Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage

Joirrnal of N~~rrroclreniislrI~ Raven Press, Ltd.. New York (0 1993 International Societ! for Neurochemistry

Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage

*-fKazunori Nakajima, *Kazuhiro Ikenaka, *Tetsushi Kagawa, "yun Aruga, *Junji Nakao, *Kensuke Nakahira, *Chiyo Shiota, SSeung U. Kim, and TKatsuhiko Mikoshiba

*Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka Universily, Osaka, Japan; +Department of Molecular Neurohiology, The Institute of Medical Science, The University of Tokyo, Tokyo, Japan; and $Division of Neurology, Department ofhfedicine, University of British Columbia, Vancouver, British Columbia, Canada

Abstract: Myelin basic protein (MBP), a major protein of myelin. is thought to play an important role in myelination, which occurs postnatally in mouse. Here we report that the MBP gene is expressed from the 12th embryonic day in mouse brain and that most of the predominant embryonic isoforms are not those reported previously. These isoforms have a deletion of a sequence encoded by exon 5 from the well-known isoforms. These isoforms show a unique developmental profile, i.e., they peak in the embryonic stage and decrease thereafter. In jimpy, a dysmyelinating mutant, the level of these isoforms remains high even in the older ages.

These results suggest that MBPs have heretofore unknown functions unrelated to myelination before myelinogenesis begins. The possible presence of I8 isoforms of MBP mRNA, which are classified into at least three groups with different developmental profiles, is also reported here. Key Words: Myelin ba:sic protein-Developing nervous system -Jimpy mutant---Fluorescence-activated cell sorting. Na- kajima K. et al. Novel isoforms of mouse myelin basic protein predominant1:y expressed in embryonic stage. J. Neuro- chern. 60, 1554-1563 (1993).

Myelin is a unique multilayered membrane structure that surrounds and insulates axons to facilitate the conduction of neuronal impulses. This is produced by oligodendrocytes in the CNS and by Schwann cells in the PNS. Myelin basic protein (MBP) constitutes approximately 30% of the myelin proteins in the CNS and 1-10% in the PNS (Sabri et al., 1974; Benjamins and Morell, 1978). The function of MBP is not well established, but its localization to the cytoplasmic aspect of the myelin membrane and its ability to make a homodimer suggest that it makes myelin lamellae compact by linking the apposed cytoplasmic surfaces of oligodendrocytes. In mouse, myelination occurs during the first 3 weeks after birth with the maximal rate at an age of about 20 days postnatal (P20) (Norton and Poduslo, 1973). Expression of the MBP gene is developmentally regulated, and the maximal expression is reported to occur at about P18

(Campagnoni et al., 1978; Carson et al., 1983; Zeller et al., 1984; Sorg et al., 1987).

Mouse MBP is known to have at least five major isoforms I21.5, 18.5, 17 (1 and 2), and 14 kDa] encoded by separate mRNAs that are produced by alter- nate use of seven exons (shown in Fig. 3) (de Ferra et al., 1985; Newnnan et al., 1987; Mikoshiba et al., 199 1). Minor isoforms that utilize novel exons (exons 0 and 1 a, or exon 5a) have also been reported recently (Kitamura et al., 1990; Aruga et al., 1991). The presence of a 20-kLIa isoform has also been suggested from the results of immunoblots of mouse CNS (Campagnoni, 1 '9 8 8).

As a model of cell type-specific gene expression, we have been studying the regulatory mechanisms of the MBP gene with in vitro assay systems, such as in vitro transcription (Miura et al., 1989, 1991; Tamura et al., 1989, 1990; Aoyama et al., 1990; Tamura and Miko-

Received August 4, 1992; revised manuscript received Sep- teniber 28, 1992: accepted September 30, 1992.

Address correspondence and reprint requests to Dr. K. Ikenaka at Division of Regulation of Macromolecular Function, Institute for Protein Research, Osaka University, 3-2 Yamada-Oka, Suita, Osaka, 565, Japan.

The present address of Dr. C. Shiota is Department of Pathology, Wakayama Medical School, Wakayama, Japan.

Abbreviations used: AMV-RT, avian myeloblastosis virus reverse transcriptase; DMEM, Dulbecco's modified Eagle's medium; E, embryonic day; FACS, fluorescence-activated cell sorter; FCS, fetal calf serum; GalC, galactocerebroside; HPRI, human placental RNase inhibitor; MBP, myelin basic protein; P, postnatal day; PCR, polymerase chitin reaction; PI, propidium iodide; RT-PCR, reverse transcription-polymerase chain reaction.

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NOVEL MBP ISOFORMS PRODUCED IN EMBRYONIC BRAIN 1555

shiba, 1991). Recently, we have developed a novel promoter assay system using a retrovirus vector, which enables us to detect promoter activity in indi- vidual brain cells (Ikenaka et al., 1992). By using this system, we have shown that the 1.3-kb promoter region of MBP is sufficient to confer oligodendrocyte [galactocerebroside (Ga1C)-positive cell]-specific expression of the gene during the active myelinating stage.

As described above, to date expression of MBP in the mouse CNS has been studied mainly in the postnatal stage, because MBP is believed to have an important role in myelination. However, in the course of studying promoter activity in primary cultures of brain cells, we found that the MBP promoter is active in brain cells obtained from embryonic mouse. Thus, we performed polymerase chain reaction (PCR) to investigate whether MBP mRNA is present in embryonic mouse brain, and we found that the MBP gene is indeed expressed from the 12th embryonic day (E 12). In addition, most of the MBP isoforms predomi- nantfy expressed in embryonic mouse brain were not those reported previously. In this study, we report the structures and expression profiles of these novel MBP isoforms and discuss the cell type producing these mol- ecules.

MATERIALS AND METHODS

Promoter assay in brain cells in primary culture with recombinant retrovirus vector

The details of the method to detect promoter activity in primary culture with recombinant retrovirus vector were reported previously (Ikenaka et al., 1992). Briefly, the cells collected from embryonic mouse cerebrum were grown in a 1:l mixture of Ham’s F-12 (Flow Laboratories) and Dul- becco’s modified Eagle’s medium (DMEM/high glucose; Hazleton Biologics) containing 10% fetal calf serum (FCS). After 2 days in vitro, the cells were incubated in viral super- natants with 8 pg/ml Polybrene for 3 h, and the medium was replaced with fresh medium. After the appropriate number of days in vitro, the cells were fixed and processed for X-Gal staining as described previously (Ikenaka et al., 1992).

Reverse transcription, PCR, and detection of the amplified products

Total RNAs were extracted by the guanidinium thiocya- natemethodfromwhole brainsofE12, E14,E15,E16,E18, P2, P8, and P18 ICR mice, sciatic nerves ofadult ICR mice, and whole brains of P2, P9, and P18 jimpy mice and their littermates on a B6CBA genetic background. The jimpy mice were identified by gene analysis (T. Kagawa, K. Iken- aka, M. Yamada, K. Shimizu, T. Hayakawa, and K. Miko- shiba, in preparation). Reverse transcription was carried out in a 2 0 4 reaction mixture containing 2 p g of total RNA, 50 mM Tris-HC1 (pH 8.3), 6 m M MgCI,, 40 m M KC1, 1 mM dithiothreitol, each dNTP at I .25 mM, 30 units of human placental RNase inhibitor (HPRI; TaKaRa), 112.5 pmol of random hexamers (TaKaRa), and 22 units of avian myeloblastosis virus reverse transcriptase (AMV-RT; Seikagaku

Kogyo). As for P8 (ICR) and PI8 (B6CBA) mice, 0.2,0.02, and 0.002 pg of total brain RNA were also used to examine the dose dependency. In these cases, yeast tRNA was used to adjust the total quantity to 2 pg. Two micrograms of yeast tRNA was used for the negative control. In the experiments with ICR mice RNA, 200 pmol of p(dT),, primer (Boehr- inger Mannheim) was also used instead of random hexamers, and 33 units of AMV-RT was used. The reaction mixture was incubated for 10 rnin at room temperature, and then for 1.5-3 h at 42°C. After being denatured at 95°C for 10 min, the reaction mixture was scaled up to 100 pl with the addition of 5 units of Replitherm thermostable DNA polymerase (Epicentre Technologies), 10 nmol of each dNTP, 100 pmol each of a pair of the PCR primers, and 8 pl of 10 X Replitherm buffer, and then PCR was performed. The primers used in the PCR were as follows: C6 primer, 5’-GCATCCTTGACTCCATCG-3’; and NC07 primer, 5’-GTCTCGCCATGGGAGATCC-3’ (the positions of the primers are shown in Fig. 3). The PCR was carried out for 30 or 20 cycles (see below) as follows: 94°C for I min, 54°C for 2 rnin, and 72°C for 3 min, with the final extension step for 10 min.

The PCR products were separated by electrophoresis through a 5% polyacrylamide gel and were electrotransferred to a Zeta-Probe blotting membrane (Bio-Rad). The membrane was hybridized with 32P-labeled oligonucleotides in a solution containing 5X Denhardt’s solution (Den- hardt, 1966), 0.9 MNaCI, 90 mMTris-HC1 (pH 7.5),6 m M EDTA, 10% dextran sulfate, and 0.5% sodium dodecyl sulfate. This was then washed and exposed to a Kodak X- OMAT AR film at -70°C or to an imaging plate of the Bio-Image Analyzer BAS2000 (Fuji). The oligonucleotides used in this experiment were as follows: C7 probe: 5‘-CATG- TGGCACAGCC-3’; C 1 2 probe: S-TGTGAGTCCTTGCC- A-3’; C46 probe: 5’-CTCGGCCCCCTTCCCTTG-3 and C47 probe: 5’-TCTTCCTCCCTTCCCTTG-3‘.

The protocol applied to cells isolated by a fluorescence- activated cell sorter (FACS) was as follows. The cells (GalC- positive fraction: about 3.3-5 X lo4 cells; GalC-negative fraction: 5 X lo5 cells) were lysed in a solution containing 20 m M Tris-HC1 (pH KO), 20 m M EDTA, and 2% sodium dodecyl sulfate. Proteinase K was added to a final concen- tration of 0.5 mg/ml, and the mixture was incubated over- night at 60°C. Phenol/ether extraction and isopropanol pre- cipitation were carried out with 20 pg of yeast tRNA, and one-third of the pellet was used for reverse transcription. Reverse transcription and PCR were performed in the same manner as described above, except that the HPRI was increased to 40 units and 24 units of AMV-RT were used. Random hexamers were used for the reverse transcription.

Cloning and sequencing For cloning the MBP isoforms, 1 pg of total RNA of El6

ICR mice brains was dissolved in 5 pl of a solution containing 10 m M Tris-HC1 (pH 8.8), 0.5% Nonidet P-40, 0.14 M NaCI, 1.5 m M MgCI,, and 100 units of HPRI, and then incubated for 5 min on ice. The reaction mixture was scaled up to 20 pl by adding 15 pl of a solution containing 50 m M Tris-HC1 (pH 8.3), 6 mMMgCI,, 40 mMKC1, 1 mMdithio- threitol, each dNTP at 1 mM, 100 pmol of NC07 primer, and 9.3 units ofAMV-RT. After incubation for 1 h at 42”C, 80 pl of PCR solution containing 2.5 units of Replitherm DNA polymerase, 10 nmol of each dNTP, 100 pmol of NCOl primer, and 8 gl of 10 X Replitherm buffer was

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1556 K. NAKAJZMA ET AL.

added. The sequence of NCOl primer was 5'-CAAGTAC- CATGGACCATGA-3'. The PCR was carried out for 30 cycles as follows: 94°C for 1 min, 55°C for 2 min, and 72°C for 3 min, with the final extension step for 10 min. A 0.1 pl sample of this mixture was then subjected to further PCR with C5 and C6 primers in a 100-pl reaction mixture containing 5 units of Replitherm DNA polymerase, 25 nmol of each dNTP, 100 pmol of each of the primers, and 10 pl of I0 X Replitherm buffer. The sequence of C5 primer was 5'- GAGCGGCTGTCTCTACC-3'. The PCR was performed for 20 cycles and, after adding 5 units of enzyme, a further I5 cycles were performed with the same program as shown above. The ends of the PCR products were blunted with T, DNA polymerase, and the fragments were inserted into the EcoRV site of the pBluescript vector (Stratagene). The clones with a fragment of an expected size were blotted onto a Zeta-Probe membrane and hybridized with 32P-labeled C7 or C12 probes. The positive clones were treated with 100 pg/pI RNase A for 30 min at 37"C, extracted with phenol/ ether, and precipitated with polyethylene glycol. Sequenc- ing of these clones was performed by the dideoxy chain termination method using the T7Sequencing kit (Pharma- cia). The MBP isoform of exon 1-3-4-7 was cloned in this series.

The isoform of exon 1-2-3-4-6-7 was cloned as follows. The PCR products described above were separated by electrophoresis through a 5% polyacrylamide gel, and the fragments of the expected sizes were purified by electroelution. PCR was performed with these fragments and C5 and C6 primers for 30 cycles, and the products were screened and sequenced as shown above.

All of the oligonucleotides, except for random hexamers and oligo dT primers, were synthesized with a Beckman system IE DNA synthesizer or an Applied Biosystems model 380B DNA synthesizer of the Research Institute for Protein Engineering, Osaka University.

Fluorescence-activated cell sorting and analysis Cerebra were dissected from P9 ICR mice, minced, and

dissociated by pipetting after treatment with 0.25% trypsin and 0.0 I % DNase I in phosphate-buffered saline. After being washed in DMEM containing 10% FCS, the cells were resuspended in the medium, filtered through a nylon mesh (70 pm pore size), and treated with a mouse monoclonal antibody to CalC (Ranscht et al., 1982) on ice. The cell suspension was washed twice over a cushion of horse serum, resuspended in the medium, and treated with fluorescein isothiocyanate-conjugated goat antiserum to mouse IgG (Cappell) on ice. The cells were washed twice over a cushion of horse serum, and resuspended at 5 X lo5 cells/ml in phosphate-buffered saline containing 10 m M HEPES, 4% FCS, and 1 pg/ml propidium iodide (PI). The cells stained only with secondary antibody and PI were used as the negative controls to determine the sorting window.

Cell sorting was carried out on a FACStar PLUS (Becton Dickinson). Fluorescein-positive, PI-negative cells and fluorescein-negative, PI-negative cells were collected as GalC- positive and GalC-negative cells, respectively. Small fractions of the collected cells were cultured in DMEM containing 10% FCS. After I day in vitro, these cultured cells were stained with mouse monoclonal antibody to GalC and bio- tinylated antiserum to mouse IgG (Vector), which was detected with an ABC-alkaline phosphatase system (Vector).

RESULTS MBP promoter is activated in primary culture of embryonic mouse brain cells

Recently, we lhave developed a novel promoter assay system using recombinant retrovirus vector (Iken- aka et al., 1992:). MBP1.3/pIP200 virus, which con- tains a 1.3-kb promoter region of the MBP gene in front of the lacZ gene (see Ikenaka et al., 1992), produced p-galactasidase specifically in GalC-positive cells, suggesting that this region is sufficient for oligodendrocyte-specific expression of the reporter gene (IucZ) during the active myelinating stage (equivalent to about P1S) (Ikenaka et al., 1992). To clarify further whether this promoter region of MBP is also developmentally regulated, we applied this system to earlier developmental stages. To normalize the differences of the transfection efficiency among culture samples and to distinguish thle cell type-specific expression of the MBP promoter from nonspecific expression, we infected the cells with SV/pIP2 1 1 virus simultaneously to serve as an internal control. This recombinant virus has the SV40 early promoter, which is active in all major cell types in the CNS (data not shown), and a nuclear location signal in front of the lac2 gene (Ya- mada et al., 199.2). The nuclear location signal in the latter virus restricts the localization of P-galactosidase to the nucleus, whereas the /I-galactosidase derived from the MBP 1.3/pIP200 virus distributes diffusely in the cytoplasm. By calculating the ratio of the num- bers of the X-Gail-positive cells in cytoplasm to those in nucleus, we studied the developmental profile of the MBP promoter activation (Table 1). The ratio of the cytoplasm-positive cells to the nucleus-positive cells was 15-19% at P1 equivalent and increased to 38% at P8 equivalent, reflecting the increase in the number of oligodendrocytes at this stage in vivo. This considerably high degree of expression ( I S - 19%) at P 1 equivalent suggests that the MBP gene is expressed at this early stage. To investigate the MBP promoter activity at a much earlier stage, we also performed the same experiment using E I 3 mouse brain. After only 3 days in vitro (equivalent to E 16), cytoplasm-positive cells were definitely observed (arrowhead in Fig. l), suggesting that the MBP gene is already expressed at E 16. These cytoplasm-positive cells in the embryonic stage were not stained with the antibodies against the glial fibrillary acidic protein or the microtubule associated protein 2, which are the cell type-specific markers for astrocytes and neurons, respectively (data not shown).

Embryonic expression of MBP is demonstrated by PCR amplification from embryonic mouse brain RNA

In order to clarify whether the MBP mRNA is really produced in the embryonic mouse brain, we performed reverse transcription-PCR (RT-PCR) amplification on tol.al brain RNAs of various stages (the positions of the PCR primers are shown in Fig. 3).


TABLE 1. Promoter assay o fMBP in primary culture of brain cells

Experiment I

Number of X-Gal- positive cells

(cells/4 fields) Ratio of

Days after SV40 MBP C/N infection (N) (C) (70)

Experiment 2

Number of X-Gal- positive cells

(cells/4 fields) Ratio of

SV40 MBP C/N (N) (C) (”/.I

1 116 17 15 48 9 19 2 121 18 15 109 22 20 4 NT NT NT 65 13 20 6 88 25 28 NT NT NT 8 141 54 38 41 18 38

Cerebra of E 17 mice were cultured at 1-3 X 1 O5 cells/cmz and double-infected with the MBP 1.3/pIP200 and the SV/pIPZ I 1 viruses after 2 days in vitro (equivalent to PO). One to 8 days later, the cells were fixed and stained with X-Gal. The P-galactosidase derived from the SV/pIP2 1 I virus locates only in the nucleus, whereas that from the MBP I .3/pIP200 virus distributes diffusely in the cytoplasm. X-Gal-positive cells of each staining pattern in four randomly selected microscopic fields were counted. The results of two inde- pendent experiments are shown. X-Gal-positive cells were not detected in noninfected cultures. C , cytoplasm-positive cells; N, nucleus-positive cells; NT, not tested.

The amplified products were separated on polyacrylamide gel and electrotransferred to a nylon membrane, which was hybridized with exon 2-specific probe (C7 probe; Fig. 2A) and then rehybridized with exon 1-exon 3 junction-specific probe (C 12 probe; Fig. 2B). The results obtained by using random hex- amen as primers for reverse transcription were similar to those obtained using oligo dT primers (the re-

FIG. 1. MBP promoter is activated in embryonic brain cells. Cere- bra of E l 3 mice were cultured and double-infected with the MBPl.3/plP200 and the SV/plP211 viruses after 2 days in vitro. On the next day (equivalent to E l 6), cells were fixed and stained with X-Gal. At this early stage, cytoplasm-positive cells were already found (arrowhead), suggesting embryonic expression of the MBP gene. Arrows indicate the nucleus-positive cells. X-Gal- positive cells were not detected in noninfected cultures (data not shown). Bar = 50 pm.

sults using random hexamers are shown in Fig. 2). As expected from the MBP promoter assay, the MBP gene was indeed expressed in the embryonic stage, that is, the amplified products from mRNAs of 2 1.5- kDa, 1 SS-kDa, 17-kDa- 1, and 14-kDa isoforms were all detected from E 12 and increased considerably during the postnatal stages (bands c and h in Fig. 2A, and e andj in Fig. 2B).

The amplified product from the 17-kDa-2 isoform mRNA was also present as band g in Fig. 2B; however, the developmental profile of this isoform was quite different from those of the other isoforms described above. Its level peaked in the embryonic stage and decreased thereafter in brain. In addition, at least three other bands (bands d and i in Fig. 2A, and k in Fig. 2B) showed developmental profiles similar to that of the 17-kDa-2 isoform. All of these are approximately 30 bp less in size than the corresponding “classical” isoforms described above, suggesting a com- mon mechanism such as specific splicing out of the same exon [exon 5 (33 bp)] in the immature stage.

Dose dependency of our PCR analysis is shown in Fig. 2 (lanes P8 X 1/10, P8 X 1/100, and P8 X 1 / 1,000). When we performed PCR for 2 1, 24, and 27 cycles to confirm the cycle-number dependency, dose dependency was determined more clearly. In these experiments, we found similar developmental patterns to the results obtained in 30-cycle PCR, although the intensity of the bands was weaker (data not shown).

Cloning and sequencing the MBP isoforms predominantly expressed in the embryonic stage

To clarify the structures of the novel isoforms predominantly expressed in the embryonic stage, we

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1558 K, NAKAJIMA ET AL.

FIG. 2. Developmental analysis of the MBP transcripts by RT- PCR. Two micrograms of RNAs of brains from various stages or adult sciatic nerves were subjected to RT-PCR; the products were hybridized with exon 2-specific (C7) probe (A), exon 1 -exon 3 junction-specific (C12) probe (B), exon 4-exon 6 junction-specific (C46) probe (C), or exon 4-exon 7 junction-specific (C47) probe (D). The lines in the left column show the positions of the amplified products detected in this experiment. The amplified products from mRNA of 21.5-kDa, 18.5-kDa, 17-kDa-1, and 14- kDa isoforms were detected from E l 2 and increased particularly during the postnatal stage (bands c, e, h, and j , respectively; the bands of lane E l2 were detected more clearly after longer exposure). The amplified products from 17-kDa-2 mRNA (band g in B and C) and three other isoforms (band d in A and C, and bands i and k in A, B, and D) showed similar developmental profiles to each other, that is, they all peaked at the late embryonic stage and decreased to very low levels at P18. Bands d and f in D showed maximal expressions at P8 and decreased thereafter. As for P8,0.2,0.02, and 0.002 pg of total brain RNA were also used. Exposure time for the sciatic nerve lane in B was slightly shorter than that of the other lanes, because the intensity of the bands was too strong to identify each band readily at the same exposure time. Positions of the size markers are shown in the right column of A.

cloned and sequenced the RT-PCR amplified products from total RNAs of El6 whole brain. PCR was carried out first with primers specific for exon 1 (NCO1 primer) and exon 7 (NC07 primer), and then with a second set of primers that are located at the more internal positions in exon 1 (C6 primer) and exon 7 (C5 primer). After screening and sequencing the clones, we found two novel isoforms of MBP with the structures of exon 1-3-4-7 and exon 1-2-3-4-6-7, which are missing exon 5 from mRNAs of the 14-kDa and 2 1.5-kDa isoforms, respectively (Fig. 3). These isoforms lacking exon 5 were thought to be the predominant isoforms in the embryonic stage.

Developmental profiles of the novel isoforms of MBP To study the developmental profiles of these novel

isoforms during the embryonic stage more clearly, we utilized oligonucleotides specific for the sequences of exon 4-exon 6 junction (C46 probe) and exon 4-exon 7 junction (C47 probe). The same membrane used for the previous experiment was rehybridized with C46 and then with C47 probes (Fig. 2C and D, respectively). The predominant bands in the embryonic stage identified in Fig. 2A and B were also detected by these probes, indicating these bands are indeed exon 5-deleted forms. Bands d and g in Fig. 2C are the amplified products from the isoforms of exon 1-2-3-4- 6-7 and exon 1-3-4-6-7 (17 kDa-2), respectively. The amounts of these isoforms reached their maximum between E 15 and El 8 as revealed by the signal inten- sities measured by Bio-Image Analyzer BAS2000 (Fuji). At P 18, when myelin is most actively synthesized, both of these isoforms were present at very low levels (Fig. 2C). Band k of Fig. 2D is derived from the exon 5-deleted variant of the 14-kDa MBP isoform (exon 1-3-4-7), and band i presumably from the isoform of exon 1-2-3-4-7 (Fig. 3). These two isoforms showed developmental profiles similar to those of the above-mentioned two isoforms without exon 5; their levels peaked between E 15 and E 18, and decreased to very low levels at P 18.

When hybridized with the C47 probe, two other bands were detected in brain (bands d’ and f i n Fig. 2D). The positicin of band d’ in Fig. 2D was very close to that of band d in Fig. 2A and C, but the developmental profiles ?were quite different from each other. The intensity of band d‘ increased in the postnatal stage, reached its peak at P8, and decreased thereafter.

MBP gene 3 4 5 6 7 Exon 1

Expected the PCR Products Sizes of

21.5 kd 480 bp (c)

Exon 1234,6,7) 447 bp (d)

18.5 kd 402 bp (-2)

(Exon 1 2 3 4 7 ) 324 bp (I)

279 bp (1)

17 kd-2

17 kd-1

14 kd

369 bp (9)

357 bp (h)

....... .... ++( . . . . . . . . . . . (Exon 1,3,4,7) 246 bp (k)

FIG. 3. Structure of the eight isoforms of MBP mRNAs. Exons 2, 5, and 6 were used alternatively to give rise to eight forms of MBP mRNAs. The “exon 5” described here is actually exon 5b (Aruga et al., 1991). The isoforms with exon 5a were not cloned from E l 6 brain RNAs. Similarly, exon l b is referred to as “exon 1.” The structures of the 5’ ilnd 3 ends of the isoforms of exon 1-2-3-4-6- 7, exon 1-2-3-4-7, ;and exon 1-3-4-7 are unclear. C6 and NC07 are the primers used for the PCR analysis. Expected sizes of the PCR products and the corresponding bands in Figs. 2, 4, and 5 are shown in the right column.

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NOVEL MBP ISOFORMS PRODUCED m EMBRYONIC BRAIN 1559

Type Scheme of the Developmental Band Protein Exon Structure Profile

c 21 .5 kd 1-2-3-4-2-6-7

I !‘I 1: e 18.5 kd 1-3-42-6-7

h 17 kd-1 1-2-3-4-57

developmental profiles in mouse brain. The d 1-2-3-4-6-7

El2 P2 P18 j 14 kd 1-3-4-A-7 1

FIG. 4. Three types of MBPs with different

isoforms detected in mouse brain were classified into three groups with different developmental profiles. Each profile is shown schematically. Band names are the same as those in Figs. 2, 3, and 5.

1-2-3-4-7

1-3-4-7

?(hybridize to

specific probe)

?(hybridize to exon 1-exon 3 junction

exon 4-exon 7 junction specific probe)

E15-18 P8 P I 8

II

d ‘ ? exon 4-exon 7 junction

f ? specific probe and

111

Band f also appeared when hybridized with the C12 probe (Fig. 2B). The developmental profile of band f was similar to that of band d‘ (Fig. 2D).

In sum, there were at least three groups of MBP isoforms with different developmental profiles (types I-III), including the “classical” isoforms with exon 5. These are summarized in Fig. 4.

MBP isoforms without exon 5 remain present at an older age in jimpy mutant than in normal mouse

In order to study the relationship between the predominant production of the isoforms without exon 5 at the early stage and proliferation or differentiation of the immature oligodendrocytes at this stage, we performed RT-PCR on the brain RNA of the dysmyelinating mutant mouse, jimpy. In this mutant, pre- mature death of oligodendrocytes occurs and most of the oligodendrocytes are known to be of the immature type even at P18 (Knapp et al., 1986; Shiota et al., 1991). Total RNAs were extracted from the whole brains of jimpy mice and their littermates, and then used for the RT-PCR analysis of MBP mRNA. Be- cause jimpy mutation is X-linked recessive, the myelin-deficient phenotype is expressed in the hemizy- gous males ( j p / Y ) . The levels of various MBP isoforms both with and without exon 5 in jimpy mouse brain were almost identical to those in normal mouse at P2 (bands c, d, h, i, e, f, g, j, and k in Fig. 5 4 c, E, and F). However, at P9 and more definitely at P 18, the levels of the isoforms without exon 5 were higher

FIG. 5. Analysis of the RT-PCR products of MBP from jimpy and its littermates. Two micrograms of total brain RNAs from jimpy and its littermates were used for RT-PCR; the products were

in jimpy brain than in normal mouse brain (ban& d,

isoforms with exon 5 became lower in jimpy than in normal mouse (bands c, h, e, and j in Fig. 5A, B, C , and D). These results suggest that the isoforms without exon 5 are associated with the genesis and differentiation Of immature oligodendrocfies~ whereas those containing exon 5 are associated with mature

hybridized with exon 2-specific (C7) probe (A, B), ‘exon 1 -exon 3 junction-specific (C12) probe (C, D). exon 4-exon 6 junction-spe-

probe (F). PCR was performed for 30 (A, C, E, and F) or 20 (B and D) cycles. As for P18 normal mouse, 0.2, 0.02, and 0.002 pg of RNAs were also used. The names of the bands are the same as those in Figs. 2-4. The levels of the isoforms without exon 5 were observed to remain high at an older age in jimpy than in normal mouse, whereas those of the isoforms with exon 5 were lower in jimpy. The positions of the size markers are shown in the right

g, f, i, and in Fig‘ 5E and F), whereas the levels Ofthe cific (C&) probe (E), or exon 4-exon 7 junction-specific (C47)

oligodendrocytes. column.

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1560 K. NAKAJIMA ET AL.

MBP isoform patterns produced in GalC-positive and GalC-negative cells

The unique developmental profile of type I1 MBP isoforms (see Fig. 4) suggests the possibility that these isoforms are produced in a cell population different from that which produces type I isoforms. To investigate this point, we isolated GalC-positive (oligodendrocytes) and GalC-negative cells from P9 mouse brain by FACS. The reason for using P9 mouse brain was that few GalC-positive cells could be isolated before this age. At P9, type I isoforms are known to be produced in oligodendrocytes. If type I1 isoforms are produced in cells of nonoligodendroglial cell lineage, then the bands of these isoforms detected at P9 should be derived from GalC-negative cells, whereas those of type I should be derived from GalC-positive cells. If type I1 isoforms are detected in GalC-positive cells, then it is strongly suggested that type I and type I1 isoforms are produced in cells of the same cell lineage (oligodendroglial cell lineage) and the production of type I1 decreases as the cells mature. The cells were double-stained with anti-GalC antibody, which labels cell surface antigen, and PI, which stains nuclei of dead cells. The results of the scan are shown in Fig. 6A and B. The events with the fluorescence intensity of fluorescein lower than level b and higher than level c were collected as the GalC-negative and GalC-positive cells, respectively. Because each fraction, espe- cially the GalC-negative one, contained cell debris, the cell number was counted and a small portion of the cells were stained with anti-GalC antibody after 1 day in culture. The GalC-negative fraction contained about 10- 15 times more cells than the positive fraction and contained less than 5% of the GalC-positive cells. The GalC-positive fraction contained approximately 10% GalC-negative cells (data not shown). The cells were lysed and the RT-PCR analysis of MBP mRNA was carried out (Fig. 6C and D). Both types of MBP, type I and type I1 (shown by arrowheads), were more abundant in cells from the GalC-positive fraction than those from the negative fraction, indicating that GalC-positive cells express both type I and type I1 isoforms at P9. These results and the developmental profile (see Fig. 4) suggest that type I1 isoforms are produced mainly in the immature cells of oligodendroglial cell lineage and decrease as the cells mature.

Several other isoforms with unknown structures were identified by the hybridization analysis

The bands of the adult sciatic nerve that were detected at approximately the same positions as the exon 5-deleted isoforms in Fig. 2B (bands g and k) did not hybridize with C46 or C47 probes (Fig. 2C and D), suggesting that these are different isoforms. Bands d and i of the adult sciatic nerve in Fig. 2A may also be different from the exon 5-deleted isoforms found in the brain. Band 1 identified by the C7 probe (Fig. 2A) was predominantly expressed in adult sciatic nerve

A.. -

c kR7

1 2 3 4 5 Z O L 504& 1 2 1.5 kd 4 m - 434-- 7/17 kd-1

267, 4

2 3 4 . k 2 13~-

D 587, 1 2 3 4 540 504& 45a-- ,18.5 kd

267, 1 1 4 kd L

234.- * 2 13,'-

434-- 1 17 kd-2

FIG. 6. Expression of the MBP gene in GalC-positive and GalC- negative cells. A: Flow cytometric analysis of the P9 brain cells treated with anti-Gale antibody, which was detected by fluorescein, and PI. Sorting windows for the GalC-positive cells (right) and for GalC-negative cells (left) are shown. 8: Histogram of the relative intensity of fluorescein of the events whose PI intensity was lower than level a in A. The solid line shows the result of the cells treated with the anti-GalC antibody, and the dotted line shows the control. L.evels band c are the same as those shown in A. C and D: Hybridization analysis of the RT-PCR products from the GalC-positive (lane 2) and GalC-negative (lane 3) fractions with exon 2-specific (C7) probe (C) or exon 1 -exon 3 junction-specific (C12) probe (D). Lane 1 shows the result from total RNA of P9 mouse brain, and lane 4 shows the negative control. The positions of the isoform!; without exon 5 (type II; identified by hybridization with C46 or C47 probes; data not shown) are indicated by arrowheads.

.I. Neirrochem.. Vol. 60, No. 4, 1993


and weakly expressed in P18 brain. Band m identified by the C47 probe (Fig. 2D) was detected only in the adult sciatic nerve. These two (1 and m) isoforms did not hybridize to the other probes described above. Band a in Fig. 2A (adult sciatic nerve and P18 brain) and band b in Fig. 2B (adult sciatic nerve and P18 and P8 brain) did not hybridize to C46 or C47 probes. The level of the intensity of band b was higher in normal mouse brain than in jimpy brain at P18 (Fig. 5C), suggesting that this isoform is mature stage-specific. In sum, there are at least seven MBP isoforms and possibly 1 1 more isoforms (bands i, d , and fin brain, and bands d, g, i, k, a, b, I, and m in sciatic nerve) at the mRNA level.

DISCUSSION

MBP isoforms produced in embryonic mouse brain In this study, we found that the MBP gene is ex-

pressed in mouse brain from a very early stage of neural development, i.e., as early as E12. MBP was found to have at least seven isoforms, including two novel isoforms identified by sequencing, and possibly an additional 11 isoforms at the RNA level. All isoforms were detected in reverse transcripts primed by oligo dT primers, as well as random hexamers; thus, it is highly likely that the transcripts are mRNAs. If the minor isoforms described previously that utilize exon 0, la, or 5a (Kitamura et al., 1990; Aruga et al., 1991) are included, then the number of MBP isoforms be- comes even larger than 18. The isoforms detected in brain were classified into at least three groups with different developmental profiles (types 1-111; summarized in Fig. 4). The predominant isoforms in the embryonic stage were identified as exon 5-deleted forms (type 11). Levels of these isoforms peaked during the late embryonic stage (E 15-E 18) and decreased to very low levels at P18. The isoforms of exon 1-2-3-4-6-7, exon 1-2-3-4-7, and exon 1-3-4-7 should give rise to proteins of approximately 20 kDa, 15.6 kDa, and 13 kDa, respectively. These may be the polypeptides observed previously as “20-kDa,” “ 1 5-kDa,” and “ 12- 1 3-kDa” MBP-related polypeptides in cell-free trans- lations or immunoblots described by others (Green- field et al., 1982; Carnow et al., 1984; Sorget al., 1986; Campagnoni, 1988). Cell type producing MBPs and possible function in embryonic stage

The developmental profiles and the experiment using jimpy mouse brain suggest that, at least after the late embryonic stage, type I isoforms are produced mainly in mature oligodendrocytes and have a function associated with myelin formation. On the other hand, type I1 isoforms are produced mainly in immature oligodendrocytes and have a function that is not associated directly with myelination. Production of both types of isoforms in cells of oligodendroglial cell lineage is supported by the results of experiments us-

ing FACS, which indicated that GalC-positive cells (oligodendrocytes) produce both types at least at P9. However, these results cannot exclude the possibility that, during the late embryonic stage, type I1 isoforms may also be produced by another type of cells that are scarcely present at P9, such as cells surrounding the immature oligodendrocytes and affecting their growth.

The cell type that produces MBPs at an earlier embryonic stage (E12-E14) remains to be clarified. If MBPs are produced in cells of the same cell lineage as those producing in the later stage, there may be a group of cells already that are committed to generate oligodendrocytes later and produce MBPs. The existence of this type of committed cells at this early stage has been suggested, although not clearly identified, by Luskin et al. (1988).

The region encoded by exon 5 is quite active meta- bolically. The serine residues in bovine MBP corresponding to serine-133 and serine-138 residues of mouse MBP are known to be phosphorylated by C kinase (1 33, 138), A kinase (1 33, 138), or Ca’+/cal- modulin-dependent protein kinase ( 138) (Carnegie et al., 1974; Turner et al., 1984; Kishimoto et al., 1985; Shoji et al., 1985, 1987). The arginine residue in human MBP corresponding to arginine- 130 of mouse MBP is a unique site for methylation, which is quite important for compact myelin formation (Baldwin and Carnegie, 1971; Amur et al., 1986). In addition, this region is known to be associated with the carbo- hydrate-binding site (Abbott et al., 1989) and main encephalitogenic region (Baldwin and Carnegie, 197 l), which has sequence homology with various viral proteins (Jahnke et al., 1985). These observa- tions strongly suggest that the function of MBP isoforms without exon 5 is different from that of isoforms with exon 5.

Developmental regulation of MBP promoter activity in brain cells in primary culture

The MBP promoter region used in the promoter assay in the present study was shown to confer brain or oligodendrocyte-specific expression in the transgenic mouse system (Katsuki et al., 1988; Kimura et al., 1989) and in the retrovirus-mediated promoter assay system (Ikenaka et al., 1992). In this study, we showed that this promoter region is also well regulated developmentally. The ratio of cytoplasm-positive cells to nucleus-positive cells increased during the stages equivalent to P6-P8, which parallels the increase in the number of oligodendrocytes in vivo. However, numerous cytoplasm-positive cells were observed at the stage of PI equivalent (1 5- 19%). It is possible that embryonic brain cells committed to oligodendroglial cell lineage already have machinery that actively initiates transcription from the MBP promoter; however, MBP mRNAs do not accumulate until oligodendrocytes mature. This hypothesis is

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1562 K. NAKAJIMA ET AL.

supported by in vitro transcription analysis of the MBP promoter, in which nuclear extracts obtained from embryonic mouse brain efficiently transcribed MBP promoter (T. Tamura, personal communica- tion). We are planning to isolate cells producing ,B-galactosidase following MBP 1.3/pIP200 virus infection by the fluorescein-di-P-D-galactopyranoside-FACS method. This should enable us to investigate if these cells are direct precursors of oligodendrocytes.

Another unknown exon may be present in type 111 isoforms

The isoforms identified as bands d and fin Fig. 2D showed different developmental profiles from the other isoforms. The sizes and the presence of exon 4-exon 7 junctional sequence suggests that these isoforms have another unknown exon of approximately the same (or slightly larger) size as exon 6.

In conclusion, we reported here the embryonic expression of the MBP gene and showed that the predominant isoforms in this stage are exon 5-deleted forms. In addition, there may be at least seven isoforms and possibly an additional 11 isoforms of mRNAs. MBP, thus, is regulated in a very compli- cated manner at each developmental stage and may have many physiological functions other than that dedicated to myelination in the developing CNS.

Acknowledgment: W e thank T. Tamura , M. Ogawa, and T. Miyata for valuable discussions, and M. Nishiwaki for technical assistance. This work was supported by a grant from the National Center for Nervous, Mental and Muscu- lar Disorders of the Ministry of Health and Welfare, and a Grant-in-Aid for Scientific Research from the Ministry of Education. We also thank Japan Intractable Diseases Re- search Foundation for research support.

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Novel Isoforms of Mouse Myelin Basic Protein Predominantly Expressed in Embryonic Stage