Differential regulation of the 2′,3′-cyclic nucleotide 3′-phosphodiesterase gene during oligodendrocyte development

Neuron,

Vol. 12, 1363-1375, June, 1994, Copyright

0 1994 by Cell Press

Differential Regulation of the 2’,3’-Cyclic Nucleotide 3’-Phosphodiesterase Gene during O ligodendrocyte Development Steven S. Scherer,* Peter E. Braun,+ Judith Grinspan,’ Ellen Collarini,§ D.-y. Wang,* and John Kamholz* *Department of Neurology Hospital of the University of Pennsylvania Philadelphia, Pennsylvania 19104 +Department of Biochemistry and Neurology McGill University 3655 Drummond Street Montreal Canada H3G lY6 *Division of Neurological Research Children’s Hospital of Philadelphia Philadelphia, Pennsylvania 19104 SDepartment of Biology Cower Street University College London London WCIE 6BT England

Summary The two major isoforms of 2’,3’-cyclic nucleotide phosphodiesterase (CNP), 48 and 46 kDa, have recently been shown to be produced from a single gene by alternative splicing. In addition, messenger RNA encoding the larger isoform is transcribed from a separate promoter, approximately 1 kb upstream from that encoding the smaller isoform. We have investigated the expression of these two CNP isoforms and have found that they are differentially expressed during the process of oligodendrocyte maturation. In oligodendrocyte precursors, only the mRNA encoding the larger protein is found. At the time of oligodendrocyte differentiation, however, both CNP mRNAs are induced. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters during the process of oligodendrocyte differentiation. Introduction Oligodendrocytes, the myelinating cells of the CNS, arise from precursors in the subventricular zones (SVZ) during late embryonic and early postnatal life (Privat, 1975). These cells are highly motile and can be recognized by their expression of a unique set of markers, including the platelet-derived growth factor a receptor (PDGF-aR), the ganglioside Co3, and a glycolipid recognized by the antibody A2B5 (Schnitzer and Schachner, 1982; Gumpel et al., 1983; Curtis et al., 1988; Levine and Goldman, 1988; Reynolds and Wilkin, 1988; Pringle and Richardson, 1993). As they migrate out of the SVZ into the brain parenchyma, oligodendrocyte precursors differentiate, and their pattern of gene expression changes. Differentiating oligodendrocytes first express sulfatide, galactocerebroside, and 2’,3’-cyclic nucleotide 3’-phosphodiester-

ase(CNP)(Mirskyetal.,1980;Mongeetal.,1986;Braun et al., 1988,199O; Hardy and Reynolds, 1991) and later express the major myelin structural proteins, proteolipid protein (PLP) and myelin basic protein (MBP), taking on their fully differentiated phenotype (Zeller et al., 1985; Dubois-Dalcq et al., 1987). A marked increase in the accumulation of CNP, MBP, and PLP and their mRNAs then occurs during myelin sheath formation. Although high levels of CNP accumulate in myelinating oligodendrocytes in parallel with the major structural myelin proteins, the onset of its expression occurs before that of either MBP or PLP. In addition, Northern blot analysis of developing brain has shown that CNP mRNA can be found as early as embryonic day 15 (E15), whereas MBP and PLP mRNAs cannot be detected until after birth (Kanfer et al., 1989). These data thus suggest that CNP gene expression is regulated differently from that of either MBP or PLP genes and that CNP may be expressed in both oligodendrocyte precursors and myelinating oligodendrocytes. Recent analyses of the structure of the CNP gene and its mRNAs are important for understanding the above data. Kurihara and coworkers (1990) have demonstrated that the two known CNP isoforms, 48 and 46 kDa in molecular weight, are encoded by a single CNP gene (Monoh et al., 1989). The larger protein, CNP-II, is identical to CNP-I except for an additional 20 amino acids at its amino terminus (Kurihara et al., 1990; Gravel et al., 1994). The transcripts encoding the two proteins differ only at their 5’ ends: a 2.6 kb transcript encodes CNP-I, whereas a 2.4 kb transcript encodes CNP-II. Transcription of the mRNA encoding CNP-II begins approximately 1 kb upstream from that encoding CNP-I at a separate promoter. The primary transcript produced from this distal site then undergoes an additional splicing event, fusing sequences encoded by an upstream exon, including an AUG codon and 60 additional base pairs of coding sequence, to sequences just upstream of the AUG of the smaller protein,addinganadditional20aminoacidsofcoding sequence (Kurihara et al., 1990; Thompson, 1992; Gravel et al., 1994). The two isoforms of CNP are thus encoded by separate, identifiable mRNAs that have been transcribed from different promoters within a single gene. A schematic representation of the CNP gene and its transcripts is shown in Figure 1. Since the two isoforms of CNP are transcribed from separate promoters and translated from separate mRNAs, the expression of each isoform might be differentially regulated, either at the transcriptional or posttranscriptional level. Differential regulation of the two CNP isoforms at distinct stages of oligodendrocyte development could explain some of the differences between the pattern of CNP expression and those of the structural myelin proteins, MBP and PLP. We have thus investigated CNP expression in devel-

NlWrlXl 1364

A. CNP-I

- 2.6 kB

B. CNP-I+II

- 2.6 kB - 2.4 kB -1.6

Figure 2. Northern pression in Neural Figure 1. Schematic Representation of the Structure End of the Mouse CNP Gene and Transcripts Adapted

from

Kurihara

of the 5

et al., 1990.

oping brain and in primary cultures of oligodendrocytes to evaluate the contribution of each of the isoforms to the profile of CNP expression. W e find that CNP-II mRNA is present at low levels in oligodendrocyte precursors, but that both CNP mRNAs are induced to much higher levels when oligodendrocytes differentiate and myelinate axons. Activation of CNP expression in vitro occurs in developing oligodendrocytes before that of either MBP or PLP; both CNP mRNAs, however, accumulate in vivo with the same time course as MBP and PLP. Expression from the two CNP promoters is thus differentially regulated during the process of oligodendrocyte development and myelination: transcription only from the distal promoter occurs in oligodendrocyte precursors, whereas transcription from both promoters occurs in differentiated, myelinating oligodendrocytes. The CNP gene is thus expressed throughout oligodendrocyte differentiation, and its expression can be used to identify oligodendrocytes and their precursors, both in vitro as we have shown and in vivo as shown by Yu and coworkers in an accompanying paper (Vu et al., 1994).

Oligodendrocytes and Schwann Cells Express Both CNP-I and CNP-II mRNAs Although CNPase activity is highest in the CNS, Schwann cells in the PNS also express relatively high amountsof CNPase(Sprinkle, 1989). In addition, many other tissues have low levels of CNPase activity (for reviews see Vogel and Thompson, 1988; Sprinkle, 1989). W e investigated the structure of the CNP transcripts found in neural and nonneural tissues using two, nonoverlapping probes derived from a fulllength rat CNP-I cDNA (pCNP7; Bernier et al., 1987): a Pstl fragment, found in both CNP-I and CNP-II transcripts, and an EcoRI-Hindlll fragment, found only in the 5’ untranslated region of the larger, CNP-I tran-

kB

Blot Analysis of CNP-I and CNP-II mRNA Exand Nonneural Adult Rat Tissues

Each lane contains 10 ug of total RNA from the indicated tissue. The blot was successively hybridized with a radiolabeled probe recognizing CNP-I only (A, EcoRI-Hindlll probe) or both CNP-I and CNP-II (B, Pstl fragment). The position of a 1.6 kb band, which may be an additional CNP transcript, is also Indicated.

script. As shown in Figure 2A, only brain and sciatic nerve contained the 2.6 kb, CNP-I transcript, which hybridized with the EcoRI-Hindlll fragment (Bernier et al., 1987). Reprobing the same blot with the Pstl fragment demonstrated two CNP transcripts in adult brain and peripheral nerve of 2.6 and 2.4 kb (Figure 28). All other adult tissues contained a single hybridizing band of approximately 2.4-2.5 kb, similar in size to that of CNP-II mRNA from either brain or sciatic nerve, as previously found by Bernier et al. (1987). CNP-I mRNA, transcribed from the more proximal of the two CNPase promoters, is thus uniquely expressed by oligodendrocytes and Schwann cells. The 2.4-2.5 kb, nonneural CNP transcript is similar in size to CNP-II mRNA, suggesting that transcription from the more distal CNP promoter may occur at low levels in many tissues. In addition to the two major CNPase transcripts described above, we found that adult brain and sciatic nerve also contained small amounts of an additional CNP transcript, approximately 1.6 kb in size (Figure 2; Figure 38). This transcript did not hybridize with EcoRI-Hindlll probe (Figure 2), but was enriched in poly(A) RNA from brain (data not shown). The structure of this 1.6 kb CNP mRNA is unknown. CNP-II mRNA Is Present before the Appearance of Mature Oligodendrocytes; Both CNP-I and CNP-II mRNAs Increase in Parallel with the Major Myelin Protein mRNAs To evaluate the timing of CNP mRNA expression during brain development, we analyzed CNP mRNA in the rat brain stem, cerebellum, and cerebrum from El6 to postnatal day 120 (P120). CNP-I mRNA was first detected at PI in the brain stem, at P5 in the cerebellum, and at PI0 in the cerebrum, at the same time

Differential 1365

Regulation

of CNP

- 1.6 kb - 2.6 kb - 2.4 kb - 3.2 kb D. PLP - 1.6 kb

SBP

- 2.2 kb

LAG

- 30kb -2545

G. GAPDH

- :.4 kb

*.I:

Figure 3. Northern

Blot Analysis

of Myelin-Specific

mRNAs

in Developing

Brain Stem, Cerebellum,

and Cerebrum

Each lane contains 10 pg of total RNA. The blots were successively hybridized with radiolabeled, cDNA probes recognizing CNP-I only (A), both CNP-I and CNP-II (B and C), PLP (D), MBP (E), MAC (F), and GAPDH (G). (B) is a longer exposure of the blot in (C). In (B), note that the2.4 kb/CNP-II transcript appears well before the other myelin-specific mRNAs, including CNP-I, as well as the putative 1.6 kb CNP transcript. The films were exposed for 16 hr (C, D: brain stem only; E, C: all panels), 2 days (D: cerebellum and cerebrum only), and 3 days (A, B, C: cerebellum and cerebrum only; F: all panels).

or slightly before the mRNAs encoding the myelin proteins PLP, MBP, and myelin-associated glycoprotein (MAC) (Figures 3C-3F). In addition, low levels of CNP-II mRNA were detected as early as El6 in cerebrum and brain stem and PI in thecerebellum (Figure 36). During postnatal development, the amounts of both CNP-I and CNP-II mRNAs changed in parallel with thoseencodingtheother myelin proteins in each of these brain regions (Figures 3C-3F). The pattern of CNP-I mRNA expression is thus identical to that of the mRNAs encoding the major myelin proteins, PLP, MBP, and MAG. In contrast, CNP-II mRNA first appears in thedeveloping brain beforeanymatureoligodendrocytes are present, then increases in parallel with the major myelin protein mRNAs during postnatal brain development. These data suggest that transcription from the distal CNP promoter is activated in oligodendrocyte precursors, whereas transcription from both promoters occurs during oligodendrocyte differentiation and myelination.

juvenile rats at P22, near the peak of myelin gene expression, as well as young adult rats at P90, after the peak of myelin gene expression. If CNP expression were regulated similarly to the genes encoding the major myelin proteins, then the steady-state level of CNP mRNA should decline after axotomy (Goto et al., 1990; Kidd et al., 1990; McPhilemy et al., 1990, 1991; Scherer et al., 1992). As shown in Figure 4, enucleation caused the steady-state levels of both CNP-I and CNP-II mRNAs to decline markedly by 12 days, both in juvenile and in adult rats, in a manner similar to the mRNAs encoding MBP, PLP, and MAC. In addition, the level of the CNP-I mRNA declined relatively more than that of the CNP-II, both in juvenile and adult rats. These results demonstrate that expression of CNP-I and CNP-II mRNAs depends on continuous axon-oligodendrocyte interactions and suggest that transcription from the proximal CNP promoter is more sensitive to axotomy than transcription from the distal promoter.

Expression of Both CNP-I and CNP-II mRNAs Is Modulated by Axons To evaluate the dependence of CNP mRNA expression on axon-oligodendrocyte interactions, we examined CNP mRNA levels in optic nerves undergoing Wallerian degeneration. We unilaterally enucleated

CNP-II Protein Is Present before the Appearance of Mature Oligodendrocytes Using antibodies that recognize both CNP-I and CNPII (anti-CNP-l+ll) or CNP-II alone (anti-CNP-II), we investigated the developmental appearance of the two isoforms by Western blot analysis. Figure 5A dem-

NellKMl 1366

Adult

Juvenile

- 2.6 kb - 2.4 kb

A. CNP --.__ - .--- ---w..i-/” ,.l____.--

-

3.2 kb

B. CNP-I+II

B. PLP

C. MBP

-1.6

kb

-2.2

kb

. D. MAG

-3.0 -2.5

kb kb

E. Cyc

-0.8

kb

Figure 4. Northern Blot Analysis of Myelin-Specific pression during Wallerian Degeneration of Juvenile Rat Optic Nerves

CNP-II

the antibody

specificity

of the

recognized

two only

antisera: the

larger,

the

anti-

48 kDa

isoform (lane 5), whereas the anti-CNP-l+ll antibody recognized both CNP isoforms (lane 4). Using the anti-CNP-I+II

antibody

with

conventional

5d

12d

“84

02

“iti

VrV “’ ”

‘.,

Figure 5. Western Blot Analysis of CNP-I and CNP-II in Developing Brain Stem and Cerebrum

Expressron

(A) Alkaline phosphatase detection of CNP isoforms at PIO. Lane 1, prestained molecular weight standards (top to bottom) 117, 75.5, 43, and 28.2 kDa; lane 2, rat cerebrum homogenate; lane 3, rat brain stem homogenate; lanes4and 5, purified bovineCNP. Lanes 2-4were subjected to immunodetection with a polyclonal rabbit antiserum against purified bovine CNP. Lane 5 was subjected to immunodetection with an affinity purified polyclonal rabbit antiserum against the amino-terminal domain of rat CNP-II and shows that only CNP-II is detected. (B and C) ECL detection of CNP isoforms expressed in rat cerebrum (C) and brain stem (BS) at PI, P5, and PI2 using a rabbit polyclonal anti-CNP antiserum against purified bovine CNP (B) or an affinity purified rabbit antiserum against the aminoterminal domain of rat CNP-II (C).

mRNA Exand Adult

Each lane contains 5 pg of total RNA isolated from the optic nerves of enucleated eyes, their unlesioned contralateral mates (Contra), or a separate group of age-matched animals (Unlesioned). The blot was successively probed with radiolabeled, cDNA probes for CNP (A), PLP (B), MBP (C), MAC (D), and cyclophilin (Cyc; E) and exposed to film for 16 hr (A, B, and C), 3 days (E), or 7 days (D).

onstrates

Id

immuno-

electroblotting with alkaline phosphatase amplification and visualization, both CNP proteins can first be detected in rat forebrain at PI0 (Figure 5A, lane 2), whereas at P5 they are faintly visible in the hindbrain (data not shown). At later stages of development, the abundance of both CNP isoforms paralleled the steady-state levels of both CNP mRNAs (data not

shown). W e also used 1251-labeled protein A to detect the CNP-containing immunocomplexes on blots (data not shown), but these studies only verified the above results and did not offer better resolution of the two isoforms. CNPase activity in protein extracts from forebrain and brain stem also paralleled this time course of CNP expression (data not shown). Application of the enhanced chemiluminescence detection method, however, enabled us to visualize CNP as early as PI in extracts from both forebrain and hindbrain (Figure 5B). This method, although exceedingly sensitive (we estimate the detection of <20 ng of CNP per brain), did not provide clear resolution of the two CNP isoformson immunoblots of whole-brain extracts, so that the CNP detected could have been either CNP-I or CNP-II. Using the anti-CNP-II antibody, however, CNP-II was clearly detected in forebrain at PI, a time when only CNP-II transcripts are present (Figure 5C). Taken together, these data suggest that CNP-II message and protein are expressed

Differential 1367

Regulation

of CNP

byoligodendrocyte precursors early in brain development, even before the onset of myelination. In addition, both CNP protein isoforms accumulate to relatively high levels during myelination in parallel with the other myelin proteins.

Both CNP lsoforms Accumulate in Myelinating Oligodendrocytes Contemporaneously with the Appearance of MBP and PLP To identify the location of appearance of the CNP isoforms during the onset of myelination, we performed an immunocytochemical analysis of developing rat brain using anti-CNP-l+ll and anti-CNP-II antibodies. The pons, cerebellum, and the rostra1 portion of the cerebrum were examined to compare our results with those of previous studies. The pons was studied in the most detail, since it contains a number of fiber tracts that myelinate at different times. Prior to the day of birth, no CNP-immunoreactive cells could be detected in any brain region (data not shown), including the SVZof thecerebellum and cerebrum, where oligodendrocyte precursors arise (Privat, 1978). Thus, even though the above data suggest that oligodendrocyte precursors express CNP-II, we could not directly demonstrate this, most likely because the amount of CNP-II expressed per cell is low, and the immunohistochemical detection method is relatively insensitive compared with chemiluminescence. Beginningat Pl,theanti-CNP-l+llantiseradetected myelin sheaths as well as single process-bearing cells, presumablyoligodendrocy-tes, both in the medial longitudinal fasciculus and in the descending trigeminal tract of the pons (Figure 6A). The number of labeled cells and myelin sheaths increased progressively from PI to PIO, not only in the medial longitudinal fasciculus and descending trigeminal tract, but throughout the pontine tegmentum; yet even by PIO, the corticospinal/bulbar tract was only slightly labeled (Figures 6D and 6H). In the cerebellum, CNP immunoreactivity was first detected at P5, particularly in the center of thevermis,neartheventricle(Figure6D).At PIO, there was extensive labeling of both cells and myelin sheaths extending centrifugally into the cerebellar hemispheres (Figures 6G and 6H). In the rostra1 forebrain, anti-CNP-l+ll staining could be detected at P5 in a few cells and myelin sheaths in portions of the lateral olfactory tract and corpus callosum. At PIO, the lateral olfactory tract and corpus callosum were extensively stained, and there were CNP-positive cells and myelin sheaths in the anterior commisure, striaturn, and cortex. The anti-CNP-II antibody gave very similar patterns of staining to those described above, although the intensity of staining was invariably less than when using the anti-CNP-l+ll antibody (Figures 6A, 6C, and 6G). Thus, in all three brain regions, the patterns of CNP immunoreactivity were similar. The higher staining intensity with the anti-CNP-l+ll antibody was probably due to the additional presence

of CNP-I in the oligodendrocytes and their myelin sheaths. To compare the onset of CNP immunoreactivity to those of the other myelin proteins, we stained adjacent sections of P5 pons with anti-CNP-l+ll, antiCNP-II, anti-MBP, or anti-PLP antibody. The pattern of immunoreactivity was essentially the same with all of these antibodies (Figures 7C-7F). To confirm that CNP, MBP, and PLP were expressed concomitantly, we performed double immunofluorescence on the same section, with a monoclonal anti-MBP antibody and either the anti-CNP-l+ll or the anti-CNP-II polyclonal antibody. The results of this experiment, shown in Figure 7, also show very similar patterns of staining. Essentially identical patterns of CNP and MBP immunoreactivity were also found in the rostra1 forebrain (data not shown). These data, taken together, demonstrate that both CNP isoforms accumulate in mature oligodendrocytes and their myelin sheaths in fiber tracts undergoing active myelination. Furthermore, the accumulation of CNP, MBP, and PLP is essentially contemporaneous in vivo. Oligodendrocyte Precursors Express CNP-II mRNA Since our Northern blot analysis of developing brain suggests that oligodendrocyte precursors express CNP-II mRNA, we attempted to identify these cells in cerebral white matter cultures by in situ hybridization using a probe which recognizes both CNP-I and CNP-II mRNAs. We carried out this experiment at a time when CNP-II mRNA was the predominant CNP transcript as determined by Northern blot analysis (data not shown). In addition, we stained separate coverslips, obtained from the same cultures, with either the A2B5 or Ranscht monoclonal antibody (R-mab) (Ranscht et al., 1982), which recognize surface epitopes that are sequentially expressed during oligodendrocyte development (Raff, 1989). The results of these experiments are summarized in Table 1, and a photomicrograph of some cells labeled by in situ hybridization is shown in Figure 8. At the time of the in situ hybridization, 33% of the cells were A2B5 positive, whereas only8% were R-mab positive (Table 1). In a set of parallel coverslips from the same cultures, 22% of the cells had an average of ten silver grains per cell. Only a few cell-associated grains were found using a sense probe (data not shown). In addition, the morphology of the labeled cells was consistent with that of cells of the oligodendrocyte lineage, and none of the flat, astrocyte-like cells contained silver grains. Since 8% of the CNPpositive cells are probably also R-mab positive, approximately one-half of the A2B5-positive, oligodendrocyte precursors (14%/33%) must express CNP transcripts. We then directly confirmed this result by first reacting the cells with either the A2B5 or R-mab antibodies and then detecting CNP transcripts by in situ hybridization. Using this method, we found that 53% of the A2B5-positive cells also expressed CNP transcripts (data not shown). These data demonstrate

Figure 6. immunohistochemical

Analysis

of Myelin

Proteins

in PI, P5, and PI0 Pons and Cerebellum

(Aand B) PI; (C-F) P5; (G and H) PI0 pons. The sectionswere incubated with anti-CNP-l+ll (A, C, and C), anti-CNP-II MBP (E), and anti-PLP (F) antibodies and developed using avidin-biotin amplification with indophane as the substrate sectionswerenotcounterstained.Themediallongitudinalfasciculus(arrowheads),thedescendingtractofthetrigeminalnerve(between parentheses), and the corticospinal tract (t) are indicated. Bar, 500 Urn (A-F); 700 pm (G and H).

(B, D, and H), antifor peroxidase. The

Differential 1369

Regulation

Figure 7. Double

of CNP

lmmunofluorescence

The sections were labeled with a and mouse anti-MBP antibodies mouse antibodies. Note that the and F). Bar, 100 urn (A-D); IO pm

Labeling

of P5 Ports

combination of rabbit anti-CNP-ii and mouse anti-MBP antibodies (A and B), or rabbit anti-CNP-l+ll (C-F), followed by rhodamine-conjugated goat anti-rabbit and fluorescein-conjugated goat antianti-CNP-l+ll antibody, but not the anti-MBP antibody, labels oligodendrocyte somata (arrows in E (E and F).

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Table 1. In Situ Hybridization Cerebral White Matter lmmunohistochemical

and lmmunohistochemical

Analysis

of CNP mRNA

in Developing

Cultures

In Situ Hybridization

Analysis

from

Studies

Cells Counted

A2B5-Positive Cells (%)

Ceils Counted

R-mab-Positive Cells (X)

Cells Counted

Cells with Grains (%)

Grains per Cell

468

154 (33)

262

21 (8)

418

93 (22)

9.9

--

The percentage of A2B5- and R-mab-positive cells in the cerebral white matter cultures was determined by counting both the total number of cells and the number of A2B5- and R-mab-staining cells per millimeter on coverslips prepared in parallel to those processed for in situ hybridization analysis. The percentage of cells with grains after in situ hybridization was determined by counting both the total number of cells and the numbers of cells with four or more grains, in 20 fields on 2 coverslips using a 63x objective on a Leitz Aristoplan microscope. The number of grains per cell was determined by counting grains in all cells’with four or more grains in the 20 fields evaluated.

that CNP mRNA is expressed by a subpopulation of A2BS-positive, oligodendrocyte precursors. Furthermore, since CNP-II mRNAis the predominant isoform detected in these cultures by Northern blot analysis, the in situ hybridization signal most likely represents hybridization to this transcript. To determine directly whether CNP-II mRNA is expressed by oligodendrocyte precursors, and to evaluate the profile of CNP mRNAexpression during differentiation, we examined the steady-state levels of CNP mRNA in cultures of growth factor-stimulated oligodendrocytes (Collarini et al., 1992). Oligodendrocyte precursors were selected by immunopanning with the A2B5 antibody and cultured in the presence of both basic fibroblast growth factor and PDGF to promote their proliferation and prevent their differentiation. When these growth factors were removed, the oligodendrocyte precursors differentiated more or

less simultaneously into galactocerebroside-positive oligodendrocytes (see Figure 1 in Collarini et al., 1992). The results of this experiment are shown in Figure 9. Prior to removal of the growth factors (time 0), when the cells were proliferating oligodendrocyte precursors, CNP-II mRNA was the only CNP transcript present. Twenty-four hours after the growth factors were removed, however, when the cells were no longer dividing, the steady-state levels of both CNP-I and CNP-II mRNAs had increased substantially. No MBP transcripts could be detected at this time. By 48 hr after growth factor removal, the levels of both CNP messages had increased even further, and MBP mRNA could now be detected. These data demonstrate that CNP-II mRNA is expressed by oligodendrocyte precursors, so that the the more distal of the two CNP promoters must be actively transcribed in these cells. When these cells differentiate, however, both CNP mRNAs accumulate to relatively high levels, suggesting that both CNP promoters have been activated. Since the increase in CNP mRNA levels occurs before that of MBP mRNA levels, the induction of CNP transcription probably occurs prior to that of MBP transcription. Differentiating

Figure 8. In Situ Hybridization Detection of CNP Transcripts Developing Cultures from Cerebral White Matter

in

Arrows indicateoligodendrocytes;“a”indicates astrocyte nuclei. Note that silver grains, representing hybridization to the labeled CNP probe, are clustered over oligodendrocytes, but not astrocytes. Bar, 10 pm.

Oligodendrocytes

Express CNP

Although we were unable to detect CNP in the developing cerebrum by immunohistochemistry, we could detect CNP-II at as early as PI using the more sensitive chemiluminescence assay. To determine directly whether oligodendrocytes or their progenitors express CNP, we examined CNP expression in the same developing cultures of cerebral white matter, described above, which contain a mixture of oligodendrocytes, oligodendrocyte precursors, and astrocytes (Grinspan et al., 1993), using the monoclonal antibody A2B5, a marker of oligodendrocyte precursors, and either the anti-CNP-l+ll or the anti-CNP-I I polyclonal antibodies. Twenty percent of the A2B5-positive cells detected were stained with the anti-CNP-l+ll antisera; no cells, however, were stained with the antiCNP-II antisera. These data demonstrate that a subpopulation of oligodendrocyte precursors expresses both A2B5 and CNP and are consistent with the in situ hybridization analysis above. Since almost 50%

Differential

Regulation

of CNP

1371

0

c

c

zi

3

more than one CNP mRNA species. This suggestion is in keeping with the recent finding that human CNP mRNA, which runs as a single, 3.0 kb band on Northern blots, consists of two mRNA species that encode both CNP isoforms (Douglas and Thompson, 1993). Since the structure of the nonneural CNP transcripts is not known, other explanations of these data are possible.

- 2.2 kb

-1.4 kb

Figure 9. Northern Blot Analysis of CNP mRNA Growth Factor-Stimulated Culturesof Developing cytes

Expression in Oligodendro-

RNA was prepared from purified oligodendrocyte precursors cultured in the presence of basic fibroblast growth factor and PDCF (lane 0), or 1 (24 hr) and 2 days (48 hr) after growth factor withdrawal. Ten micrograms of RNA was electrophoresed per lane, and the blot was sequentially probed with radiolabeled cDNAs hybridizing to both CNP isoforms (A), MBP (B), and CAPDH (C) as a loading control.

of A2B5-positive cells contained CNP transcripts detected by in situ hybridization but only 20% of these cells reacted with the anti-CNP-l+ll antisera, approximately 30% of the CNP transcript-positive cells could not be identified in this assay. These A2B5-positive, CNP-positive cells are most likely differentiating oligodendrocytes. Unfortunately, since no CNP-IIpositive cells could be detected, the CNP isoform expressed in these cells could not be determined in this experiment. The reasons for the lack of CNP-II staining are not known, but may be due to the low amount of CNP-II expressed per cell, or the low affinity of the anti-CNP antisera for CNP-II. Discussion Different CNP mRNAs Are Expressed in Neural and Nonneural Tissues As depicted in Figure 1, the two isoforms of CNP are encoded by separate mRNAs that differ at their Sends (Bernier et al., 1987; Kurihara et al., 1990). Although only myelinating glia express CNP-I mRNA, many tissues, including the thymus, express small amounts of a 2.5 kb CNP transcript (Bernier et al., 1987), similar in size to the 2.4 CNP-II mRNA. When thymus RNA is used to program an in vitro translation system, however, both CNP-I and CNP-II are produced (Bernier et al., 1987), suggesting that this 2.5 kb mRNAcontains

CNP-II but Not CNP-I mRNA Is Expressed in Oligodendrocyte Precursors Because of the differences between the onset of CNP expression and those of the other major CNS myelin proteins, we investigated the patterns of CNP protein and message expression during oligodendrocyte development, both in vivo and in vitro. In the developing brain, we found that the CNP mRNA encoding CNP-II appeared before those encoding PLP, MBP, or MAG and before the appearance of differentiated oligodendrocytes (Curtis et al., 1988; Reynolds and Wilkin, 1988; Card and Pfeiffer, 1989; Hardy and Reynolds, 1991; Levine et al., 1993). In cerebral white matter cultures, CNP mRNA was localized to cells that expressed A2B5, a marker of oligodendrocyte precursors (Raff et al., 1983). In addition, we directly demonstrated that the CNP-II message was expressed in oligodendrocyte precursors before the appearance of either CNP-I or MBP mRNA by Northern blot analysis of growth factorstimulated cultures of oligodendrocytes. These data strongly suggest that the 2.4 kb CNP-II mRNA, but not the 2.6 kb CNP-I mRNA, is expressed by oligodendrocyte precursors. Kanfer and coworkers (1989) also noted the early expression of CNP mRNA in embryonic rat brains. They described the initial expression of a 3.2 kb CNP transcript, followed by expression of a 2.6 kb CNP transcript. The level of their 3.2 kb transcript increased during development and was always more abundant than their 2.6 kb transcript. The relationship of these transcripts to the 2.6, 2.4, and 1.6 kb CNP mRNAs we and others have identified is unclear. The most likely explanation is that their 3.2 kb CNP transcript consists of both the 2.4 and the 2.6 kb CNP mRNAs, which were incompletely separated during gel electrophoresis, and that their 2.6 kb transcript is the same as our 1.6 kb CNP transcript. Thus, during embryonic development, their 3.2 kb band probably consists of only the 2.4 kb CNP message, whereas at later times it probably consists of both the 2.6 and the 2.4 kb CNP mRNAs. The Activation of CNP mRNA Expression Occurs before That of MBP and PLP mRNA Expression Anumberof studies havefound thattheonsetof CNP protein expression in oligodendrocytes occurs before that of either MBP or PLP, both in vivo and in vitro. In developing rodent brain,oligodendrocytesexpress galactocerebroside and CNP before the expression of the major myelin structural proteins (Mottet et al.,

Neuron 1372

1979; Roussel and Nussbaum, 1981; Monge etal., 1986; Reynoldsand Wilkin,1988; Hardyand Reynolds, 1991). Similarly, in cultured rodent oligodendrocytes, the onset of galactocerebroside and CNP expression are essentially contemporaneous and can bedetected before the onset of MBP (Pfeiffer et al., 1981; Knapp et al., 1988; Amur-Umarjee et al., 1990). In addition, some of the first CNP-positive cells in the cerebrum are found in the SVZ, the source of oligodendrocyte precursors, whereas MBP-positive cells are not found in the SVZ (Braun et al., 1988; Hardy and Reynolds, 1991). These observations provide strong support for the notion that the onset of CNP gene expression occurs in differentiating oligodendrocytes before that of MBP and PLP gene expression. Since all of the above studies used immunohistochemical techniques to detect the appearance of CNP, they do not directly address the timing of CNP mRNA accumulation during oligodendrocyte differentiation. We examined this issue two ways. First, in cultures of oligodendrocyte precursors grown in the presence of basic fibroblast growth factor and PDCF, only CNP-II transcripts are present; both CNP transcripts, however, are induced during the initial 24 hr after growth factorwithdrawal when theoligodendrocytes differentiate. By 48 hr, MBP transcripts can also be detected, and the levels of both CNP transcripts have further increased. Thus, both CNP transcripts are up-regulated in parallel 24 hr before the expression of the mRNAs encoding MBP. Second, in developing mixed cerebral white matter cultures, in situ hybridization analysis detected a population of R-mabnegative cells which contained CNP transcripts, indicating that CNP transcripts accumulate in oligodendrocytes before the appearance of the epitopes recognized bythisantibody. Sincewe have previously shown that both PLP and MBP mRNAs accumulate contemporaneously with the appearance of R-mab staining in these cultures (Grinspan et al., 1993), these data also demonstrate that CNP transcripts appear before MBP or PLP mRNA. Thus, in both growth factor-stimulated oligodendrocyte cultures and mixed cultures of differentiating oligodendrocytes, CNP mRNAcan bedetected in differentiatingoligodendrocytes before the appearance of either MBP or PLP and often before the appearance of galactocerebroside. Although this transient stage in oligodendrocyte development could be identified in culture, these differences were not readily apparent in Northern blot analysis of developing brain in which all of the myelin specific mRNAs increased synchronously. This discrepancy is probably due to the heterogeneity in the maturational state of oligodendrocytes in developing brain.

CNP-II Protein Probably Appears Prior to CNP-I Although CNP-II mRNA is present before CNP-I mRNA, it was difficult to detect CNP-II protein before the onset of CNP-I protein expression. Only the ECL detection techniqueallowed ustoidentifyCNP-II pro-

tein in cerebrum at PI, a time at which only CNP-II transcripts are present. Because of the technical limitations of the study, however, we could not rule out the possibilitythat CNP-I was also present at this time. Consistent with these data, Hardy and Reynolds (1991), using an optimized immunohistochemical technique, also detected CNP-positive cells in PI cerebrum. These cells probably express CNP-II, but this has not yet been demonstrated directly.

Both CNP lsoforms Accumulate in Parallel with MBP and PLP during Myelination and Decrease in Parallel with MBP and PLP after Axotomy Although the initial up-regulation of CNP-I and CNP-II mRNA expression occurs slightly before that of MBP and PLP gene expression in developing oligodendrocytes, both isoforms accumulate to high levels in parallel with the MBP, PLP, and MAC transcripts during myelination (Figure 3). In addition, we have shown that both CNP protein isoforms accumulate in parallel with their respective mRNAs in the developing brain (Figures 5-7). Finally, our immunohistochemical analysis of developing brain showed essentially identical temporal and spatial patterns of immunoreactivity with antibodies that recognized CNP-I+II, CNP-II alone, MBP, and PLP. These data demonstrate that CNP-I, CNP-II, MBP, and PLP and their respective mRNAs accumulate with similar temporal and spatial profiles during myelination. If CNP-I and CNP-II were coordinately regulated with the other myelin genes, then one might expect that they should also be similarly affected by axotomy. Removal of the retinal ganglion cells causes Wallerian degeneration of the optic nerve, which has been found to cause a substantial fall in the steady-state levels of CNP, MBP, PLP, and MAC mRNAs (Goto et al., 1990; Kidd et al., 1990; McPhilemy et al., 1990,199l; Scherer et al., 1992). Kidd et al. (1990) found that the level of CNP mRNA fell by 12 days post-lesion by slot blot analysis. Since the relative contribution of the two CNP transcripts cannot be determined by slot blotting, our Northern blot analysis confirms and extends their findings by showing a fall in both CNP-I and CNP-I I transcripts. Thus, axons are also necessary for the maintenance of normal levels of both CNP mRNAs, although they are not required for the initial expression of the CNP gene during development. Insummary,ourdatademonstratethatthetwoCNP transcripts are differentially regulated during the process of oligodendrocyte maturation. In oligodendrocyte precursors, prior to the onset of myelin-specific gene expression, only the 2.4 kb transcript, encoding the larger protein, is expressed. At the time of oligodendrocyte differentiation, however, both CNP transcripts are induced approximately 24 hr before either MBP or PLP transcript. Both CNP transcripts and proteins then accumulate to high levels in parallel with those of the major myelin protein genes, and this expression is partially dependent upon the presence of axons. CNP is thus expressed throughout the

Differential 1373

Regulation

of CNP

course of oligodendrocyte development and can be used as a marker of both oligodendrocyte precursors and differentiating and myelinating oligodendrocytes. These patterns of CNP expression are likely due to stage-specific transcriptional regulation of the two CNP promoters.

Experimental

Procedures

Surgery and Collection of Tissues We enucleated one eye using aseptic technique by introducing iridectomy scissors behind the right globe and cutting the optic nerve and the extraocular muscles; the left eye was untouched. After 12 days, the rats were sacrificed by CO2 inhalation, and the right and left optic nerves were individually isolated and immediately frozen in liquid nitrogen. We sacrificed normal rats of various ages by CO2 inhalation. Theywerethen decapitated,and thecerebral hemispheres, optic nerves, brain stem, and cerebellum wereseparated and frozen in liquid nitrogen. To obtain embryonic rat tissues, timed pregnant rats were purchased, and at the appropriate time, the mothers were sacrificed by CO? inhalation, and the same brain regions were collected. The day after mating was considered El; the day after birth was considered PI. SDS-Polyacrylamide Gel Electrophoresis and Western Blot Analysis Samples of tissue homogenates (see above) were concentrated by precipitation with trichloroacetic acid (15% final concentration) followed by centrifugation and partially delipidated by two acetone washes of the pellet at OOC. Pellets were dispersed by boiling for 5 min in electrophoresis sample buffer (Laemmli, 1970) to a final concentration of 20 mg of protein per milliliter. Insoluble material was removed by centrifugation in a microfuge. Samples of 100-200 pg of protein were applied to 8 x IO cm minigels of 7.5%, IO%, or 4%-23% gradient polyacrylamide and separated by electrophoresis (Laemmli, 1970). A modification of the immunoelectroblotting procedure of Towbin et al. (1979) was used to identify CNP. The protocol was that of the Promega Corporation (1988 catalog). Polyclonal antisera to CNP (both isoforms) have been previously described (Braun et al., 1988). Antiserum to CNP-II was prepared against a synthetic peptide representing the unique amino-terminal sequence of rat CNP-II and affinity purified on a column of the immobilized peptides (further details to be provided elsewhere). In addition to the visualization of immunocomplexes on electroblots by alkaline phosphatase reaction product, we employed the enhanced chemiluminescence procedure as described by Amersham Internationalintheinstructionsthataccompanytheir Kit RPN 2108. Cell Culture Cerebral white mattercultureswereestablished from thecorpus callosum and nearby white matter of 6-day-old Sprague-Dawley rats(Grinspan etal.,1990,1993). Fourdaysafterseedingin serumcontaining medium (the equivalent of PIO), the medium was changed to a defined medium containing 1% platelet-poor plasma supplemented with 2 rig/ml human PDCF (largely AB heterodimers; R&D Systems) as described (Grinspan et al., 1993). Cultures were refed every 3-4 days with fresh platelet-poor plasma medium with PDCF. Purified cultures of oligodendrocyte precursors were obtained as described by Collarini et al. (1992). Dissociated cells from neonatal rat brains were sequentially added to Petri dishes that were coated with anti-RAN-2 (to remove astrocytes and macrophages), anti-galactocerebroside (to remove oligodendrocytes), and A2B5 (to select oligodendrocyte precursors) antisera. The adherent cells were removed from the A2B5 dishes and grown in a modified Bottenstein and Sato medium (Richardson et al., 1988), supplemented with 10 rig/ml recombinant human fibroblast growth factor and PDGF-AA (Peprotech).

Northern Blot Analysis RNA was isolated from tissues by CsCl? gradient centrifugation (Chirgwin et al., 1979) and from cultured cells by guanidiumthiocyanate method (Chomczynskyand Sacchi, 1987). Equal samples (IO pg) of total RNA were electrophoresed in 1% agarose/ 2.2 M formaldehyde gels, transferred to nylon membranes (Duralon, Stratagene) in 6x SSC, and ultraviolet cross-linked (0.12 J). Blots were prehybridized, hybridized, and washed using standard techniques (Sambrook et al., 1989). The following probes were utilized: a 0.85 kb Pst fragment and a 0.4 kb EcoRI-Hindlll fragment from a rat CNP cDNA (Bernier et al., 1987); a 1.4 kb fragment from a rat PLP cDNA (Kamholz et al., 1992); a 1.5 kb EcoRl fragment from a rat MBP cDNA, isolated in our laboratory; a 0.9 kb EcoRl fragment from a rat MAC cDNA (Salzer et al., 1987); a 1.3 kb cDNA encoding rat glyceraldehyde 3-phosphate dehydrogenase (GAPDH; Fort etal., 1985); and a full-length cDNA clone encoding rat cyclophilin (Danielson et al., 1988). Plasmid inserts were isolated after restriction endonuclease digestion, separated by agarose gel electrophoresis, and purified by electrolution. ‘*P-labeled cDNA probes with specific activities of 2 x IO9 to 5 x IO9 cpm/mg were prepared by primer extension with random hexamers using the Prime-a-Gene kit (Promega) according to the manufacturer’s instructions. lmmunohistochemistry, Immunocytochemistry, and In Situ Hybridization Brains were fixed in Bouins solution, embedded in paraffin, sectioned transversely at 6 wrn, and mounted on slides. After deparaffinization, endogenous peroxidase was blocked by incubation in hydrogen peroxide/methanol, and nonspecific binding was blocked by preincubation of the sections with 2% fetal calf serum. The following primary antibodies were used: a rabbit polyclonal antiserum that recognizes both CNP-I and CNP-II (gift of Dr. F. A. McMorris), a rabbit polyclonal antiserum that recognizes proteolipid protein (gift of Dr. A. H. Koeppen), a mouse monoclonal antiserum that recognizes MBP (clone #2; Serotec), and a rabbit polyclonal antiserum, prepared against a peptide unique to CNP-II, that recognizes CNP-II but not CNP-I. After incubating the sections overnight at 4OC with the primary antibody, they were washed extensively and incubated with rhodamine-, fluorescein-, or avidin-conjugated goat anti-rabbit or anti-mouse IgG, followed when appropriate by avidin-peroxidase complex, and reacted with indophane as a substrate (Biomeda). Some sections were counterstained with hematoxylin. To control for nonspecific staining, the primary antiserum was omitted in the overnight incubation. To compare A2B5 and CNP immunoreactivities in cultured cerebral white matter cells, unfixed cells were incubated at 4OC for 25 min with undiluted A2B5 antibody (Eisenbarth et al., 1979), rinsed, and then incubated with fluorescein-conjugated goat anti-mouse IgM (Cappel Labs) diluted I:50 at 4OC for 25 min. Then the cells were rinsed, fixed in ice-cold methanol for 10 min, incubated with anti-CNP antibody (Sigma) diluted I:500 for 25 min at room temperature, and finally rinsed and incubated in biotinylated goat anti-mouse IgG-1 (Sigma) diluted I:100 in defined medium for 30 min at room temperature. The cells were rinsed, incubated in streptavidin-rhodamine diluted I:100 for 20 min at room temperature, rinsed, and mounted in aquamount (American Scientific). To determine the number of doubly labeled cells, 21 randomly selected fields were examined with a 63x oil immersion lens (equivalent to a 1 mm2 area). To compare CNP-II and CNP-I immunoreactivities, the cells were fixed for 10 min in Bouins, rinsed, and sequentially labeled with a rabbit polyclonal antibody against CNP-II, followed by a mouse monoclonal antibody that recognizes both CNP-I and CNP-II (Sigma). The coverslips were rinsed and incubated in rhodamine- and fluorescein-conjugated goat anti-rabbit and antimouse antisera, respectively, and mounted as above. In situ hybridization was performed according to the method of Oronzi-Scott et al. (1988) with minor modifications. Coverslips were fixed in 4% formaldehyde in phosphate-buffered saline, dehydrated, and stored under 70% ethanol until use. Sense and antisense cRNA probes labeled with YYabeled UTP were syn-

NEWrOn 1374

thesized from CNP cDNA templates in pBluescript vectors using either T3 or T7 RNA polymerase. Coverslips were hybridized for 3.5 hr at 52OC in 2x SSC, 50% formamide, 10 m M dithiothreitol, salmon sperm DNA (l mglml), yeast transfer RNA (1 mglml), bovine serum albumin (2 mglml), and 1.0 x IO6 cpm of probe per coverslip. After hybridization, the coverslips were incubated with ribonuclease A (100 fig/ml) in 2x SSC for 30 min at 37°C and washed several times in 2x SSC, followed by a high stringency wash in 0.1 x SSC at room temperature and an overnight wash in 2x SSC and 10 m M dithiothreitol. The coverslips were dehydrated, mounted faceup on glass slides with permount, dipped in NTBL photographic emulsion (Kodak), and stored at 4X After 10 days, the coverslips were developed, fixed, and counterstained with toluidine blue. Twenty random fields were scanned using a 100x oil immersion lens and the total number of cells, the number of cells containing four or more grains, and the number of grains per cell were determined for each coverslip. Acknowledgments We thank Drs. F. A. McMorris and A. H. Koeppen for their generous gifts of antibodies against CNP and PLP, respectively. We also thank Elsa Horvath (McGill University) for excellent technical assistance. This work was supported by National Institutes of Health grants NS11037 (J. K.), NSO8075 (J. K. and J. G.), and NS01565 (S. S.), Multiple Sclerosis Society of Canada and Medical Research Council grants (P. B.), National Multiple Sclerosis Society grants (J. K. and J. C.), and the Medical Research Council of Great Britain (E. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC Section 1734 solely to indicate this fact. Received

December

27, 1993; revised

March

and migration

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J. Neu-

Danielson, P. E., Forss-Peter, S., Brow, M. A., Calavetta, L., Douglass, J., Milner, R. A., and Sutcliffe, J. C. (1988). plB15: a cDNA clone of the rat mRNA encoding cyclophilin. DNA 7, 261-267. Douglas, A. J., and Thompson, R. J. (1993). Structure of the myelin membrane enzyme 2’,3’-cyclic nucleotide 3’phosphodiesterase-evidence for 2 human messenger RNAs. Biochem. Sot. Trans. 21, 295-297. Dubois-Dalcq, M., Behar, T., and Lazzarini, R. A. (1987). Emergence of three myelin proteins in oligodendrocytes cultured without neurons. J. Cell Biol. 702, 384-392. Eisenbarth, G. S., Walsh, F. S., and Nirenberg, clonal antibody to a plasma membrane antigen Natl. Acad. Sci. USA 76, 4913-4917.

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