Myelin transcription factor 1 (MyT1) immunoreactivity in infants with periventricular leukomalacia

Developmental Brain Research 140 (2003) 85–92 www.elsevier.com / locate / devbrainres

Research report

Myelin transcription factor 1 (MyT1) immunoreactivity in infants with periventricular leukomalacia Aya Hirayama a,b , *, Akira Oka c , Masayuki Ito d , Fumi Tanaka b,e , Yumi Okoshi b , Sachio Takashima f a

Department of Pediatrics, Akita University, Akita, Japan National Institute of Neuroscience, National Center of Neurology and Psychiatry ( NCNP), Tokyo, Japan c Division of Child Neurology, Tottori University School of Medicine, Tottori, Japan d Department of Mental Retardation and Birth Defect Research, NCNP, Tokyo, Japan e Department of Pediatrics, Tohoku University, Hiyagi, Japan f Yanagawa Institute for Developmental Disabilities, International University of Health and Welfare, Fukuoka, Japan b

Accepted 17 October 2002

Abstract Myelin transcription factor 1 (MyT1) is a zinc-dependent, DNA-binding protein, and is known to be expressed in early progenitors of oligodendrocytes. We examined the immunoreactivity of MyT1 in developing human brains and brains with periventricular leukomalacia (PVL) to understand the relationship between the expression of MyT1 and myelination in PVL brains. MyT1-positive glial cells were first detected at 19 gestational weeks (GWs) and then gradually increased until 26–29 GWs in the control group. Then they decreased and became very rare at 1 year of age. The expression of MyT1 immunoreactivity shifted from the nucleus to the cytoplasm of the glial cells in the developmental time course. In the chronic stage of PVL, MyT1-positive cells were significantly increased around necrotic foci and some of the regions were coincident with increasing MBP and PLP immunoreactivity. These results may reflect myelin repair on dysmyelination around PVL areas. Therefore, MyT1 may play an important role in the myelin repair in PVL regions.  2002 Elsevier Science B.V. All rights reserved. Theme: Development and regeneration Topic: Glia and other non-neuronal cells Keywords: Myelination; Myelin basic protein; Oligodendrocyte; Periventricular leukomalacia; Transcription factor

1. Introduction Hypoxic–ischemic brain damage is an important neurological problem during the perinatal period. In particular, periventricular leukomalacia (PVL) is becoming increasingly recognized as an important cause of cerebral palsy. There have been several reports of myelination observed in magnetic resonance imaging (MRI) in children with PVL or cerebral palsy. Van de Bor et al. have reported delayed myelination in patients with PVL diagnosed by means of *Corresponding author. Department of Pediatrics, Akita University, 1-1-1 Hondo, Akita, Akita 010-8543, Japan. Tel.: 181-18-884-6159; fax: 181-18-836-2620. E-mail address: [email protected] (A. Hirayama).

ultrasonography [1], but other investigators have described normal myelination in PVL brains [2,3]. Several authors emphasized that infants with extensive PVL exhibit more delayed myelination or dysmyelination [4]. Neuropathological studies have shown that myelination is mainly impaired in the necrotic and gliotic periventricular white matter of widespread PVL brains [5]. Myelin transcription factor 1 (MyT1) is named for its ability to recognize the proteolipid protein (PLP) gene, the most abundantly transcribed central nervous system myelin gene [6]. MyT1 is a zinc-dependent, DNA-binding protein of the Cys 2 –His–Cys class. The pattern of MyT1 expression suggests that MyT1 may be instrumental in early stages of oligodendrocyte development and myelin production [7].

0165-3806 / 02 / $ – see front matter  2002 Elsevier Science B.V. All rights reserved. PII: S0165-3806( 02 )00585-0

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The purpose of this study is to clarify the development of MyT1 in the cerebral hemispheres of human brains, and the relationship between expression of MyT1 and myelination in PVL brains.

2. Materials and methods

antibodies, followed by visualization with an enhanced chemiluminescence detection kit (ECL Western blotting analysis system; Amersham). Afterwards, the membranes were stained with Coomassie brilliant blue R-250, in order to confirm that the sample loaded onto each lane contained an equal amount of protein. For negative control experiments, the primary anti-MyT1 antisera were substituted by preimmune or preabsorbed sera.

2.1. Human cerebral specimens 2.4. Immunohistochemistry for MyT1 We examined 29 infants with PVL, who were born at 24–41 gestational weeks (GWs) and died at less than 11 months. Twenty age-matched controls without leukomalacia, intracranial hemorrhages or anomalies were also selected from the autopsy files. They were born at 19–41 GWs; three cases were stillborn and the others survived for 1 h to 1 year. We obtained parental informed consent for autopsy and research. Neuropathological examination was performed on large coronal specimens of the cerebral hemispheres, which were stained with hematoxylin and eosin (H–E), and luxol fast blue. For Western blotting, frozen samples of cerebral white matter obtained from the frontal lobes of the control subjects were used. Postmortem examination was performed within 24 h of death.

Immunohistochemical studies were performed with antibodies against glial fibrillary acidic protein (GFAP), myelin basic protein (MBP), proteolipid protein (PLP) and myelin transcription factor 1 (MyT1), on 4-mm thick sections of formalin-fixed and paraffin-embedded tissues. The sections were deparaffinized in xylene and then rehydrated in ethanol. Microwave irradiation was performed to retrieve the antigen after endogenous peroxidase activity had been blocked with 0.3% H 2 O 2 in methanol. The sections were then incubated with 10% normal goat or rabbit serum (depending on the first antibodies) to block

2.2. Antiserum production Antisera were raised in Japanese white rabbits against two peptides synthesized by solid-phase techniques according to the reported sequence [6]. They comprised amino acid residues 130 to 145 plus the -amino terminal cysteine (peptide 24; CVPPYGSYRPNVAPRHT), and 579 to 595 plus the amino terminal cysteine (peptide 25; CFAGKKGKLSGDEVLSPK). After immunization, collection, titration, and absorption were performed as described previously [8], and two antisera (24-2 against peptide 24, and 25-1 against peptide 25) were produced.

2.3. Western blotting Western blotting was performed as described previously [9]. Briefly, frontal white matter tissues from control patients aged 21, 30 GWs, 5 months and 11 years were thawed, and then proteins were extracted with Tris–saline buffer containing 1% Triton X-100. After centrifugation, supernatants were collected, and then protein assaying was performed by the method of Bradford [9]. Samples (30 mg / lane) were separated on 10% sodium dodecyl sulfate (SDS) polyacrylamide gels. After they had been electrophoretically transferred to nitrocellulose membranes (Hybond ECL; Amersham Life Science, Buckinghamshire, UK), the membrane was blocked with 5% skim milk. The membranes were then probed overnight at 4 8C with antiMyT1 antiserum (diluted 1:500). Then, incubation was performed with peroxidase–conjugated anti-rabbit IgG

Fig. 1. Immunoblot analysis of the frontal white matter of controls at 21 gestational weeks (GWs) with MyT1 antisera (25-1) and (24-2) (A), and of developing brains at 21 GWs, 30 GWs, 5 months and 11 years with MyT1 antiserum (24-2) (B). Both antisera recognized a band approximately corresponding to 110 kDa, which corresponded to the molecular weight of MyT1(A). The band of MyT1 seen at 21 GWs was not observed after 30 GWs (B). MyT1, Myelin transcription factor 1, preim, preimmune sera; ab, preabsorbed sera.

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Table 1 Expression of myelin transcription factor 1 in the deep white matter GWs

n

19–21 22–25 26–29 30–34 35–37 38–40 1–5 Ms 6 Ms–1 Y

2 2 2 3 3 3 3 1

Deep white matter Total

Nucleus

Nucleus1cytoplasm

Cytoplasm

11 111 111 11 11 1 1 1

1 11 1 1 – – – –

– 1 1 1 1 1 – –

– 1 1 1 1 1 1 1

Myelination on MBP stains

1 1 1 1

GWs, Gestational weeks; MBP, myelin basic protein; Ms, months; Y, year.

nonspecific binding. Between the steps, the sections were thoroughly washed with phosphate-buffered saline (PBS), pH 7.4. The sections were then incubated with anti-GFAP antibodies (Dako, Carpenteria, CA, USA, diluted 1:2000, 30 min at room temperature), anti-MBP antibodies (Boehringer, Mannheim, Germany, diluted 1:500, 1 h at room temperature), anti-PLP antibodies (AGMED, Bedford, MA, USA, diluted 1:50, overnight at 4 8C), and anti-MyT1 (25-1) antibodies recognizing an epitope located in the carboxyl-terminal fragment corresponding to amino acid residues 579–596 (developed in our laboratory; diluted 1:1000, overnight at 4 8C), followed by biotinylated second antibodies (depending on the first antibodies) and peroxidase-conjugated streptoavidin (Nichirei, Tokyo, Japan, prediluted). Between the steps, the sections were thoroughly washed with PBS, pH 7.4. The immunoproducts were

visualized using diaminobenzidine (Dojin, Osaka, Japan; 0.02%) as a chromogen. We compared the number of MyT1 positive cells in the deep white matter of the parietal lobes. The degree of immunostaining was evaluated as the number of positive cells per high-magnification view for MyT1 classified into five grades: –, 0 / 6.25310 4 mm 2 ; 1, 1 to 5 / 6.25310 4 mm 2 ; 11, 6 to 10 / 6.25310 4 mm 2 ; 111, 11 to 15 / 6.25310 4 mm 2 ; 1111, more than 15 / 6.25310 4 mm 2 .

3. Results The specificity of the MyT1 (25-1) and (24-2) antisera on Western blotting of white matter samples is shown in Fig. 1A. Both antisera recognized a band approximately

Fig. 2. Immunohistochemical expression of MyT1 (25-1) in the deep white mater (A, B, D), and the germinal layer (G) of controls, at 19 GWs (A, B, E), and 40 GWs (D). MyT1-immunoreactivity was first detected only in the nuclei of glial cells at 19 GWs (B), and then shifted to the cytoplasm as they increased. The number of MyT1-positive cells decreased gradually after 30 GWs (D). MyT1-immunoreactivity was also detected in the germinal zone (E). Immunohistochemical expression of MyT1 (24-2) in the deep white mater of controls, at 19 GWs (C). The immunohistochemistry with Ab 24-2 showed similar findings to those of Ab 25-1. A, 340; B, C, D, 3200; E, 3100. V, Lateral ventricle; DW, deep white matter.

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corresponding to 110 kDa, which corresponded to the molecular weight of MyT1. The band was not observed with preimmune antisera and preabsorbed antisera. The expression of MyT1 (24-2) in the cerebral white matter of developing human brains observed on Western blotting of white matter samples is shown Fig. 1B. The band of MyT1 seen at the age of 21 GWs was not observed after the age of 30 GWs, indicating upregulation of the protein in the early fetal period. The expression of MyT1 in the cerebral white matter of developing human brains is shown in Table 1. These are data obtained in immunohistochemical studies with antiserum MyT1 (25-1). Using an antiserum (24-1 or 25-1), almost identical results were obtained (Fig. 2). In the white matter, a shift of MyT1 localization from the nuclei to the cytoplasm was observed in the developmental time course. MyT1-positive glial cells in the control group were first detected at 19 GWs only in the nuclei of the glial cells, and then MyT1 immunoreactivity shifted to the cytoplasm at

23 GWs as the cells gradually increased. Some cells exhibited MyT1 immunoreactivity in both the nucleus and cytoplasm, and others only in the cytoplasm. The number of MyT1-positive cells peaked at 26–29 GWs and then decreased gradually. Only a few MyT1-positive cells were found at 1–5 months and they were very rare at 1 year of age (Fig. 2B, D). MyT1-positive cells were also detected in the germinal layer (Fig. 2E). Microscopically, myelination was observed at 35 weeks gestation in the deep white matter on MBP stains (Table 1). The pathologic features of PVL on H–E staining are summarized in Table 2. All samples with PVL could be divided into three stage groups according to the tissue reaction on H–E staining. The definition of each stage was as follows. Acute PVL, tissue with coagulation necrosis and / or microglial activation but without astrocytosis. Subacute PVL, tissue with astrocytosis but without any of the chronic changes. Chronic PVL, tissue with at least one of the chronic changes, such as neovascularization, calcifi-

Table 2 Pathology features of periventricular leukomalacia No.

Age

GWs

Distribution

Hematoxylin and eosin staining Coagulation necrosis

Axonal swelling

Reactive astrocyte

Foam cell

Neovascularization

Cavity formation

Acute 1 2 3 4 5 6 7

2 days 3 days 13 days 1 day 2 days SB 1 day

26 32 34 36 38 39 40

W F W W F F F

1 1 1 1 1 1 1

– – 1 1 1 – –

– – – – – – –

– – – – – – –

– – – – – – –

– – – – – – –

Subacute 8 9 10 11 12 13 14 15 16 17

5 days 18 h 0 day 7 days 1h 3 days 6 days 0 day 8 days 1M

24 27 34 35 36 38 39 40 39 38

W W F W W F F F W F

1 1 – – – 1 1 – 1 –

– – 1 1 – 1 1 – 1 –

1 1 1 1 1 1 1 1 1 1

– – 1 1 – 1 1 1 1 1

– – – – – – – – – –

– – – – – – – – – –

Chronic 18 19 20 21 22 23 24 25 26 27 28 29

1 M 2 days 1 M 24 days SB 3 Ms 2 Ms 3 Ms 2 Ms 6 Ms 3 Ms 7 Ms 8 Ms 11 Ms

24 24 35 26 29 29 41 27 40 31 33 28

W W F F F W W W F W W D

– – – – – – – – – – – –

– – – 1 – 1 1 – 1 – – –

1 1 1 1 1 1 – 1 – 1 1 1

1 – 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1

1 1 1 – 1 – – 1 – – – –

GWs, Gestational weeks; SB, stillbirth; F, focal; W, widespread; D, diffuse; M, month; PVL, periventricular leukomalacia; MBP, myelin basic protein.

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cation and cavity formation. They were also divided into three groups, i.e., focal (F), widespread (W), and diffuse (D), according to the distribution of tissue necrosis, as described previously [10]. The MyT1 reactivity in the PVL cases, is shown in Table 3, and the mean numbers of MyT1-immunoreactive cells in the deep white matter of controls and PVL regions are shown in Fig. 3. In the acute stage, MyT1-positive cells were more decreased in PVL areas than in controls (Fig. 4A). In the subacute stage, MyT1-positive cells were more decreased or equal to in controls from 22 to 37 GWs, but were more increased at the corrected ages of 38 to 40 GWs. In the chronic stage, MyT1-positive cells were significantly more increased around necrotic foci at the age from 1 to 8 months than in controls (Fig. 4B). As shown in Fig. 4, MyT1 expression was found in both the nuclei and cytoplasm of glial cells. In chronic PVL, GFAP-positive cells were also increased in and around necrotic foci, but the distribution of their expression was different from that of MyT1 expression. MBP expression was also more Table 3 Expression of myelin transcription factor 1 No.

Myelin transcription factor 1 PVL area

non-PVL area

Control

Acute 1 2 3 4 5 6 7

1 1 1 1 1 1 1

11 1 11 1 1 11 1

111 111 11 11 1 1 11

Subacute 8 9 10 11 12 13 14 15 16 17

1 1 1 111 1 1 1 111 11 11

11 1 1 11 1 1 1 111 11 11

11 111 11 11 11 1 1 11 1 1

Chronic 18 19 20 21 22 23 24 25 26 27 28 29

111 1111 1111 111 11 111 1111 1111 11 1111 111 1

11 1 1 1 1 1 1 1 1 1 111 1

111 111 11 11 11 1 1 1 1 1 1 1

PVL, Periventricular leukomalacia; MBP, myelin basic protein.

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increased around PVL areas than in the other white matter in cases 22–26, suggesting transient overexpression of MBP, and consistent with the increase of MyT1-positive cells (Fig. 5). We also performed the immunochemistry with anti-PLP antibodies (data not shown), and the overexpression of PLP was observed as well as that of MBP.

4. Discussion Our previous study revealed that impairment of myelination in PVL brains occurred in widespread necrotic or diffuse gliotic regions, especially regarding the lipid components of myelin rather than MBP. In these regions, ferritin-containing oligodendrocytes are decreased in number. These findings suggest that the impairment of myelination may be related to a dysfunction of iron metabolism or the degeneration of oligodendrocytes [5]. In the present study, impairment of myelination was observed in most chronic and widespread PVL regions on MBP staining. Excitotoxins may also play a role because cultured oligodendroglia are highly vulnerable to glutamate-induced cell death [11,12]. Back and Volpe revealed that PVL results in a chronic disturbance of myelination, which suggests that oligodendrocyte progenitors are a major target cell of ischemic injury in PVL [13,14]. Use of antibodies specific to oligodendrocyte progenitors has demonstrated that late oligodendrocyte progenitors are the predominant oligodendrocyte stage in human cerebral white matter during the time peak of incidence of PVL [15]. Late oligodendrocyte progenitors were the major oligodendrocyte lineage stage killed by apoptosis, whereas early oligodendrocyte progenitors and more mature oligodendrocytes were highly resistant in neonatal rat models of hypoxic–ischemic injury [16]. MyT1 is the prototypic member of a new class of DNA-binding proteins in the ‘zinc-finger’ superfamily of transcription factors [6]. Western blotting showed that MyT1 (24-2) antiserum recognized a single band at 110 kDa, but MyT1 (25-1) antiserum recognized two bands, one weak band at 110kDa and a second band at around 130 kDa. Although our antibody was made excluding the amino acids of MyT1-like (MyT1l) protein, the antiserum MyT1 (25-1) may cross-react with MyT1l protein. The novel gene, MyT1l, is highly homologous to the original representative of this class, MyT1 [6]. Unlike MyT1, Myt1l has not been detected in the glial lineage [7]. But the protein contains numerous potential phosphorylation sites that could increase the relative mobility of the protein on denaturing gels. During normal development in the postnatal rat brain, MyT1 is present in the nuclei of early progenitors of oligodendrocytes and continues to be present in the nuclei of differentiated oligodendrocytes, but then shifts to the cytoplasm and progressively decreases as the mature oligodendrocytes accumulate myelin-specific proteins [7].

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Fig. 3. The mean number of MyT1 (25-1) immunoreactive cells in the deep white matter of controls and periventricular leukomalacia (PVL) cases (A). Immunohistochemical expression of MyT1 (25-1) in case 25 with chronic PVL (B, C). B, 340; C, 3200. V, Lateral ventricle; N, necrosis.

Our results demonstrated that the MyT1 localization shifted from the nuclei to the cytoplasm in the developmental time course. MyT1-positive glial cells in the control group were first detected at 19 GWs and then gradually increased until 26–29 GWs. Then they decreased and were very rare by one year of age. We also detected MyT1-immunoreactivity in the germinal layer. MyT1-immunoreactivity in neuroepithelial germinal zones of the central nervous system (CNS) has been reported in tissues at embryonic [9], early postnatal [7], and adult [17] ages. The developmental expression and localization of MyT1 indicates that it may play a role in the development of neurons and oligodendroglia, and the persistence of MyT1 expression in oligodendrocyte progenitors within the white matter and in the germinal zone suggests a potential role for MyT1 in CNS regenerative responses [18]. In the chronic stage of PVL, MyT1-positive cells were

significantly more increased around necrotic foci during early infancy. Some of the regions were coincident with increasing MBP immunoreactivity around necrotic foci. This finding might reflect remyelination or hypermyelination. Remyelination in demyelinating diseases such as multiple sclerosis (MS) has been reported. However, remyelination in MS is often incomplete and limited. Also, studies on the adult rat spinal cord have revealed that remyelination is carried out by oligodendrocyte pregenitor cells [19]. Our results might suggest that myelin repair occurs on dysmyelination around PVL areas and is related to the expression of MyT1. This hypothesis is supported by results indicating the reappearance of MyT1 in response to demyelination in rodents [20]. By using a neonatal rat models, a reactive response was observed in both late oligodendrocyte progenitors and immature oligodendrocytes in perinatal brain that is related to the oligoden-

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Fig. 4. Immunohistochemical expression of MyT1 (25-1) in case 7 with acute PVL (A), case 20 with chronic PVL (B), and the control case (C). The control case died at 40 GWs (C). In the acute stage, MyT1-positive cells were more decreased in PVL areas than in controls. In the chronic stage, MyT1-positive cells were significantly increased around PVL. 350. N, Necrosis.

Fig. 5. Histopathological findings on H–E stain and immunohistochemical expression of MyT1 (25-1) and MBP in case 25 with PVL. There is remote PVL with astrogliosis and foam cells (A). MyT1-positive cells were increased around PVL areas (arrows) (B). MBP expression was also were increased around PVL areas (arrows) than in the other white matter, and consistent with the increase of MyT1-positive cells (C). A, 350; B, C, 310.

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drocyte stages present at the time of hypoxic–ischemic injury [16]. Further understanding of the myelin repair process might lead to new treatments aimed at inducing remyelination in PVL. Our neuropathological and immunohistochemical studies have shown that oligodendrocyte progenitors are increased in the chronic stage of PVL, and some cases are consistent with the overexpression of MBP and PLP. Therefore, MyT1 may play an important role in myelin repair in PVL regions.

Acknowledgements We thank Drs. Y. Kida, A. Nishida, M. Kajiwara and H. Horie for their kind collaboration; Professor G. Takada for his support.

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