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Antisera specific for cell lines with mixed neuronal and glial properties
DEVELOPMENTALBIOLOGY 83, 146-153 (1981)
Antisera Specific for Cell Lines with Mixed Neuronal and Glial Properties SONE-SEERE WILSON, E. EDWARD BAETGE, 1 AND WILLIAM B. STALLCUP2 Neurobiology Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, California 92138 Received June 13, 1980; accepted in revised form September 29, 1980 Antisera were raised against examples of "pseudo-neuronal" and "pseudo-giiar' cell lines--so named because they exhibited some but not all of the characteristics of neuronal and glial cell lines, respectively. The antisera defined several antigens that provide clues to the relationship of these unusual cell lines to the more classical nerve and glial lines. For example, the N4 antigen was expressed by each of the three pseudo-neuronal cell lines and by seven of the ten neuronal cell lines but by none of the pseudo-giial or giial cell lines. This further established a relationship of pseudo-neurons to neurons that was first indicated by the finding of similar Na + and K + channels on the two cell types. Nevertheless, the N5 antigen, found only on the three pseudo-neuronal lines, emphasized the fact that two cell types are distinguishable. The NG1 and NG2 antigens were found only on the pseudo-neuronal and pseudo-glial cell lines, further establishing the distinctions between these cell lines and the more straightforward neuronal and giial lines. NG1 was present on one pseudo-neuron and two of the seven pseudo-gila, while NG2 was found on all three pseudoneurons and four of the psuedo-glia that lacked NG1. This distribution of NG1 and NG2 reinforced our impression that pseudo-neurons and pseudo-gila might be related to each other, possibly as part of a developmental lineage. Since both of these antigens are present in normal rat brain, as determined by quantitative absorption, it may be possible to map their location at the cellular level using immunohistochemical techniques.
INTRODUCTION
For example, the B19, B82, and B108 cell lines all failed to generate action potentials, did not express N1, N2, or N3, and carried the G2 antigen: criteria which would normally lead to classification of these cell lines as glia. Yet each of these cell lines responded positively in assays of veratridine-induced ZZNa+uptake, 3 evidence that the cells had voltage-dependent Na + channels comparable to those found in the neuronal lines (Stallcup and Cohn, 1976; Stallcup, 1977a). Furthermore, B19, B82, and B108 appear to have voltage-dependent K + channels similar to those found in the neuronal lines (Arner and Stallcup, 1981). The physiological similarity of B19, B82, and B108 to the neuronal lines has prompted us to call them "pseudo-neurons." K + channels were also found in seven other cell lines previously defined as glia based on their failure to generate action potentials, their failure to respond positively in the ~Na + uptake assays, and their expression of the G1 and/or G2 antigens. We have designated these seven cell lines "pseudo-gila" to distinguish them from the more clear-cut glial lines that did not have K § channels. However, as discussed in the preceding paper (Arner and Stallcup, 1981), the pseudo-glial lines could
We have shown that antisera raised against cloned cell lines from the rat central nervous system can be used to define antigens that are cell type specific (Stallcup and Cohn, 1976). Cell lines defined as neuronal by electrophysiology and by measurements of veratridineinduced 22Na+ influx were found to carry three nervespecific antigens: N1, expressed by each of 6 neuronal lines; N2, found on 4 of the 6 neuronal lines; and N3, expressed by 2 of the 6 neuronal lines. Antisera against glial (i.e., nonexcitable) cell lines defined two glial-specific antigens: G1, found on 8 of 14 glial cell lines and G2 found on 9 of the 14 glial lines (3 lines expressed both G1 and G2). We have since derived a number of other cell lines from rat brain and again found that those lines with voltage-dependent Na § channels (i.e., the neuronal ones) express various combinations of N1, N2, and N3 while those lines without Na § channels (i.e., the glial ones) express G1 or G2 (Bulloch et al., 1977). There were some cell lines that could not be unambiguously classified as neuronal or glial by the above criteria. Either they carried neither neuronal- nor glialspecific antigens or they expressed antigens that were apparently inappropriate for their physiological type.
8 Although B19 was originally found to lack Na + channels, subsequent testing of cells from the original freezing has revealed positive 2~Na+ uptake in response to 2 • 10-4 M veratridine. All of the other cell lines have also been retested since our initial report (Stallcup and Cohn, 1976), and our findings in these cases remain unchanged.
1 Present address: Cornell University, Graduate School of Medical Sciences, 1300 York Ave., New York, N. Y. 10021. 2 To whom inquiries should be addressed. 146 0012-1606/81/050146-08502.00/0 Copyright 9 1981by AcademicPress,Inc. All rightsof reproductionin anyformreserved.
WILSON, BAETGE,AND STALLCUP
be (1) neuronal cells that lack Na § channels, (2) a novel type of glial cells that have K § channels, (3) representatives of a class of incompletely differentiated stem cells that can give rise in vivo to mature nerves or glia, or (4) cells that, because they are neoplastic, display unusual and misleading combinations of neuronal and glial properties. In this investigation we have tried to gain a better understanding of the nature of these pseudo-neuronal and pseudo-glial cell lines and their relation to more clear-cut nerve and glia by characterizing additional antigens found on the cell surfaces. In particular we have utilized antisera against two of the pseudo-neurons (B82 and B108) and two of the pseudo-giia (B9 and B49). These serological results bear out our earlier experience (Stallcup and Cohn, 1976) that one can use antisera to define cell surface markers for a group of neural cells that have similar physiological traits (e.g., the pseudo-neurons). In addition these markers provide clues to relationships that exist between groups of cells (e.g., between pseudo-giia and pseudo-neurons) and may help to define the lineage of cells in a given developmental pathway.
MATERIALS AND METHODS
Cell culture. The origin and maintenance of the cell lines used in these experiments have been described in the preceding paper (Arner and Stallcup, 1981). Antisera. Antisera were raised in New Zealand white rabbits by eight weekly intravenous immunizations with suspensions of 2 X 1 0 7 viable cells. The crude antisera obtained from pooled bleeds were processed as described previously (Stallcup and Cohn, 1976) with a few modifications. Briefly, (1) the vG fraction from the serum was purified by ammonium sulfate precipitation. (2) The -/G was diluted with phosphate-buffered saline (PBS) to a concentration of 3 mg/ml and spun at 100,000g for 10 min in a Beckman Airfuge. (3) Specificity was achieved by absorbing the supernatant from this centrifugation with the desired cell lines, using 1 vol of packed cells per 2 vol of antiserum. Absorptions were carried out with a given cell line until the reactivity of the antiserum did not change with further absorptions. In general this required three absorptions with that cell line. (4) The specificity of the antiserum was tested at each stage in a two-step radioimmune binding assay
ISIB
B108 B82 ZKC.2
ISIB
CK2D B35
B19 XKM XKC.7
f
Bl08
B103 Bll
? 0 I i
147
Antiserafor Nerve-Glial Cell Lines
x
a z
0 re
3E a,
c,)
y
~
:::
B92,B49,B50 B9,Bgo, 50 pg yG
I00
SW.16
50 pg
I00
vG
FIG. 1. Antiserum raised against B108. The indirect radioimmune binding assay has been described previously (Stallcup and Cohn, 1976). The amount of 125I-labeled goat anti-rabbit ~'G (cpm) bound to monolayer cultures of the various cell lines in 16-ram Linbro wells is plotted as a function of the amount of primary rabbit ~'G added. (A) Anti-B108 absorbed with ]39, ]392, and ]349 yields anti-N4. (B) Anti-B108 absorbed with Bg, B92, B49, and ]3103 yields anti-N5, e, B108; O, B82; A, B35; n, B103; A, Bll; I, B92; V, B49; X, B9; @, B90; O, ZKC.2; ~, XKC.7; V, SW16; | B50; ~), B19; [], XKM; *, CK2D.
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DEVELOPMENTAL BIOLOGY
VOLUME 83, 1981
B82
15
15
BIOS
!s
CK20
ZKC.2
B103
~5
10
10
Bll
b
_o x
x
r~ z
,..t Z
0 m
0
m
10
ft.
L)
5 B49, B92 Bg, B90
50
100
pg yG
~
B82
B103
50
100
pg u
FIG. 2. Antiserum raised against B82. Details in Fig. 1. (A) Anti-B82 absorbed with B9, B92, and ]349 yields anti-N4. (B) Anti-B82 absorbed with B9, B92, B49, and B103 yields no reactivity, e, B108; O, B82; A, [335; [], B103; 4, Bll; i B92; V, B49; • B9; 0, B90; ~, B19; O, ZKC.2; ~1, XKM; *, CK2D.
using the rabbit ~fG and a z~SI-labeled goat antibody against rabbit vG. Quantitative absorptions. Detection of the presence of an antigen in normal rat brain was accomplished by quantitative absorption experiments using the appropriate antiserum, as described for the N3 and G2 antigens (Stallcup, 1977b). The ability of homogenized brain tissue to absorb activity from a given antiserum was compared to the absorbing capacity of cell lines known to be positive or negative for the antigen in question. In the case of the NG1 antigen, the B9 cell line, which is positive for NG1 (NGI+), was used as the positive control in absorptions, while the B103 cell line, which is negative for NGI(NG1-), was used as the negative control. For the NG2 antigen the B49 cell line (NG2 +) and the B103 cell line (NG2-) were used as the positive and negative control, respectively. In the case of the N4 and N5 antigens that B108 cell line (N4+N5 +) and the B9 cell line (N4-N5-) were used for the control absorptions. RESULTS AND DISCUSSION
Since the B19, B82, and B108 cell lines were found to have Na + and K + channels comparable to those found
in neuronal cell lines, we tried to identify surface antigens shared by these "pseudo-neurons" with the neuronal cell lines. To do this we made antisera against B82 and B108 and absorbed the sera with both pseudoglial and glial cell lines (which incidently share the G2 antigen with B19, B82, B108). Figures 1A and 2A show that absorption of either of these two antisera with the cell lines B9 (pseudo-glial), ]349 (pseudo-giial), and B92 (glial) left a reagent that reacted not only with the three pseudo-neurons B19, ]382, and B108, but also with B35, B103, Bll, ZKC, CK2D, XKM, and XKC. Since these seven cell lines were among those classified as neuronal on the basis of electrophysiology and 22Na+ uptake measurements, we called this antigen N4. The distribution of the N4 antigen (see Table I for a summary) supports the relationship between the neurons and pseudo-neurons that was first suggested by the similarity of their ion channels. The N4 marker effectively separates the neurons and pseudo-neurons from the glia and pseudogila, a distinction that is also made by the 22Na+ uptake assay. The fact that N4 is not expressed by three of the neuronal cell lines that have Na + channels (B50, B104, and SW16) indicates that the N4 determinant is probably not carried by the Na + channel itself. Consistent
WILSON, BAETGE, AND STALLCUP
149
Antisera for Nerve-Glial Cell Lines
TABLE 1 SUMMARY OF NEURONAL AND GLIAL PROPERTIES
Electrical properties Antigens Cell line
ActiOn potential a
Na + channel b
G r o u p 1: N e u r o n s Bll B35 B50 B103 B104 XKM XKC ZKC SW16 CK2D Group 2~ Psuedo-neurons B19 B82 B108 G r o u p 3: P s e u d o - g l i a ( A ) B49 Blll C6
K+ channel c
N1,N2,N3 b
G1,G2 b
N4
N5
+ +
+ + +
--
+ _ +
-_ -
+ +
+ + +
--
+ + +
--
+
+
--
+
--
+ + +
----
+ + +
+ + +
+ + +
m
+ + +
NG2
w
+ + +
m
m
R
m
D
--+
R
R
m
R
R
R
D
w
G r o u p 4: G l i a B15 B23 B27 B90 B92 ~CFA 3T3
+ + +
D
m
D
+ + +
+ + + +
+ +
+ w
D
m
R
~HC (B) B9 BEll B28
NG1
m
+ + +
m
m
D
Note. The criteria for g r o u p i n g the cell lines into four c a t e g o r i e s - - n e u r o n s , pseudo-neurons, pseudo-glia, and g l i a - - a r e derived f r o m the studies listed in footnotes a, b, and c. As discussed in the introduction, pseudo-neuronal cell lines resemble n e u r o n a l cell lines in t h a t they have both Na + and K § channels, yet they have not been f o u n d to g e n e r a t e action potentials or to e x p r e s s the N1, N2, and N3 a n t i g e n s characteristic of the nerve cell lines. Pseudo-glial cell lines s h a r e the G1 and G2 a n t i g e n s w i t h glial cell lines, b u t the pseudo-glia have K + channels w h e r e a s the glia have n e i t h e r Na + or K + channels. The pseudo-glial lines are f u r t h e r subdivided into g r o u p s 3A and B on the b a s i s of the properties of the K + channels. The K + c h a n n e l s of cells in g r o u p 3A can be blocked by t e t r a e t h y l a m m o n i u m chloride and by 4aminopyridine, while the K + channels of g r o u p 3B cells are blocked by neither. As discussed in the text, the r e s u l t s of the p r e s e n t s t u d y f u r t h e r highlight and define the r e l a t i o n s h i p s between the four g r o u p s of cell lines. a S c h u b e r t et al. (1974). b Stallcup and Cohn (1976); Bulloch et al. (1977). c A r n e r and Stallcup (1981).
with this is the finding that the anti-N4 serum does not block veratridine and scorpion venom-induced 22Na+ uptake in the B103 cell line (Fig. 3), although it is possible that the antibody could bind to the channel without affecting its function. Further absorption of the anti-B82 serum with the neuronal B103 cell line led to a complete loss of reac-
tivity (Fig. 2B); i.e., at 100 ~g of rabbit v-globulin all cell lines exhibited only the background binding of roughly 3000-4000 cpm that is characteristic of our assay (see also Figs. 1A and 2A for other examples of this background binding). On the other hand, parallel absorption of the anti-B108 serum with B103 cells left a small but reproducible degree of reactivity against the
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DEVELOPMENTALBIOLOGY 4
~3
! ~2
minutes
FIG. 3. Effect of anti-N4 serum on tuNa+ uptake. Z2Na+ uptake in B103 cells was induced with veratridine and scorpion venom as described previously (Stallcup, 1977a). In parallel another set of B103 cells was preincubated at 37~ for 45 min with the anti-N4 serum at a 1:4 dilution (sufficient to saturate the cells in a binding assay) and then assayed. No effect on ~Na § uptake was observed. O, 2 m M ouabain; 0, 2 mM ouabain + 0.2 raM veratridine + 20 ~g/ml scorpion venom; [3, 2 mM ouabain + anti-N4; ~, 2 m M ouabain + 0.2 mM veratridine + 20 ttg/ml scropion venom + anti-N4.
VOLUME83, 1981
three pseudo-neurons B19, B82, and B108 (Fig. 1B); we designated this antigen N5. All other cell lines tested showed only the characteristic 3000- to 4000-cpm background binding such as seen in Fig. 2B. Thus although there are many similarities between the neurons and pseudo-neurons, there are also differences. The pseudoneurons express the N5 antigen, but lack the NI, N2, and N3 antigens found on the neuronal lines (Stallcup and Cohn, 1976). These antigenic differences are interesting in light of the major physiological distinction between the neurons and pseudo-neurons, namely, that although the latter cell lines had Na + and K + channels they failed to generate action potentials (Schubert et al.,1974). Although the failure to spike could be due to technical problems in making satisfactory penetrations of the cellswith microelectrodes, the parallels between the serological data and the electrophysiological data (summarized in Table 1) suggest that the failure to observe the expected action potential is not random. Perhaps there is a more interesting basis for the failure
BEll
30
15
13 JEll
10
20
'o
o
a Z
a Z -t
-I<
x
0,,t =Z o. 0
0
=E o. (J
10
B103 192. B 2 8 ~7 E49. B 8 2 50 pg u
100
50
100
pg yG
FIG. 4. Antiserum raised against B9. Details as in Fig. 1. (A) anti-B9 absorbed with B103, B65, and Bg0 yields Anti-G2. (B) Anti-B9 absorbed with B103, ]365, B90, B92, and t349 yields anti-NG1, e , B E l l ; r~, B9; A, B108; ~7, B82; I , B28; O, B92; O, ]349; 4, B65; )<, B103; v , B90; ~, B35.
WILSON, BAETGE, AND STALLCUP
40: B49
I S C6 / ~ , /
3O
Bill
's z
~ 2o
al Z a.
/ ~
f I0
~ 20
9 30
40
50
BBo
60
/~g TG FIG. 5. Antiserum raised against B49. Details in Fig. 1. Anti-B49 absorbed with B103 and B65 yields anti-NG2. $, B49; O, C6; n Bl11; I , B19; O, ~HC; A, B82; V, B65; V, B92; O, B103; A, B90; e , B108.
of the pseudo-neurons to spike--possibly a difference in the Na + channel that is not reflected in the ~ a + uptake assay or possibly the immaturity of some other membrane property that is required to initiate a regenerative spike. These three cell lines do not represent an isolated phenomenon. Veratridine-activated, tetrodotoxin-sensitive Na + channels have also been found in several other types of cells that normally show no evidence of Na + spikes. These include embryonic cardiac muscle cells (Galper and Catterall, 1978; Fosset et al., 1977), a bladder cell line (Romey et al., 1979), pancreatic fl cells (Donatsch et al., 1977), and crab sensory neurons (Lowe et al., 1978). The anti-B82 and B108 antisera failed to define any antigens shared by the pseudo-neurons with glial or pseudo-glial cell lines. Absorption of the anti-B82 serum with the neuronal lines B35 and B103 resulted in complete loss of reactivity, while absorption of anti-B108 with either B35 or B103 led to a serum with anti-N5 specificity. Thus by these criteria, also, the pseudo-neuronal lines were more closely related to neuronal cells than to glial or pseudo-glial cells. However, this did not explain the observation that B19, B82, and B108 each expressed the G2 antigen. We thought that perhaps this problem stemmed from the anti-G2 serum having more than one specificity and that the paradox might be resolved by further absorbing the anti-G2 serum in such a way that B19, B82, and B108 would no longer be re-
151
Antiserafor Nerve-Glial Cell Lines
active. This strategy was only partially successful. The anti-G2 serum, prepared by absorbing the anti-B9 serum with the neuronal lines B103 and B65 and with the glial line B90 (Fig. 4A), could be further absorbed with the B49 and B92 cell lines to yield an antiserum with reactivity against only three lines--B9, B E l l , and B108 (Fig. 4B)--and attempts to absorb further with B108 completely abolished reactivity. We called this antigen NG1 since B108 was pseudo-neuronal and B9 and B E l l were pseudo-glial. This situation had a parallel in the analysis of an antiserum made against another pseudo-glial cell line, B49. Absorption of this antiserum with the two neuronal lines B103 and B65 resulted in a reagent that reacted with only seven cell lines--B49, C6, Bl11,/~HC, B19, B82, and B108 (Fig. 5). The first four of these lines were pseudo-glial and the latter three were pseudo-neuronal, so we called this antigen NG2. Attempts to absorb further with B82 or B108 at this point led to complete loss of activity. Thus at the limit of their specificity both the anti-B9 serum and the anti-B49 serum reacted with combinations of pseudo-glial and pseudo-neuronal lines. The distributions of these two antigens are summarized in Table 1. It is particularly striking that NG1 and NG2 were not found on any of the cell lines that could be classified as unambiguously neuronal or glial. This supports and emphasizes previous distinctions concerning
B
9
9 x
5 minutes
10 minutes
FIG. 6. Effect of anti-NG1 and anti-NG2 sera on 8SRb+ efflux. S~Rb+ efflux from B9 and ]349 cells was induced with 100 m M KC1 as described previously (Arner and Stallcup, 1981). In para[lel other cultures of B9 and B49 were preincubated at 37~ for 45 min with 1:4 dilutions of the anti-NG1 and anti-NG2 sera, respectively, and then assayed for efflux. No effect was seen on the B9 cells, and with the ]349 cells the KCl-induced efflux remained unchanged after correcting the data for a small decrease in the rate of background efflux. (A) B9 (+anti-NG1). (B) B49 (_+anti-NG2). O, 2 m M ouabain; @, 2 m M ouabain + 100 m M KCI; n, 2 m M ouabain + antiserum; O, 2 m M ouabain + 100 m M KCI + antiserum.
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DEVELOPMENTAL BIOLOGY
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NG 2 Z i-
~4 o3 w :>
~2
N3
_1 LU n,.
o
J
J a,tul~ DAYS
FIG. 7. Schedule for appearance of antigens in r a t brain. Quantitative absorption experiments were performed as described previously (Stallcup, 1977b) to assess the presence of antigens in brain homogenates from rats of different ages. "Relative amount of antigen" present is plotted in arbitrary units and is m e a n t to refer to each antigen individually. T h a t is, i~ is not implied t h a t the antigens are present in roughly equal amounts; they have only been plotted in this way to facilitate comparison of the curves. The actual level of antigen in t e r m s of the number of molecules is not known in any of these cases.
pseudo-neurons and pseudo-gila made on the basis of other data, namely, the presence of the N5 antigen and lack of action potentials in the pseudo-neurons, separating them from neurons, and the presence of K + channels in the pseudo-gila, separating them from the gila. Furthermore, among the pseudo-glial cell lines the serological distinction between NG1 + cells and NG2 + cells is paralleledby a physiological distinction between the K* channels of these cells. NG2-bearing pseudo-gila have K + currents that can be blocked by tetraethylammonium or 4-aminopyridine, but not by scorpion venom. These cells are designated as constituting Group 3A in Table 1, as defined in the preceding paper (Arner and Stallcup, 1981). NGl-bearing pseudo-glial cells, on the other hand, exhibit K + currents that cannot be blocked by tetraethylammonium, 4-aminopyridine, or scorpion venom. These cells are in Group 3B in Table 1. These findings once again follow our general theme that it is possible to define cell surface markers for physiologically distinct groups of cells. On the chance that the anti-NG1 and anti-NG2 antisera actually recognized the K + channels of the Group 3B and Group 3A pseudo-glia, respectively, we tested the ability of the antisera to block KCl-stimulated S6Rb+ efflux from B9 and B49 cells (see Arner and Stallcup, 1981). Figure 6 shows that SeRb+ effluxwas unaffected by the antiserum in each case. Thus the antisera either do not recognize K + channels or else do not interfere with K + channel function. The findingthat pseudo-neuronal lines,with both N a + and K + channels, share antigens with pseudo-gliallines
with only K + channels lends some support to an earlier hypothesis of ours that pseudo-neurons and pseudo-gila might represent cells of common lineage arrested by the process of transformation at different stages of differentiation (Arner and Stallcup, 1981). The acquisition or loss of Na + channels could be the process which links these two cell types in a developmental sequence. Consistent with the idea that these cell lines are incompletely differentiated precursor cells rather than cells in terminal stages of differentiation is the finding that one of the pseudo-glial lines, C6, can be induced by special culturing conditions to differentiate further to a form that closely resembles mature astrocytes (Silbert and Goldstien, 1972; Bissell et al., 1974). It would be of great interest to know whether this differentiation, marked by morphological changes and the appearance of the astrocyte-specific glial fibriUary, acidic protein (Bignami et al., 1972), is accompanied by changes in surface antigens and ion channels, but to date we have been unable to induce C6 cells to differentiate in sufficient numbers to make very satisfactory determinations of these parameters. The few differentiated C6 cells t h a t we have observed (five or six out of several hundred examined, as judged by immunofluorescence with a rabbit antibody against the glial fibrillary acidic protein) seemed to retain the NG2 marker, as judged by immunofluorescence with a mouse antibody against NG2 (see Stallcup 1981) for a description of this antibody and the double labeling protocol). No information on the presence of ion channels could be obtained.
WILSON, BAETGE, AND STALLCUP
Further support for our hypothesis that pseudo-neurons and pseudo-glia are incompletely differentiated precursors of nerve and glia would be gained by finding that some of the other cell lines can be induced to differentiate along other pathways, i.e.,to mature neuronal cells.Evidence from other sets of neural cell lines suggests that this m a y occur in some cases (DeVitry, 1977; T o m o z a w a and Sueoka, 1979). While it m a y be possible to obtain useful data of this type from further examination of the cell lines,a complementary and perhaps more direct approach is to turn to the normal brain. Quantitative absorption experiments show that both the N G 1 and N G 2 antigens are present in normal rat brain. Figure 7 illustrates the appearance of NG2 in the middle of the second week of embryogenesis, while NG1 appears a few days later. Both antigens can be found in the adult brain; thus it is possible that these markers are retained by mature cells that are fully differentiated. The "timetables" for NG1 and NG2 are contrasted with that of one of the neuronal antigens, N3, which can be detected only in the fetus (Stallcup, 1977b). The quantitative absorption technique does not reveal a detectable amount of either the N4 or N5 antigen in normal rat brain. Since this technique is relatively insensitive, we do not know whether N4 or N5 are totally absent from normal brain or merely present in small quantities. Nevertheless, the presence of NG1 and NG2 in rat brain shows that these two antigens, which define relationships between pseudo-neuronal and pseudo-glial cell lines, are not tumor-specific markers. This substantially eases our worry concerning possible artifacts stemming from the neoplastic nature of the cell lines and, moreover, offers hope that the antigens can be mapped at the cellular level to reveal something about the identity and developmental fate of the cell types that express these markers in the normal brain. This approach is the topic of the following paper (Stallcup, 1981). We thank Dr. David Schubert and Dr. Jim Patrick for their helpful discussions concerning this work. This research was supported by NIH Grant NS/CA 16112 to W.B.S.
REFERENCES ARNER,L. S., and STALLCUP,W. B. (1981). Rubidium efflux from neural cell lines through voltage-dependent potassium channels. Develop. Biol. 83, 138-145.
Antiserafor Nerve-Glial Cell Lines
153
BIGNAMI, A., ENG, L., DAHL, D., and UYEDA, C. (1972). Localization of the glial flbrillary acidic protein in astrocytes by immunofluorescence. Brain Re~ 43, 429-435. BISSELL, M., RUBINSTEIN,L., BIGNAMI, A., and HERMAN, M. (1974). Characteristics of the rat C6 glioma maintained in organ culture systems. Production of glial flbrillary acidic protein in the absence of glioflbrillogenesis. Brain Res. 82, 77-81. BULLOCH,K., STALl,UP, W., and COHN, M. (1977). The derivation and characterization of neuronal cell lines from rat and mouse brain. Brain Rea 135, 25-36. DEVITRY, F. (1977). Growth and differentiation of a primitive neuronal cell line after in vivo transplantation into syngeneic mice. Nature (London) 267, 48-50. DONATSCH,P., LOWE, D., RICHARDSON,B., and TAYLOR,P. (1977). The functional signifcance of sodium channels in pancreatic beta-cell membranes. J. Physio~ 267, 357-376. FOSSET, M., DE BARRY, J., LENOIR, M., and LAZDUNSKI, M. (1977). Analysis of molecular aspects of Na + and Ca ++ uptakes by embryonic cardiac cells in culture. J. BioL Chem. 252, 6112-6117. GALPER, J., and CATTERALL,W. (1978). Developmental changes in the sensitivity of embryonic heart cells to tetrodotoxin and D600. Develop. Biol. 65, 216-227. LOWE, D., BUSH, B., and RIPLEY, S. (1978). Pharmacological evidence for 'fast' sodium channels in non-spiking neurons. Nature (London) 274, 289-290. ROMEY, G., JACQUES,Y., SCHWEITZ,H., FOSSET, M., and LAZDUNSKI, M. (1979). The sodium channel in non-impulsive cells: Interaction with specific neurotoxins. Biochim~ Biophys. Acta 556, 344-353. SCHUBERT,D., HEINEMANN,S., CARLISLE,W., TARIKAS,H., KIMES, B., PATRICK, J., STEINBACH,J., CULP, W., and BRANDT,B. (1974). Clonal cell lines from the rat central nervous system. Nature (London) 249, 224-227. SILBERT, S., and GOLDSTEIN,M. (1972). Drug-induced differentiation of a rat glioma in vitro. Cancer Res. 32, 1422-1427. STALLCUP, W. (1977a). Comparative pharmacology of voltage-dependent Na + channels. Brain Res. 135, 37-58. STALLCUP, W. (1977b). Nerve and glial-specific antigens on cloned neural cell lines. In "Progress in Clinical Biological Research, Vol. 15, "Cellular Neurobiology" (Z. Hall, R. Kelly, and C. F. Fox, eds.), pp. 165-178. Liss, New York. STALLCUP.W. B. (1981). The NG2 antigen, a putative lineage marker: Immunofluorescent localization in primary cultures of rat brain, Develop. Biol. 83, 154-165. STALLCUP, W., and COHN, M. (1976). Correlation of surface antigens and cell type in cloned cell lines from the rat central nervous system. Exp. Cell Res. 98, 285-297. TOMOZAWA, Y., and SUEOKA, N. (1979). In vitro segregation of different cell lines with neuronal and glial properties from a stem cell line of rat neurotumor RT4. Proc. Nat. Acad. Sci. USA 75, 63056309.
Antisera Specific for Cell Lines with Mixed Neuronal and Glial Properties SONE-SEERE WILSON, E. EDWARD BAETGE, 1 AND WILLIAM B. STALLCUP2 Neurobiology Laboratory, The Salk Institute for Biological Studies, P.O. Box 85800, San Diego, California 92138 Received June 13, 1980; accepted in revised form September 29, 1980 Antisera were raised against examples of "pseudo-neuronal" and "pseudo-giiar' cell lines--so named because they exhibited some but not all of the characteristics of neuronal and glial cell lines, respectively. The antisera defined several antigens that provide clues to the relationship of these unusual cell lines to the more classical nerve and glial lines. For example, the N4 antigen was expressed by each of the three pseudo-neuronal cell lines and by seven of the ten neuronal cell lines but by none of the pseudo-giial or giial cell lines. This further established a relationship of pseudo-neurons to neurons that was first indicated by the finding of similar Na + and K + channels on the two cell types. Nevertheless, the N5 antigen, found only on the three pseudo-neuronal lines, emphasized the fact that two cell types are distinguishable. The NG1 and NG2 antigens were found only on the pseudo-neuronal and pseudo-glial cell lines, further establishing the distinctions between these cell lines and the more straightforward neuronal and giial lines. NG1 was present on one pseudo-neuron and two of the seven pseudo-gila, while NG2 was found on all three pseudoneurons and four of the psuedo-glia that lacked NG1. This distribution of NG1 and NG2 reinforced our impression that pseudo-neurons and pseudo-gila might be related to each other, possibly as part of a developmental lineage. Since both of these antigens are present in normal rat brain, as determined by quantitative absorption, it may be possible to map their location at the cellular level using immunohistochemical techniques.
INTRODUCTION
For example, the B19, B82, and B108 cell lines all failed to generate action potentials, did not express N1, N2, or N3, and carried the G2 antigen: criteria which would normally lead to classification of these cell lines as glia. Yet each of these cell lines responded positively in assays of veratridine-induced ZZNa+uptake, 3 evidence that the cells had voltage-dependent Na + channels comparable to those found in the neuronal lines (Stallcup and Cohn, 1976; Stallcup, 1977a). Furthermore, B19, B82, and B108 appear to have voltage-dependent K + channels similar to those found in the neuronal lines (Arner and Stallcup, 1981). The physiological similarity of B19, B82, and B108 to the neuronal lines has prompted us to call them "pseudo-neurons." K + channels were also found in seven other cell lines previously defined as glia based on their failure to generate action potentials, their failure to respond positively in the ~Na + uptake assays, and their expression of the G1 and/or G2 antigens. We have designated these seven cell lines "pseudo-gila" to distinguish them from the more clear-cut glial lines that did not have K § channels. However, as discussed in the preceding paper (Arner and Stallcup, 1981), the pseudo-glial lines could
We have shown that antisera raised against cloned cell lines from the rat central nervous system can be used to define antigens that are cell type specific (Stallcup and Cohn, 1976). Cell lines defined as neuronal by electrophysiology and by measurements of veratridineinduced 22Na+ influx were found to carry three nervespecific antigens: N1, expressed by each of 6 neuronal lines; N2, found on 4 of the 6 neuronal lines; and N3, expressed by 2 of the 6 neuronal lines. Antisera against glial (i.e., nonexcitable) cell lines defined two glial-specific antigens: G1, found on 8 of 14 glial cell lines and G2 found on 9 of the 14 glial lines (3 lines expressed both G1 and G2). We have since derived a number of other cell lines from rat brain and again found that those lines with voltage-dependent Na § channels (i.e., the neuronal ones) express various combinations of N1, N2, and N3 while those lines without Na § channels (i.e., the glial ones) express G1 or G2 (Bulloch et al., 1977). There were some cell lines that could not be unambiguously classified as neuronal or glial by the above criteria. Either they carried neither neuronal- nor glialspecific antigens or they expressed antigens that were apparently inappropriate for their physiological type.
8 Although B19 was originally found to lack Na + channels, subsequent testing of cells from the original freezing has revealed positive 2~Na+ uptake in response to 2 • 10-4 M veratridine. All of the other cell lines have also been retested since our initial report (Stallcup and Cohn, 1976), and our findings in these cases remain unchanged.
1 Present address: Cornell University, Graduate School of Medical Sciences, 1300 York Ave., New York, N. Y. 10021. 2 To whom inquiries should be addressed. 146 0012-1606/81/050146-08502.00/0 Copyright 9 1981by AcademicPress,Inc. All rightsof reproductionin anyformreserved.
WILSON, BAETGE,AND STALLCUP
be (1) neuronal cells that lack Na § channels, (2) a novel type of glial cells that have K § channels, (3) representatives of a class of incompletely differentiated stem cells that can give rise in vivo to mature nerves or glia, or (4) cells that, because they are neoplastic, display unusual and misleading combinations of neuronal and glial properties. In this investigation we have tried to gain a better understanding of the nature of these pseudo-neuronal and pseudo-glial cell lines and their relation to more clear-cut nerve and glia by characterizing additional antigens found on the cell surfaces. In particular we have utilized antisera against two of the pseudo-neurons (B82 and B108) and two of the pseudo-giia (B9 and B49). These serological results bear out our earlier experience (Stallcup and Cohn, 1976) that one can use antisera to define cell surface markers for a group of neural cells that have similar physiological traits (e.g., the pseudo-neurons). In addition these markers provide clues to relationships that exist between groups of cells (e.g., between pseudo-giia and pseudo-neurons) and may help to define the lineage of cells in a given developmental pathway.
MATERIALS AND METHODS
Cell culture. The origin and maintenance of the cell lines used in these experiments have been described in the preceding paper (Arner and Stallcup, 1981). Antisera. Antisera were raised in New Zealand white rabbits by eight weekly intravenous immunizations with suspensions of 2 X 1 0 7 viable cells. The crude antisera obtained from pooled bleeds were processed as described previously (Stallcup and Cohn, 1976) with a few modifications. Briefly, (1) the vG fraction from the serum was purified by ammonium sulfate precipitation. (2) The -/G was diluted with phosphate-buffered saline (PBS) to a concentration of 3 mg/ml and spun at 100,000g for 10 min in a Beckman Airfuge. (3) Specificity was achieved by absorbing the supernatant from this centrifugation with the desired cell lines, using 1 vol of packed cells per 2 vol of antiserum. Absorptions were carried out with a given cell line until the reactivity of the antiserum did not change with further absorptions. In general this required three absorptions with that cell line. (4) The specificity of the antiserum was tested at each stage in a two-step radioimmune binding assay
ISIB
B108 B82 ZKC.2
ISIB
CK2D B35
B19 XKM XKC.7
f
Bl08
B103 Bll
? 0 I i
147
Antiserafor Nerve-Glial Cell Lines
x
a z
0 re
3E a,
c,)
y
~
:::
B92,B49,B50 B9,Bgo, 50 pg yG
I00
SW.16
50 pg
I00
vG
FIG. 1. Antiserum raised against B108. The indirect radioimmune binding assay has been described previously (Stallcup and Cohn, 1976). The amount of 125I-labeled goat anti-rabbit ~'G (cpm) bound to monolayer cultures of the various cell lines in 16-ram Linbro wells is plotted as a function of the amount of primary rabbit ~'G added. (A) Anti-B108 absorbed with ]39, ]392, and ]349 yields anti-N4. (B) Anti-B108 absorbed with Bg, B92, B49, and ]3103 yields anti-N5, e, B108; O, B82; A, B35; n, B103; A, Bll; I, B92; V, B49; X, B9; @, B90; O, ZKC.2; ~, XKC.7; V, SW16; | B50; ~), B19; [], XKM; *, CK2D.
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DEVELOPMENTAL BIOLOGY
VOLUME 83, 1981
B82
15
15
BIOS
!s
CK20
ZKC.2
B103
~5
10
10
Bll
b
_o x
x
r~ z
,..t Z
0 m
0
m
10
ft.
L)
5 B49, B92 Bg, B90
50
100
pg yG
~
B82
B103
50
100
pg u
FIG. 2. Antiserum raised against B82. Details in Fig. 1. (A) Anti-B82 absorbed with B9, B92, and ]349 yields anti-N4. (B) Anti-B82 absorbed with B9, B92, B49, and B103 yields no reactivity, e, B108; O, B82; A, [335; [], B103; 4, Bll; i B92; V, B49; • B9; 0, B90; ~, B19; O, ZKC.2; ~1, XKM; *, CK2D.
using the rabbit ~fG and a z~SI-labeled goat antibody against rabbit vG. Quantitative absorptions. Detection of the presence of an antigen in normal rat brain was accomplished by quantitative absorption experiments using the appropriate antiserum, as described for the N3 and G2 antigens (Stallcup, 1977b). The ability of homogenized brain tissue to absorb activity from a given antiserum was compared to the absorbing capacity of cell lines known to be positive or negative for the antigen in question. In the case of the NG1 antigen, the B9 cell line, which is positive for NG1 (NGI+), was used as the positive control in absorptions, while the B103 cell line, which is negative for NGI(NG1-), was used as the negative control. For the NG2 antigen the B49 cell line (NG2 +) and the B103 cell line (NG2-) were used as the positive and negative control, respectively. In the case of the N4 and N5 antigens that B108 cell line (N4+N5 +) and the B9 cell line (N4-N5-) were used for the control absorptions. RESULTS AND DISCUSSION
Since the B19, B82, and B108 cell lines were found to have Na + and K + channels comparable to those found
in neuronal cell lines, we tried to identify surface antigens shared by these "pseudo-neurons" with the neuronal cell lines. To do this we made antisera against B82 and B108 and absorbed the sera with both pseudoglial and glial cell lines (which incidently share the G2 antigen with B19, B82, B108). Figures 1A and 2A show that absorption of either of these two antisera with the cell lines B9 (pseudo-glial), ]349 (pseudo-giial), and B92 (glial) left a reagent that reacted not only with the three pseudo-neurons B19, ]382, and B108, but also with B35, B103, Bll, ZKC, CK2D, XKM, and XKC. Since these seven cell lines were among those classified as neuronal on the basis of electrophysiology and 22Na+ uptake measurements, we called this antigen N4. The distribution of the N4 antigen (see Table I for a summary) supports the relationship between the neurons and pseudo-neurons that was first suggested by the similarity of their ion channels. The N4 marker effectively separates the neurons and pseudo-neurons from the glia and pseudogila, a distinction that is also made by the 22Na+ uptake assay. The fact that N4 is not expressed by three of the neuronal cell lines that have Na + channels (B50, B104, and SW16) indicates that the N4 determinant is probably not carried by the Na + channel itself. Consistent
WILSON, BAETGE, AND STALLCUP
149
Antisera for Nerve-Glial Cell Lines
TABLE 1 SUMMARY OF NEURONAL AND GLIAL PROPERTIES
Electrical properties Antigens Cell line
ActiOn potential a
Na + channel b
G r o u p 1: N e u r o n s Bll B35 B50 B103 B104 XKM XKC ZKC SW16 CK2D Group 2~ Psuedo-neurons B19 B82 B108 G r o u p 3: P s e u d o - g l i a ( A ) B49 Blll C6
K+ channel c
N1,N2,N3 b
G1,G2 b
N4
N5
+ +
+ + +
--
+ _ +
-_ -
+ +
+ + +
--
+ + +
--
+
+
--
+
--
+ + +
----
+ + +
+ + +
+ + +
m
+ + +
NG2
w
+ + +
m
m
R
m
D
--+
R
R
m
R
R
R
D
w
G r o u p 4: G l i a B15 B23 B27 B90 B92 ~CFA 3T3
+ + +
D
m
D
+ + +
+ + + +
+ +
+ w
D
m
R
~HC (B) B9 BEll B28
NG1
m
+ + +
m
m
D
Note. The criteria for g r o u p i n g the cell lines into four c a t e g o r i e s - - n e u r o n s , pseudo-neurons, pseudo-glia, and g l i a - - a r e derived f r o m the studies listed in footnotes a, b, and c. As discussed in the introduction, pseudo-neuronal cell lines resemble n e u r o n a l cell lines in t h a t they have both Na + and K § channels, yet they have not been f o u n d to g e n e r a t e action potentials or to e x p r e s s the N1, N2, and N3 a n t i g e n s characteristic of the nerve cell lines. Pseudo-glial cell lines s h a r e the G1 and G2 a n t i g e n s w i t h glial cell lines, b u t the pseudo-glia have K + channels w h e r e a s the glia have n e i t h e r Na + or K + channels. The pseudo-glial lines are f u r t h e r subdivided into g r o u p s 3A and B on the b a s i s of the properties of the K + channels. The K + c h a n n e l s of cells in g r o u p 3A can be blocked by t e t r a e t h y l a m m o n i u m chloride and by 4aminopyridine, while the K + channels of g r o u p 3B cells are blocked by neither. As discussed in the text, the r e s u l t s of the p r e s e n t s t u d y f u r t h e r highlight and define the r e l a t i o n s h i p s between the four g r o u p s of cell lines. a S c h u b e r t et al. (1974). b Stallcup and Cohn (1976); Bulloch et al. (1977). c A r n e r and Stallcup (1981).
with this is the finding that the anti-N4 serum does not block veratridine and scorpion venom-induced 22Na+ uptake in the B103 cell line (Fig. 3), although it is possible that the antibody could bind to the channel without affecting its function. Further absorption of the anti-B82 serum with the neuronal B103 cell line led to a complete loss of reac-
tivity (Fig. 2B); i.e., at 100 ~g of rabbit v-globulin all cell lines exhibited only the background binding of roughly 3000-4000 cpm that is characteristic of our assay (see also Figs. 1A and 2A for other examples of this background binding). On the other hand, parallel absorption of the anti-B108 serum with B103 cells left a small but reproducible degree of reactivity against the
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DEVELOPMENTALBIOLOGY 4
~3
! ~2
minutes
FIG. 3. Effect of anti-N4 serum on tuNa+ uptake. Z2Na+ uptake in B103 cells was induced with veratridine and scorpion venom as described previously (Stallcup, 1977a). In parallel another set of B103 cells was preincubated at 37~ for 45 min with the anti-N4 serum at a 1:4 dilution (sufficient to saturate the cells in a binding assay) and then assayed. No effect on ~Na § uptake was observed. O, 2 m M ouabain; 0, 2 mM ouabain + 0.2 raM veratridine + 20 ~g/ml scorpion venom; [3, 2 mM ouabain + anti-N4; ~, 2 m M ouabain + 0.2 mM veratridine + 20 ttg/ml scropion venom + anti-N4.
VOLUME83, 1981
three pseudo-neurons B19, B82, and B108 (Fig. 1B); we designated this antigen N5. All other cell lines tested showed only the characteristic 3000- to 4000-cpm background binding such as seen in Fig. 2B. Thus although there are many similarities between the neurons and pseudo-neurons, there are also differences. The pseudoneurons express the N5 antigen, but lack the NI, N2, and N3 antigens found on the neuronal lines (Stallcup and Cohn, 1976). These antigenic differences are interesting in light of the major physiological distinction between the neurons and pseudo-neurons, namely, that although the latter cell lines had Na + and K + channels they failed to generate action potentials (Schubert et al.,1974). Although the failure to spike could be due to technical problems in making satisfactory penetrations of the cellswith microelectrodes, the parallels between the serological data and the electrophysiological data (summarized in Table 1) suggest that the failure to observe the expected action potential is not random. Perhaps there is a more interesting basis for the failure
BEll
30
15
13 JEll
10
20
'o
o
a Z
a Z -t
-I<
x
0,,t =Z o. 0
0
=E o. (J
10
B103 192. B 2 8 ~7 E49. B 8 2 50 pg u
100
50
100
pg yG
FIG. 4. Antiserum raised against B9. Details as in Fig. 1. (A) anti-B9 absorbed with B103, B65, and Bg0 yields Anti-G2. (B) Anti-B9 absorbed with B103, ]365, B90, B92, and t349 yields anti-NG1, e , B E l l ; r~, B9; A, B108; ~7, B82; I , B28; O, B92; O, ]349; 4, B65; )<, B103; v , B90; ~, B35.
WILSON, BAETGE, AND STALLCUP
40: B49
I S C6 / ~ , /
3O
Bill
's z
~ 2o
al Z a.
/ ~
f I0
~ 20
9 30
40
50
BBo
60
/~g TG FIG. 5. Antiserum raised against B49. Details in Fig. 1. Anti-B49 absorbed with B103 and B65 yields anti-NG2. $, B49; O, C6; n Bl11; I , B19; O, ~HC; A, B82; V, B65; V, B92; O, B103; A, B90; e , B108.
of the pseudo-neurons to spike--possibly a difference in the Na + channel that is not reflected in the ~ a + uptake assay or possibly the immaturity of some other membrane property that is required to initiate a regenerative spike. These three cell lines do not represent an isolated phenomenon. Veratridine-activated, tetrodotoxin-sensitive Na + channels have also been found in several other types of cells that normally show no evidence of Na + spikes. These include embryonic cardiac muscle cells (Galper and Catterall, 1978; Fosset et al., 1977), a bladder cell line (Romey et al., 1979), pancreatic fl cells (Donatsch et al., 1977), and crab sensory neurons (Lowe et al., 1978). The anti-B82 and B108 antisera failed to define any antigens shared by the pseudo-neurons with glial or pseudo-glial cell lines. Absorption of the anti-B82 serum with the neuronal lines B35 and B103 resulted in complete loss of reactivity, while absorption of anti-B108 with either B35 or B103 led to a serum with anti-N5 specificity. Thus by these criteria, also, the pseudo-neuronal lines were more closely related to neuronal cells than to glial or pseudo-glial cells. However, this did not explain the observation that B19, B82, and B108 each expressed the G2 antigen. We thought that perhaps this problem stemmed from the anti-G2 serum having more than one specificity and that the paradox might be resolved by further absorbing the anti-G2 serum in such a way that B19, B82, and B108 would no longer be re-
151
Antiserafor Nerve-Glial Cell Lines
active. This strategy was only partially successful. The anti-G2 serum, prepared by absorbing the anti-B9 serum with the neuronal lines B103 and B65 and with the glial line B90 (Fig. 4A), could be further absorbed with the B49 and B92 cell lines to yield an antiserum with reactivity against only three lines--B9, B E l l , and B108 (Fig. 4B)--and attempts to absorb further with B108 completely abolished reactivity. We called this antigen NG1 since B108 was pseudo-neuronal and B9 and B E l l were pseudo-glial. This situation had a parallel in the analysis of an antiserum made against another pseudo-glial cell line, B49. Absorption of this antiserum with the two neuronal lines B103 and B65 resulted in a reagent that reacted with only seven cell lines--B49, C6, Bl11,/~HC, B19, B82, and B108 (Fig. 5). The first four of these lines were pseudo-glial and the latter three were pseudo-neuronal, so we called this antigen NG2. Attempts to absorb further with B82 or B108 at this point led to complete loss of activity. Thus at the limit of their specificity both the anti-B9 serum and the anti-B49 serum reacted with combinations of pseudo-glial and pseudo-neuronal lines. The distributions of these two antigens are summarized in Table 1. It is particularly striking that NG1 and NG2 were not found on any of the cell lines that could be classified as unambiguously neuronal or glial. This supports and emphasizes previous distinctions concerning
B
9
9 x
5 minutes
10 minutes
FIG. 6. Effect of anti-NG1 and anti-NG2 sera on 8SRb+ efflux. S~Rb+ efflux from B9 and ]349 cells was induced with 100 m M KC1 as described previously (Arner and Stallcup, 1981). In para[lel other cultures of B9 and B49 were preincubated at 37~ for 45 min with 1:4 dilutions of the anti-NG1 and anti-NG2 sera, respectively, and then assayed for efflux. No effect was seen on the B9 cells, and with the ]349 cells the KCl-induced efflux remained unchanged after correcting the data for a small decrease in the rate of background efflux. (A) B9 (+anti-NG1). (B) B49 (_+anti-NG2). O, 2 m M ouabain; @, 2 m M ouabain + 100 m M KCI; n, 2 m M ouabain + antiserum; O, 2 m M ouabain + 100 m M KCI + antiserum.
152
DEVELOPMENTAL BIOLOGY
VOLUME83, 1981
NG 2 Z i-
~4 o3 w :>
~2
N3
_1 LU n,.
o
J
J a,tul~ DAYS
FIG. 7. Schedule for appearance of antigens in r a t brain. Quantitative absorption experiments were performed as described previously (Stallcup, 1977b) to assess the presence of antigens in brain homogenates from rats of different ages. "Relative amount of antigen" present is plotted in arbitrary units and is m e a n t to refer to each antigen individually. T h a t is, i~ is not implied t h a t the antigens are present in roughly equal amounts; they have only been plotted in this way to facilitate comparison of the curves. The actual level of antigen in t e r m s of the number of molecules is not known in any of these cases.
pseudo-neurons and pseudo-gila made on the basis of other data, namely, the presence of the N5 antigen and lack of action potentials in the pseudo-neurons, separating them from neurons, and the presence of K + channels in the pseudo-gila, separating them from the gila. Furthermore, among the pseudo-glial cell lines the serological distinction between NG1 + cells and NG2 + cells is paralleledby a physiological distinction between the K* channels of these cells. NG2-bearing pseudo-gila have K + currents that can be blocked by tetraethylammonium or 4-aminopyridine, but not by scorpion venom. These cells are designated as constituting Group 3A in Table 1, as defined in the preceding paper (Arner and Stallcup, 1981). NGl-bearing pseudo-glial cells, on the other hand, exhibit K + currents that cannot be blocked by tetraethylammonium, 4-aminopyridine, or scorpion venom. These cells are in Group 3B in Table 1. These findings once again follow our general theme that it is possible to define cell surface markers for physiologically distinct groups of cells. On the chance that the anti-NG1 and anti-NG2 antisera actually recognized the K + channels of the Group 3B and Group 3A pseudo-glia, respectively, we tested the ability of the antisera to block KCl-stimulated S6Rb+ efflux from B9 and B49 cells (see Arner and Stallcup, 1981). Figure 6 shows that SeRb+ effluxwas unaffected by the antiserum in each case. Thus the antisera either do not recognize K + channels or else do not interfere with K + channel function. The findingthat pseudo-neuronal lines,with both N a + and K + channels, share antigens with pseudo-gliallines
with only K + channels lends some support to an earlier hypothesis of ours that pseudo-neurons and pseudo-gila might represent cells of common lineage arrested by the process of transformation at different stages of differentiation (Arner and Stallcup, 1981). The acquisition or loss of Na + channels could be the process which links these two cell types in a developmental sequence. Consistent with the idea that these cell lines are incompletely differentiated precursor cells rather than cells in terminal stages of differentiation is the finding that one of the pseudo-glial lines, C6, can be induced by special culturing conditions to differentiate further to a form that closely resembles mature astrocytes (Silbert and Goldstien, 1972; Bissell et al., 1974). It would be of great interest to know whether this differentiation, marked by morphological changes and the appearance of the astrocyte-specific glial fibriUary, acidic protein (Bignami et al., 1972), is accompanied by changes in surface antigens and ion channels, but to date we have been unable to induce C6 cells to differentiate in sufficient numbers to make very satisfactory determinations of these parameters. The few differentiated C6 cells t h a t we have observed (five or six out of several hundred examined, as judged by immunofluorescence with a rabbit antibody against the glial fibrillary acidic protein) seemed to retain the NG2 marker, as judged by immunofluorescence with a mouse antibody against NG2 (see Stallcup 1981) for a description of this antibody and the double labeling protocol). No information on the presence of ion channels could be obtained.
WILSON, BAETGE, AND STALLCUP
Further support for our hypothesis that pseudo-neurons and pseudo-glia are incompletely differentiated precursors of nerve and glia would be gained by finding that some of the other cell lines can be induced to differentiate along other pathways, i.e.,to mature neuronal cells.Evidence from other sets of neural cell lines suggests that this m a y occur in some cases (DeVitry, 1977; T o m o z a w a and Sueoka, 1979). While it m a y be possible to obtain useful data of this type from further examination of the cell lines,a complementary and perhaps more direct approach is to turn to the normal brain. Quantitative absorption experiments show that both the N G 1 and N G 2 antigens are present in normal rat brain. Figure 7 illustrates the appearance of NG2 in the middle of the second week of embryogenesis, while NG1 appears a few days later. Both antigens can be found in the adult brain; thus it is possible that these markers are retained by mature cells that are fully differentiated. The "timetables" for NG1 and NG2 are contrasted with that of one of the neuronal antigens, N3, which can be detected only in the fetus (Stallcup, 1977b). The quantitative absorption technique does not reveal a detectable amount of either the N4 or N5 antigen in normal rat brain. Since this technique is relatively insensitive, we do not know whether N4 or N5 are totally absent from normal brain or merely present in small quantities. Nevertheless, the presence of NG1 and NG2 in rat brain shows that these two antigens, which define relationships between pseudo-neuronal and pseudo-glial cell lines, are not tumor-specific markers. This substantially eases our worry concerning possible artifacts stemming from the neoplastic nature of the cell lines and, moreover, offers hope that the antigens can be mapped at the cellular level to reveal something about the identity and developmental fate of the cell types that express these markers in the normal brain. This approach is the topic of the following paper (Stallcup, 1981). We thank Dr. David Schubert and Dr. Jim Patrick for their helpful discussions concerning this work. This research was supported by NIH Grant NS/CA 16112 to W.B.S.
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