depending on the culture medium. Nature (London) 303, 390-396. RAFF, M. C., ABNEY, E. R., and MILLER, R. H. (1984a). Two glial cell lineages diverge prenatally in rat optic nerve. Dev. BioL 106, 5360. RAFF, M. C., WILLIAMS, B. P., and MILLER, R. H. (1984b). The in vitro differentiation of a bipotential glial progenitor cell. EMBO J. 3, 1857-1864. RANSCHT, B., CLAPSHAW, P. A., PRICE, J., NOBLE, M., and SEIFERT, W. (1982). Development of oligodendrocytes and Schwann cells studied with a monoclonal antibody against galactocerebroside. Proc. Natl. Acad. Sci. USA 79, 2709-2713. SKOFF, R., PRICE, D., and STOCKS, A. (1976a). Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. I. Cell proliferation. J. Comp. NeuroL 169, 291-312. SKOFF, R., PRICE, D., and STOCKS, A. (1976b). Electron microscopic autoradiographic studies of gliogenesis in rat optic nerve. II. Time of origin. J. Camp. Neural 169, 313-333. TOKUYASU, K. T. (1973). A technique for ultracryotomy of cell suspensions and tissues. J. Cell Biol. 57, 551-565. VALAT, J., PRIVAT, A., and FULCRAND, J. (1983). Multiplication and differentiation of glial cells in the optic nerve of the postnatal rat. Anat. Embryol. 167, 335-346. VAUGHN, J. E. (1969). An electron microscopic analysis of gliogensis 94, 293-324. in rat optic nerves. 2. Zellforsch.