Journal of Neuroscience Research 27: 144-152 (1990)

Isolation of cDNAs From a Mouse Astroglial Cell Line by a Subtracted cDNA Library T.A. Rhyner, E. Lecain, J. Mallet, and B. Pessac Ccntrc de Biologie Cellulaire, CNRS UPR 3 101, Ivry-sur-Seine cedex (E.L., B.P.) and Laboratoire de Neurobiologic Cellulaire et MolCculairc, CNRS, Gif-sur-Yvette (T.A.R., J.M.) France

Astrocytes belong to the glial cell population and represent a major subclass of the CNS. Although different subtypes of astrocytes have been described according to their morphological characteristics, biochemical markers of each subtype of astrocytes are not yet available. We have thus undertaken to compare gene expression pattern of different astroglial subtypes. In this study we have taken advantage of two astroglial cell clones derived from 8 day postnatal mouse cerebellar explants and which might be the in vitro equivalents of the velate protoplasmic (D19) and of the Golgi-Bergmann (C8S) astrocytes (Alliot and Pessac, Brain Res., 306: 283-291, 1984). We have constructed a subtracted cDNA library derived from cytoplasmic poly(A)+ RNAs of the D19 cell line. This library was enriched 12-fold for D19 specific sequences by subtractive hybridization with an excess of cytoplasmic poly(A)+ RNAs purified from the C8S astroglial clone. This subtracted library was differentially screened with cDNA probes derived from D19 and CSS cell lines; both probes were subtracted with C8S poly(A)+ RNAs. Eight cDNA clones corresponding to transcripts overexpressed in D19 were selected. Three cDNAs encode for smooth muscle actin, one for fibronectin and one for polyadenylate binding protein. The three other gene products have not been previously reported. The in vivo distribution pattern of these sequences in various mouse adult tissues shows that all these transcripts are expressed in the cerebellum andlor in the brain. Key words: central nervous system (CNS), protoplasmic astrocytes, fibronectin, actin IKTRODUCTTON The glial cells of the central nervous cyTteni (CNS), which include astrocytes. oligodendrocytes, and the microglia, outnumber the neurons and can make up to 50% of the total cell volume. The astrocytcs, which represent around one half of the total glial population, comprise distinct morphological cell types located in the grey as well as in the white mattcr A5trocytes have a 0 1990 Wiley-Liss, Inc.

variety of active roles in maintaining normal brain physiology (Kimelberg and Norenberg, 1989). For example, astrocytcs arc known to occupy a critical position in the metabolism of neurotransmitters. Also, some astrocytes known as radial glia have a key role in development of thc brain. In addition. it is known that astrocytes are crucial in regulating the composition of the extracellular milieu and in particular the potassium concentration (for review, see Fedoroff and Vernadakis, 1986). The role of astrocytes can be best approachcd in thc cerebellum, where the development and the architectonics of the neuroglia have been well described. The cerebellar cortex contains two major types of astroglia. the velate protoplasmic astrocytes which are localized in the granular layer where they compartmentalize the somas of the granule cells, and the Golgi cpithclial cells which extend slender processes, the Rcrgmanri fibers, to the surface of the folia (Palay and Chan-Palay, 1974). Anatomical studies suggest that the velate protoplasmic astrocytes and the Golgi epithelial cclls originate from the primary germinal zone early in cerebellar development. Although it has not been established if they derive from the same precursor, Palay and Chan-Palay regard the Bergniann glia and the velate protoplasmic astrocytcs as variants of a single cell type. While it has been shown that granule cells migrate along Bergmann fibers, no role of protoplasniic astrocytes in the cytoarchitecture or in the differentiation process of the cerebellar neurons has yet becn described in vivo. The morphological characteristics that distinguish the different astrocytes must be determined by unique patterns of gene expression which can be investigated by various methods. Previous studies have attempted to

Received March 7, 1990: revised April 23, 1990; accepted May 2, 1990. Address reprint requests to Dr. B. Pessac, Centre de Biolopie Cellulaire CNRS. 67 rue Maurice Gunsbourg. F-94205 Ivry-sur-Seinc cetiex. France. -1 .A. Rhyner's present address is Pharmakologisches lnstitut der Universitiit Zurich, Zurich. Switzcrland.

This work resulted from an equal contribution by thc first two authors.

Astroglial Cell cDNAs Isolation

generate monoclonaI antibodies specific for each astrocytic cell type. While one antibody has been reported to recognize essentially the Bergmann fibers (Schachner et al., 1981), no such antibody has been described that binds specifically to the protoplasmic astrocytes of the cerebellar cortex. Another approach is to investigate the pattern of mRNA expression of the different astroglial cell types. One might expect differences in gene expression between morphologically distinct astroglial cells. These differences in the pattern of mRNA exprcssion might be the result of distinct levels of transcription of the same genes in different astrocytes. It is also possible that each astroglial cell type expresses a distinct set of genes. Furthermore, these genes might already be identified and their transcripts known to be expressed in cell types other than astrocytes. These issues can be addressed by taking advantage of astroglial cell lines which we have previously derived from mouse cerebella (Alliot and Pessac, 1984). They have been previously designated, respectively, as type I1 and type 111 or C8S and D19. One of these inimortalized clones (D19) is comparable to the velate protoplasmic astrocytes, while another clone (C8S) closely resembles the Golgi epithelial cells. Both astroglial clones support the survival of embryonic cerebellar neurons. In addition, when they are cocultured on the “velate protoplasmic” astroglial clone, the majority of the neuronal cells acquire the granule cell-like phenotype (Alliot et al., 1988). We have constructed a subtracted cDNA library from D19 poIy(A)+ RNAs, in order to identify mRNAs specifically expressed in this cell line. We report the isolation of 8 independent recombinant cloncs which are expressed in the CNS, in astrocyte-enriched cell cultures, as well as in non-nervous tissues. Three clones encode respectively fibronectin and smooth muscle vascular a-actin, and the poly A binding protein. The other clones correspond to transcripts uncharactcrized to date.

MATERIALS AND METHODS Preparation of RNAs D19 and C8S cells were cultured in basal medium Eagle (Gibco) containing 10% fetal calf serum. Cells were collected at confluence by trypsinization and suspended in PBS. After centrifugation for 10 mn at 500g, the cell pellet was immediately processed for RNA preparation or stored at -80°C. To prepare cytoplasmic RNA of the D19 cell line, the cell pellet was suspended in 250 mM saccharose, SO mM Tris-HC1 (pH 7 4 , 75 mM KCI, 5 mM MgCI2, 1 mM dithiothreitol, and homogenized with a Dounce pestle 2 min at 4°C. The suspension was centrifuged twicc at 1,5001: and the pellets comprising nuclei were discarded.

145

Guanidinium thiocyanatc was added to the supernatant to a final concentration of 5 M. Cytoplasmic RNAs were then prepared according to the procedure described by Civelli et al. (1982). The total RNAs from mouse (C57 BI 6iJ) tissues and organs were purified using the same procedure. The tissue was frozen in liquid nitrogen followed by its homogenization for 3 min with a Warying blendor in 5 M Guanidinium thiocyanate, 100 mM TrisHC1 (pH 7.5), 20 mM EDTA. The poly(A)-enriched RNAs (poly(A)+ KNAs) were selected by oligo (dT) cellulose chromatography (Aviv and Leder, 1972).

Construction of the Subtracted cDNA Library The subtracted cDNA library was constructed as previously described by Rhyner et al. (1986). The level of enrichment of the cDNA populations was estimated through the calculation of the ratio of the total mass of ss-cDNA eluted from the hydroxylapatite columns (PB 120 and PB 400) and the mass of unhybridized ss-cDNA eluted in the PR 120 fractions. Double stranded cDNA synthesis. After subtraction, the dried pellet of unhybridized ss-cDNA was dissolved into water, disaggregated 1 min at 95”C, and chilled in dry ice. The synthesis of the second strand was performed in HEPES 100 mM (pH 6.9); 10 mM MgC1,; 70 mM KCI; 1 mM each dATP, dTTP, dCTP, dGTP; 800 pCiiml [a-32P]dCTP, 2.5 mM DTT, and 800 Uiml Klenow fragment of Eschrrichia coli DNA polymerase I. After 20 hr of incubation at 15”C,the reaction was stopped by addition of EDTA to a final concentration of 10 mM. The cDNA was phenolichloroform-extracted and purified by filtration on a Biogel P10 (Biorad) column. The synthesis of double-stranded (ds) cDNA was completed with reverse transcriptase 400 Uiml in 100 mM Tris-HC1 (pH 8.3); 140 mM KCI; 10 mM MgCl,; 1 mM each dATP, dCTP, dGTP, dTTP; 28 mM P-mercaptoethanol, by incubation for 2 hr at 37°C. The reaction was stopped by addition of EDTA to a final concentration of 10 mM. The sample was phenoUchloroform-extracted, ethanol prccipitated. and digested with 120 Uiml S, nuclease in 50 mM sodium acetate (pH 4.5), 300 mM NaCl, 1 mM ZnSO, and 1 mM Z,,C1,, 30 min at 37°C. The sample was phcnoli chloroform-extracted and loaded on a Biogel P10 column. The ds-cDNA was made blunt-end in 30 mM Trisacetate (pH 7.9); 66 mM potassium acetate: 10 m M magnesium acetate; 100 p g h l BSA; 500 p M DTT; 240 p M each dATP, dCTP, dCTP. dTTP containing 200 Uiml T, DNA polymerase. After incubation for 20 min at 37”C> the reaction was terminated by addition of EDTA to a final concentration of 80 mM. The sample was phenol/ chlorofonn-extracted and ethanol-prccipitated. Preparation and ligation of Barn HI linkers. The ds-cDNA was inserted into the Bam HI site of pSPTl8

146

Khyner et al.

(Pharmacia) according to the procedure described by cyloplxsmic poly(h) .'- RNA of D19 xstrocytic cell line Bahl et al. (1976). First, 100 pgiml of Barn HI linkers I were heat denatured, chilled on dry ice, and phosphorysinglc s t r a n d c d cDNA lated in 66 mM Tris-HC1 (pI1 7.6), 800 pCiiml [LU-~'PP] cy1opl;isinic poly(A) RSA ATP, 10 niM MgCI,, 10 mM DTT, 100 pgiml BSA or C8S astrocytic ccll line containing 1,000 Uiml T4 polynuclcotide kinase. After sublraclivc Ilybrirlimlion an incubation of 30 min at 37"C, a chase was performed (ERot= 5700 Ms) for I hr at 37°C with addition of ATP and T4 polynucleotide kinase to final concentrations of 100 p M and 200 Uiml, respectively. unliyliridizcd cDNA The phosphorylated Barn HI linkers (80 pgiml) I were ligated to the ds-cDNA in 10 mM MgCI,, 66 niM ds cDNA Tris-HCI (pH 7.6), 10 mM DTT, 100 pgiml BSA, 1 mM ATP, and 250 Uiml T, DNA ligase, overnight at 15°C. The sample was then treated for 2 hr with 20 U of Barn 12 000 rccambinants HI (Boehringer) in the buffer recommended by the manDiTTcrential colony l~ybricliz:~tio~i wit11 cDNA clones ufacturer. Digested linkers as well as short ds-cDNA 3 cco rccon:binants were eliminated by filtration on Ultrogel AcA 34 (IBF). The gel suspension was equilibrated in 10 mM Tris-HCI s u b t r a c t e d CSS c D S A p r o b c (pH 8.0), 300 mM NaCl. 1 mM EDTA, 0.0S% SDS. s u b t r a c t e d U 1 9 c U K A pl-obc The ds-cDNh sample containing 2 mM EDTA and Fig. 1. Scheme of the strategy to generate the subtracted 0.001 9% Bromophenol blue wax loadcd on a 300 x 1 mm cDNA library. packed column. The fractions containing ds-cDNA cxcluded from thc gel were ethanol precipitated. The sam72 ple was then dissolved in water and inserted into the Bani insertions P-labelled by nick translation (Amcrsham HI site of the pSPT I8 vector as described (Rahl et al., kit). had a specific activity above 10' cpmipg of DNA. Filters were washed with SSC X 0.1-SDS 0.1 Ti, 3 x 20 1976). mn at 50°C. E. coli MC 1061 (Casadaban and Cohen, 1980) cells were transformed with the recombinant plasmids according to the procedure described by Hanahan ( 1983) Sequence Determination The nucleotidc sequence determination was perand transformants selected on ampicilline (50 pgiml) formed according to the dideoxynucleotide chain termiplates . nation method in M 13 (Sanger et al., 1977) or in pspT I8 (Chen and Seeburg, 198s) vectors. Ambiguities originatScreening of the Library ing from band compressions were removed by using 'The library was screened with subtracted cDNA deoxy-7-deasaguanosine triphosphate instead of dGTP probes as previously described (Khyner et al., 19%). (Mizusawa et al., 1986).

t

-

+

1

t

t

f

Northern Blot Analysis The RNA samples were pretreated, run on a denaturing agarose gel and transferred on a nitrocellulose filter according to the method described by Faucon-Biguct et al. (1986). The filter was prehybridized at least 4 hr at 42°C in 50% formamide, Denhardt X 5 (Denhardt x 50 is 1 % Ficoll, 1% polyvinylpyrolidone, 1% BSA), SSC X 5 , (SSC X 20 i s 3 M Nacl. 300 mM sodium citrate, pH 7.0), 50 mM phosphate buffer, pH 7.0, 0.3%)SDS, denatured herring sperm DNA 20 pg/ml. Hybridization was performed in 50% formaniide, SSC x 5 , Denhardt x I , 20 mM phosphate buffer, pH 7.0, 10% dextran sulfate, 0.3'10 SDS, 20 pgiml denatured herring sperm DNA, and 10' cprn/ml nick-translated and denatured DNA probe. The probes used. i.c., recombinant plasmids or the

I

RESULTS Construction and Screening of the Subtracted cDNA Library In order to select mRNAs characteristic of the D I9 ccll line: likely to be of moderate to low abundance, we have generated a subtracted cDNA library following the subtraction procedurc oullined in Figure 1, which rem o w s abundant sequences. Six micrograms o f cytoplasiiiic poly(A)+ RNAs yielded 1.3 pg of 3'P-labeled single-stranded cDNAs (ss-cDNA). The Ss-cDNAs were then hybridixd under stringent conditions with a large excess of cytoplasmic poly(A)+ RNAs from the C8S cell linc. After 36 hr of hybridization, an cquivalent Rot (ERot) of 5,700 Ms was reached using the corrections for salt concentration reported by Britten ct al. (1974). The

Astroglial Cell cDNAs Isolation

mixture was loaded on a hydroxylapatite column. The 120 mM phosphate buffer fractions which included the unhybridized ss-cDNA contained 8.5% of the eluted radioactivity. The 91 .S% of the eluted counts were present in the 400 mM phosphate buffer fractions which contained the hybridized ss-cDNA. Therefore, the D19 subtracted cDNA library was cnriched about 12-fold in sequences specific for the D19 cell line transcripts. About 100 ng of ss-cDNA were obtained, transcribed into a double-stranded form, and inserted into the plasmid pSPT 18. This procedure yielded 12,000 recombinants. The sizes of cDNA insertions of this D19 subtracted cDNA library ranged from 100 to 300 base pairs (bp). Three thousand recombinants from this library were manually arranged on filters and were screened with cDNA probes. To select cDNA clones specifically expresscd in the D19 astroglial cell line relative to the C8S cell line, we performed a differential scrcening procedure deliberately limited to cDNA probes prepared from D19 and C8S poly(A) RNAs. The specific and control probes were derived from D19 and C8S poly(A)+ RNAs, respectively. To increase thc hybridization efficiency of low and moderately abundant sequences, we used subtracted probes (Rhyner et al., 1986). The specific probe was a subtracted D19 cDNA probe prepared with the same poly(A)+ RNAs batch that was used to gencratc the library. Subtraction of this cDNA probe was performed with C8S poly(A) ' RNAs to deplete the cDNA population of abundant and common sequences. To reach a comparable colony hybridization sensitivity with the control C8S cDNA probe, wc have generated a selfsubtracted C8S cDNA. Thus, the two probes differed exclusively by the origins of the RNA templates. A 5 7-fold enrichment for spccific and low abundant sequences was obtained at an ERot of about 3,000 Ms for both probes. Both radiolabelled probes had the same specific and total activities in order to reliably compare the intensity of hybridization signals. Among the 3,000 recombinants screened, only 400 colonies yielded a detectable signal with the D19 substracted cDNA probe. The clones which were not detected under these conditions correspond to sequences present in the probe at a too low level to generate a positive hybridization signal relative to the background signal. This preliminary differential screening yielded 100 clones, which responded differently with the two probes. These clones were subjected to a second differential screening experiment with the same probes and the same conditions. This step enabled us to select eight clones hybridizing at a higher level of intensity with the D19 probe. These cDNAs were candidates to encode specific markers of the D19 astroglial cell line and were further analyzed. +

147

Northern Blot Analysis With the D19 and C8S Cell Lines mRNAs To ascertain that these selected clones designated as p4, p5, p7, p14, p44, p47, p.57, and p80 are differentially expressed, mRNAs from both cell lines were probcd by Northern blot analysis. Figure 2 indicates that these clones hybridized to transcripts that are overexpressed in the D19 cells. The hybridization patterns of each of five clones were clearly unique while p7. p44, and p80 showed an identical hybridization pattern with the same mKNA sizes. ~

Characterization of the Cloned Sequences In order to know if these clones correspond to previously reported sequences, we determined their primary nucleotide structure. Indeed, p.5 encodes the poly A binding protein, p80 encodes an actin isoform, and p47 encodes firbonectin while the other sequences did not reveal any significant homology with any cDNA described to date when compared with databases (GENBANK; EMBI,) (Fig. 3). Clone p5 shows a high degree of homology with the 3' UI'K of the human poly A binding protein (Grange et al.. 1987). The size of the mRNA is 3.1 kb, and this messenger is expressed in all the tissues investigated and in secondary cultures of astrocytes. The sequence of clone pX0 is identical to that of mouse smooth muscle a-actin recently published by Min et al. (1988). To ascertain whether this actin isoform is expresscd in astrocytes. niRNAs from secondary cultures of 8 day postnatal cerebellar astrocytes were probed with p80. Table 1 shows that these cells express both cytoplasmic and smooth muscle actin isoforms. The p47 clone which is 100 bp in length is aligned with that of rat fibronectin mRNAs in Figure 3 (the mouse fibronectin sequence has not yet been described). Although the sequence homology maps in the 3' untranslated region (3' UTR) of the rat mKNA (Schwartzbaucr et al., 1983), the two nucleic acid sequences differ only in 18% of positions. Such a high degree of conservation in the 3' U'TK between fibronectin sequence from rat and human sequences has been reported by Schwarzbauer (1983). Furthermore, the si7e of the transcript recognixed by p47 is in agreement with that reported for human fibronectin (Kornblith et al., 1983). In addition, expression of fibronectin expressed in astrocytes cultures has been previously reported (Liesi et al., 1986). l'hc three other sequences have not been previously described. We therefore investigated whether corresponding transcripts are expressed in the adult mouse CNS. The results of Table 1 show that all the recombinants hybridize with transcripts expressed in the forebrain and in astrocyte-enriched cultures. All transcripts

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Rhyner et al.

Fig. 2. Northern blot analysis with RNAs from the D19 and C8S astroglial cell lines probed with isolated cDNA recombinants. 3 pg of poly(A)+ RNAs were loaded on each lane. except for the p57 lanes where 1 p g of poly(A)+ was loaded. All the autoradiograms were exposed 1 week at -70°C with an intensifying screen.

MOUSE

10 20 30 40 50 60 GAGACACTTGTAGCCAACTAGGAGG~TGTACTGAATGCTAGTACCC~GACCTTGAGCAGG~GTC

.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ............

GTAGCCAACCACGATGCAATGAATTGAATGGTAGTACCCAGTTA 2650 2 670 2690

RAT

70

80

90

MOUSE

ACCCAGACACCTCTGCTTTCTTTTGCCG

RAT

AACCASACAGTCTGCTTTCTTTTGCCG 2710 2730

a

.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. ........

10 MOUSE

20

30

40

50

60

70

AACTTTGAACCTTATGTACCGAGCAAATGCCAGGTCTAGGTCTAGC~CAG--TGCTAGTCCTAGATTACTTGATTT~C~C(A)26

.. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. .. HUMAN

b

.. .. ..

AACTTTGAACCTTATGTACCGAGCAAATGCCAGGTCTAGGTCTAGC~CAT~TGCTAGTCCTAGATTACTTATTGATTT~A~C.. 2540

2560

2580

2600

Fig. 3. a: Correspondence between 3' UTR of the rat fibronectin (Schwait7bauer et a ] . , 1983) and the mouse p47 cDNA. b: Correspondence between the 3' LJTR of the human polyadenylate binding protein (tirange et al., 1087) and the mouse cDNA.

Astruglial Cell cDNAs Isolation

149

TABLE I. Distribution and Kelative Abundance Corresponding to the Transcripts in Different Tissues and in Secondary Astrocvte Cultures* cDNA clones

S i x of transcripts (Kb)

Cerebellum

Forebrain

4.8 3. I 2 8 2.7 2 1.4

+ ++ ++ + 0 ++ +

++ ++ ++ +

P4 P5 P‘4

P47 P57 P80

*

+t

+

Liver

+ + i

+++

Digestive tract

0

?

-L

t+ t++

+

Spleen

+ i i + ++ + 0 ++ +

+ +++ ++ +

Skclettal rnuxles

Heart

++ +

++ ++ +++ +

0 0

0

0 i

+++

0

++

Secondary culturcs of astrocytes

+++ +++ +++ t ++ t ++ +++

*The autoradiograms were exposed during it week at - 7 0 T with an intensifying screen. Each lane w a loaded with 3 p g of poly(A)+ or with 20 pg of total RNAs for skeletal muscle. heart. and hecondary culturc of astrocytes, except for clone p14. where 60 p g of total RNAs were loaded. + + + = strong hybridization signal; + + = moderate hybridization signal: = weak hybridilation signal; = = barely detectable hybridization signal

-

also hybridize to mRNAs of non-nervous tissues. However, each clone has a distinct pattern of expression (Fig. 4). Clone p4 hybridizes to a 4 . 8 kb transcript and is present in liver, intestine, spleen, and heart. Notably, the inRNA p4 was not detectable in striated skeletal muscles. Clone p14 recognizes a single transcript of 2.0 kb. It is the only cloned transcript that is expressed in all the tissues tested, including striated muscles. Clone p57 hybridizes to a 2.7 kb niRNA. In contrast to the other mKNAs described above, the distribution is restricted to forebrain and intestine. In addition, a detectable signal was seen in secondary cultures of astrocytes. In these tissues and astrocytes. the expression of this mRNA is very low. Taken together the results show that recombinant clones isolated from the astroglial D 19 cDNA library with probes only derived from astroglial cell lines correspond to inRNAs expressed in the CNS as well as in other tissues of the adult mouse. Furthermore, these inRNAs are present in enriched astroglial cultures. suggesting that they may also be expressed in astrocytes in vivo.

DISCUSSION Numerous attempts have been made to generate probes specific for nervous cell populations in the CNS. Advantage has been taken of mutant mice in which a distinct cell type is considerably reduced or missing. The mouse cerebellum is particularly well suited to these studies since many mutations have been described (Sidman, 1983). A first approach was directed to the preparation of antibodies specific for the Purkinje cells. The antiserum raised against an enriched preparation of isolated rat Purkinje cells was made specific through a series of absorptions with membranes from different tis-

sues (Mallet et al., 1979; Woodhams et al.: 1979). Recently, Nordquist et al. ( I 988) and Oberdick et al. ( 1988) have analyzed cell-type-specific markers at the gene expression level. They have used “Purkinje cell degeneration” and lurcher mutant mice respectively to isolate transcripts specific for Purkinje cells. These different investigations were made possible by the availability of mutations affecting a single neuronal type of the cerebellum. However, this approach cannot be used to investigate gene expression specificity of the different types of astrocytes since no mutant affecting astroglial cells has been described. Therefore, we have used an alternative experimental system: we have taken advantage of immortalized cell lines with astrocytic characteristics, which we had derived from postnatal mouse cerebellum. The D19 and C8S cell clones may be regarded, respectively, as the in vitro counterpart of the velate protoplasmic astrocytes, while the C8S astroglial clone may be the in vitro equivalent of the Golgi epithelial cells and their processes, the Bergmann fibers (Alliot and Pessac, 1984). Therefore, these cell lines appear to be a convenient paradigm to isolate RNAs specific for distinct astroglial cell types. We report here the isolation and preliminary characterization of cDNAs corresponding to genes abundantly expressed in the astroglial cell line D19. The C8S and D19 cell lines both belong to the astrocytic lineage, and are likely to be highly similar at the transcriptional level. Thcrcfore, to facilitate analysis of specific messages, the subtracted cDKA library derived from D19 cytoplasmic poly(A)+ RNAs was enriched by hybridization of D 19 sequences with an excess of C8S poly(A) RNAs. The 12-fold enrichment obtained through a single step of subtrdctivc hybridization for the construction of the D 19 cDNA library greatly facilitated the screening. We performed a differential screening with 3.000 clones which might represent 36,000 clones of a nonsubtracted



150

Rhgner et al. Ut v)

3

Ut c1 I

3

4.8-

P4

a

*

2 .o-

PI4

2.7-

C

*

library. The stringent hybridization conditions used here resulted in fairly small inserts which were adequate for screening and for Northern blot analysis. To improve the sensitivity of the screening, wc used subtracted D19 and C8S cDNA probes as the enriched probes have a higher concentration of rare to moderately abundant sequences. As anticipated, the large majority of the analyzed sequences were found to be expressed at a similar level in both cloned cell lines. We have found that 8 recombinants. out of 3,000 recombinants screened, corresponding to six distinct mKNAs arc clearly ovcrexpressed in the D19 cell line versus the CXS cell line. Thus we conclude that these two astroglial cell lines appear to express the same set of genes, but sume of them are transcribed with very different degrees of expression. Among the cDNAs we have isolated. three encode smooth muscle vascular a-actin, and two cDNAs map, respectively, to the 3‘ UTR of fibronectin and of poly A binding protein RNA. The three other cDNAs investigated have not yet been described and are expressed in forebrain and/or in cerebellum of adult mouse. The sequence of clone p5 shows a very unusual high degree of homology with the 3’ UTR of the human polyadenylate binding protein transcript. This may suggest that this region plays a role in the regulation of the expression of the protein. This protein would be involved in the regulation of the half-life of poly(A)+ (Bernstein and Ross, 1989). It is not clear why it is overexpressed in the D19 versus C8S cell line. The overexpression of fibroncctin by thc D I9 vcrsus the CXS cell lines may be related to their morphologies and cell adhesion properties. While D19 cells are flat, spread, and strongly attached, C8S cells are fusiform, weakly anchored to the plastic, have a tendency to aggregate at high density, and never totally cover the bottom of the flask. The presence of fibronectin immunoreactivity has also been described in the embryo and in the first postnatal days in rodent cerebral (Stewart and Pcarlman, 1987) and cerebellar cortices (Hatten et al., 1983) in unidentified cell types. In the adult CNS, fibronectin has been detected in the blood vessels, meninges, and in choroid plexus (Schachncr et al., 1978). It has also been shown that primary cultures of “flat-like” astrocytcs can synthesize this protein (Liesi et al., 1986). It remains to establish whether the fi bronectin transcript Northern blot distribution of the transcripts corresponding to p4, p14, and p57 cDNAs in different adult mousc tissues and in secondary astrocyte cultures. 3 p g of poly(A) + or 20 p g of total RNAs(*) were loaded on each lane, except for clone p13 where 60 k g of total RNAs(*) wcre loaded. Cbm: cercbellum; Fb: forebrain; Spl: spleen; Int: intestine; Liv: liver; Hea: heart: Sk Mus: skeletal muscles: Ast cult: secondary cultures of astrocytes. Fig. 4.

P 57 Fig. 4.

Astroglial Cell cDNAs Isolation

revealed by Northern blot in brain reflects the synthesis of fibronectin by a subset of adult astrocytes. The smooth muscle a-actin isofon11 has been described in vivo in smooth muscle vascular cells, and in vitro in Rat-2 and in NTH 3T3 cells (Leavitt et a]. , 1985). However, the data reported here have shown that secondary cultures of astrocytes express both cytoplasmic and smooth muscle a-actin isoforms. These cultures. which coniprise large flat astrocytcs, are thus similar to D19 astrocytcs. In this respect, it is of interest to note that smooth muscle actin has been correlated with the flat morphology of NTH 3T3 and Rat-2 cells (Leavitt et al., 1985). Moreover. actin is a protein of the cytoskeleton and interacts with fibronectin in the extracellular matrix (Singer, 1979). One can thus hypothesize that fibroncctin and smooth muscle actin, which are co-expressed in the D19 astroglial ccll line, interact to maintain the morphology of this cell. It will now be of irnportancc to investigate whether smooth muscle actin is indeed expressed in vivo in this subclass of astrocytcs and if this gene product constitutes a biological marker for subclasses of astrocytes. The roles of the three other clones remain to be established. The corresponding transcripts were all overexpressed in the D19 astroglial ccll line as compared to the C8S cell line. The distribution pattern in vivo has revealed that thc corresponding transcripts of the three clones are all expressed in the forebrain and/or in the cerebellum and in different peripheral tissues. In addition, these transcripts are all expressed in secondary cultures of cerebellum highly enriched in flat-like astrocytes, suggesting that they might be expressed in astrocytes in vivo. This result is not unexpected sincc the D19 cDNA library was only screened with probes derived from astroglial cell lines and not with probes constructed from the CNS or from non nervous tissues. This is in contrast with the approaches of Travis et al. ( 1987) where only transcripts specific for the CNS were deliberately selected. Indeed, many markers are sharcd by nervous and non-nervous cells. T h u s, the Thy I protein, which was initially described in a subset of thymocytes, is expressed by Purkinje cells in the cerebellum (Williams et al., 1976; Bolin and Rouse, 1986). Furthermore. it has been recently shown that the RNA encoding calbindin Il,,Kis identical in the Purkinje cells of the cerebellum and in the kidney (Nordquist ct a]., 1988). Similarly, the mRNAs reported here may be markers for a subset of astrocytes as well as for other cell types in non-nervous tissues. Moreover, the samc protein may play different functions according to the tissue in which it is expressed. For example, Wistow et al. (1987) have shown that e-crystallin appears to be identical to duck lactate dchydrogcnase B4 and Piatigorski et al. (1988) reported that duck 8-crystallin and thc mctabolic enzyme

151

argininosuccinate lyase are certainly encoded by the same gene. In order to investigate the putative role(s) of thc proteins encoded by the new transcripts reportcd here, experimcnts are in progress to localize their sites of expression in the mouse CNS and peripheral tissucs at different stages of the development. Such experiments could prove to be a productive merger of molecular and neurobiological approaches towards investigating the mechanisms underlying CNS ontogenesis.

ACKNOWLEDGMEIVTS The authors wish to thank Franpise Alliot for her help and advice in tissue culture all over the years: Patricia Gaudoin for typing the manuscript and Michel Loucttc for the photographic work. This work was supported by the C.N.R.S., the Ministere de la Recherche et de la Technologic (No. 87.C.0527), Fondation pour la Recherche MCdicale. the F.E.G .E.F.L.U.C., and A.K.C. (No. 6777).

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Isolation of cDNAs from a mouse astroglial cell line by a subtracted cDNA library.

Astrocytes belong to the glial cell population and represent a major subclass of the CNS. Although different subtypes of astrocytes have been describe...
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