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Endocrinology Vol. 145, No. 11 5013-5020
Copyright © 2004 by The Endocrine Society

Thyroid Hormone Regulates Oligodendrocyte Accumulation in Developing Rat Brain White Matter Tracts

Christopher M. Schoonover, Melissa M. Seibel, Dawn M. Jolson, Mary Jo Stack, Rounak J. Rahman, Sidney A. Jones, Cary N. Mariash and Grant W. Anderson

Department of Medicine (C.M.S., M.M.S., M.J.S., R.J.R., S.A.J., C.N.M.), University of Minnesota, Minneapolis, Minnesota 55455; Department of Biological Sciences, University of California-San Diego (D.M.J.), San Diego, California 92093; and Department of Pharmacy Practice and Pharmaceutical Sciences (G.W.A.), University of Minnesota College of Pharmacy, Duluth, Minnesota 55812

Address all correspondence and requests for reprints to: Grant W. Anderson, Ph.D., College of Pharmacy, Duluth, 354 Kirby Plaza, 1208 Kirby Drive, Duluth, Minnesota 55812-3095. E-mail: ander163{at}umn.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Thyroid hormone (TH) is necessary for normal axonal myelination. Myelin basic protein (MBP) is a structural protein essential for myelin function. In this study, we demonstrate that perinatal hypothyroidism regulates MBP mRNA levels via indirect mechanisms. We observed decreased MBP mRNA accumulation in the hypothyroid rat brain at postnatal (PN) d 10 and 50. Acute TH replacement did not rescue hypothyroid MBP mRNA levels at PN5, 10, or 50. TH is necessary for normal intrahemispheric commissure development including the anterior commissure (AC) and the corpus callosum (CC). We determined that perinatal hypothyroidism decreases AC area and cellularity in the developing rat brain by PN10 and 50. In the developing CC, hypothyroidism initially increases area and cellularity by PN5, but then ultimately decreases area and cellularity by PN50. MBP-expressing oligodendrocytes are a recognized target of TH and are responsible for myelination within intrahemispheric commissures. We found that hypothyroidism reduces the number of mature oligodendrocytes within both the AC and CC. This reduction is noted at PN5, 10, and 50 in the AC and by PN10 and 50 in the CC. Together, these data suggest that TH regulates MBP mRNA levels through indirect mechanisms. These data demonstrate the complex mechanisms whereby TH regulates myelination in the developing brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE (TH) is required for normal fetal and neonatal brain development (1, 2, 3, 4, 5). Hypothyroidism during brain development results in permanent functional deficits. TH controls brain development by regulating the transcription of specific genes expressed in the developing brain (6). Late brain development is particularly sensitive to TH levels. In rats, this TH-sensitive period corresponds to approximately the first month of postnatal life (7). During this time, neurons begin the process of maturation. This maturation process includes the myelination of neuronal axons by the oligodendrocyte.

In the mammalian brain, cortical areas of the two hemispheres are reciprocally connected by intrahemispheric commissures (white matter tracts) including the corpus callosum (CC) and the anterior commissure (AC). Information transfer via intrahemispheric commissures is critical for higher brain functions. Commissures arise embryonically in the rat and develop postnatally (8). During normal brain development, commissure axons project to their appropriate targets and become myelinated.

TH is necessary for normal intrahemispheric commissure development (9, 10, 11, 12). The number of myelinated axons in hypothyroid commissures is decreased (9, 11). Additionally, the thickness of the myelin sheath surrounding myelinated axons is significantly reduced in the hypothyroid animal (13). TH does not appear to control axonal generation because the total number of axons is unaffected by the thyroidal state (9, 11, 12). These data suggest that hypothyroidism interferes with the normal processes that contribute to axonal myelination within intrahemispheric commissures.

The oligodendrocyte is responsible for myelin formation within intrahemispheric commissures. The final coordination of an appropriately myelinated axon is accomplished through complex regulation of oligodendrocyte proliferation, differentiation, gene expression, and cell death. TH regulates oligodendrocyte development at several levels (14). First, TH initiates oligodendrocyte maturation. In the absence of TH, the oligodendrocyte precursor cell [oligodendrocyte-type II astrocyte (O-2A)] proliferates indefinitely in response to specific growth factors in vitro (15, 16, 17). However, in the presence of TH the O-2A cell terminates cell division and differentiates into a mature oligodendrocyte. Thus, hypothyroidism may result in a decreased number of mature oligodendrocytes by suspending O-2A cell precursor differentiation. A second role of TH in oligodendrocyte development is to enhance oligodendrocyte survival. Recently, our laboratory has demonstrated that TH is able to protect developing oligodendrocytes from apoptosis in vitro (18). Thus, TH appears to regulate both oligodendrocyte proliferation and survival.

In vivo studies also support this hypothesis because decreased numbers of mature oligodendrocytes are observed within hypothyroid rat optic nerves (19). Interestingly, hyperthyroidism has also been associated with decreased numbers of mature oligodendrocytes in the rat brain (20). The effects of TH on oligodendrocyte development are likely multifactorial and may include TH-dependent effects on O-2A cell proliferation and developing oligodendrocyte survival. Importantly, however, the in vivo effects of hypothyroidism on oligodendrocyte development within intrahemispheric commissures are undefined.

A third role of TH is to regulate myelin production in the developing oligodendrocyte (6, 14). Myelin is a multilamellar, protein-containing membrane that insulates mature axons and facilitates conduction of nerve impulses. Rodent myelin production is reduced in the hypothyroid neonate (21, 22, 23, 24, 25, 26). Interestingly, myelin levels remain reduced in the adult brain of animals rendered continuously hypothyroid from birth (23, 24).

MBP is one of the essential proteins that compose myelin. The regulation of MBP by TH is well described (6, 14). MBP mRNA levels are reduced in the brains of neonates and adults rendered hypothyroid from birth (27, 28, 29, 30, 31). However, published data suggest that the MBP gene is refractory to TH administration in the early neonatal (30, 32) and adult brain (29). If the MBP gene is unresponsive to TH in the adult brain, why then do adult animals rendered hypothyroid from birth (23, 24) exhibit decreased myelin production?

We hypothesize that the effects of hypothyroidism on oligodendrocyte proliferation and survival contribute to observed decreases in myelin and MBP mRNA levels during brain development. We demonstrate here that hypothyroidism leads to a reduction in oligodendrocyte numbers within brain white matter tracts. Oligodendrocyte cell number reductions are concomitant with reductions in MBP gene expression. These data support a role for TH in regulating oligodendrocyte accumulation during brain development. They further suggest that TH-dependent control of oligodendrocyte accumulation during development indirectly determines total MBP mRNA content in the developing brain.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Sprague Dawley rat pups (Charles River Laboratories, Kingston, NY) were rendered hypothyroid by ad libitum delivery of methimazole-treated water (0.025%) to midgestation dams. Acute TH injections included ip delivery of 2 µg/g body weight of T3 and T4. Euthyroid controls were injected with saline plus 2% BSA (vehicle). Brains were harvested at postnatal days of development as indicated. The brains were then used for mRNA preparation or histologic analysis. All animal studies were conducted in accordance with the principles and procedures outlined in the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. These procedures were approved by the local Institutional Animal Care and Use Committee.

RNA extraction and RT-PCR
Total RNA was extracted from rat brains using a QIAGEN RNeasy midikit (QIAGEN, Valencia, CA). Quantitative, real-time, RT-PCR was performed using intron-spanning primer sequences targeting the 5' end of the MBP mRNA (nucleotides 405–608, 5' end: 5'-GACTCACACACGAGAACT-3'; 3' end: 5'-CCAGCTAAATCTGCTGAG-3'). The primer set used for assaying MBP mRNAs consists of the 5' primer in exon 3 and the 3' primer in exon 7. The MBP splicing patterns have been well described (33). All known MBP splice variants include exons 3 and 7.

RT-PCRs were conducted using a Roche LightCycler (Roche Applied Science, Indianapolis, IN). Reagents used for the reactions were provided in the Roche SYBR Green I RNA Amplification Kit. One hundred nanograms of total RNA were used for each reaction. RT-PCR was be performed as follows: reverse transcription of template RNA for 30 min at 42 C; denaturation of the cDNA/RNA hybrid for 30 sec at 95 C; followed by 35 cycles of cDNA amplification consisting of a 15-sec denaturation at 95 C, primer annealing for 20 sec at 54 C, and product elongation for 15 sec at 72 C. The amplification process was monitored in real-time via fluorescence data acquisition at the end of each amplification cycle at a temperature slightly lower than the temperature required to melt the PCR product (83 C). Threshold cycle values were determined in the log-linear amplification phase using LightCycler Software (version 3.5; Roche Diagnostics GmBH, Mannheim, Germany), and plotted vs. log RNA content.

Histology
Rat brains were harvested and immediately fixed in 4% paraformaldehyde overnight at 4 C. After paraffin embedding, 8-µm sagittal sections were prepared beginning at the brain midline. All analyzed brain sections were located within 100–200 µm from the midline. We further ensured that all analyzed brain sections were taken from the same brain region by examining specific brain structures. Specifically, we assessed the shape and size of the third ventricle flanking the hippocampus and the CA1, CA3, and CA4 regions of the hippocampus. We chose sections for use in our comparative studies that were structurally similar within these brain regions. Figure 1Go shows a stained section representative of the brain region assessed in our comparative studies.



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  		FIG. 1. H&E-stained brain section representative of the brain region used in the comparative histological and in situ hybridization studies. CA3, Field CA3 of Ammon’s horn; CA4, field CA4 of Ammon’s horn; DG, dentate gyrus; 3V, third ventricle.

 
Cell counting
Sagittal sections of the developing rat brain were stained with hematoxylin and eosin (H&E). Microscopic images were digitally captured using Adobe Photoshop 5.0 (Adobe Systems, San Jose, CA). AC white matter tract nuclei were identified by their dark blue staining pattern and were enumerated by direct counting of the preserved images. The number of CC white matter tract nuclei was estimated as follows. The average total cellular density of each digitally preserved callosum was determined by sampling the density of nine 5.5-mm2 sections within each individual callosum. The nine sample areas represented rostral, medial, and caudal aspects of each individual callosum. The average nuclear density of each individual callosum was then multiplied by the known callosal area to provide an estimate for the total number of nucleated cells within each CC. The accuracy of this estimation was confirmed by direct counting of representative slides. The relative diameter of H&E-stained nuclei within the PN50 AC was not markedly different between hypothyroid (0.0647 ± 0.0109) and euthyroid (0.0697 ± 0.0142) animals.

For the in situ hybridization experiments, proteolipid protein (PLP)-positive cells were visualized by their purple stain after treatment with BM purple reagent (Roche Molecular Biochemicals). PLP-positive cells were identified in the AC by their dark purple, cytoplasmically focused stain and were enumerated by direct counting. PLP-positive cells were identified in the CC by their dark purple cytoplasmic staining and were enumerated by automated cell counting using NIH Image 1.62. The relative diameter of PLP-positive cells within the PN50 AC was not different between hypothyroid (0.2532 ± 0.0449) and euthyroid (0.2532 ± 0.0.0518) animals.

Area calculation
Area calculations for the CC and AC were measured using NIH Image 1.62. Briefly, the commissures were identified by distinctive staining patterns and morphology after H&E staining. Area measurements were obtained by digitally tracing the outline of the desired structure after establishing a known distance-to-pixel ratio.

In situ hybridization
Nonisotopic in situ hybridization on hypothyroid and euthyroid rat brains was performed using a modification of the Wilkinson protocol (34). Digoxigenin probes were prepared using either SP6 or T7 promoter-driven synthesis (Roche Molecular Biochemicals). Slides were treated with histisol and rehydrated with graded ethanol. After protinase K treatment (10 µg/ml at room temperature for 7 min), the slides were hybridized with a digoxigenin-labeled riboprobe overnight at 65 C in hybridization buffer. The slides were then washed four times in MABT at room temperature followed by incubation with MABT [100 mM maleic acid (pH 7.5), 150 mM NaCl, 0.1% Tween 20] containing 2% blocking reagent and 20% fetal calf serum for 1.5 h at room temperature. The slides were subsequently incubated overnight at room temperature with antidigoxigenin activator protein-conjugated antibody (Roche Molecular Biochemicals) (1 µl antibody/2 ml). The slides were again washed four times in MABT at room temperature and equilibrated in alkaline phosphatase buffer for 10 min before exposure to BM purple (Roche Molecular Biochemicals) color development reagent. Slides were fixed in 4% paraformaldehyde after 1–3 d of color development.

Statistics
Statistical significance was determined by parametric analysis where appropriate. We first subject the data to a Levine test to ensure that the variances between the groups were not different. We then performed a two-way ANOVA with interaction using age and thyroidal status for main effects. If the ANOVA showed a significant difference (P < 0.05) for either of the main effects, or the interaction, a post hoc test using the Scheffé method was used to compare means between groups. In the event that one or more groups had no variance, we used the nonparametric Kruskal-Wallis test to compare differences between groups.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TH regulation of MBP mRNA levels during rat brain development
Rat pups were rendered hypothyroid from gestational d 14 and brains harvested at the noted postnatal days. MBP mRNA levels were assessed by quantitative real-time RT-PCR (Fig. 2AGo). MBP mRNA levels were not altered in the hypothyroid PN6 brain. However, hypothyroid rats displayed a significant decrease in MBP mRNA expression on PN10 (P < 0.01). This difference in MBP mRNA levels persisted in the adult PN50 brain (P < 0.01). Forty-eight hours of TH treatment of hypothyroid rats did not significantly alter MBP mRNA levels. To further assess whether TH acutely up-regulates MBP mRNA levels, PN10 rats were acutely treated with TH and total brain mRNA was harvested at the noted time points post treatment (Fig. 2BGo). PN10 was chosen for this study because the data presented in Fig. 2AGo indicated a slight increase in MBP mRNA levels when hypothyroid animals were treated for 48 h with exogenous T3. We observed no statistically significant increases in MBP mRNA levels when PN10 hypothyroid rats were acutely treated with T3 (Fig. 2BGo).



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  		FIG. 2. Effects of thyroidal status on rat brain MBP mRNA levels. A, TH transiently regulates MBP mRNA levels during postnatal development. Hypothyroid rat dams were treated with methimazole-treated water (0.025%) from midgestation until the time the rats were killed. Acute TH treatment consisted of ip TH delivery (2 µg/mg body weight T3 and T4) or vehicle at 48 and 24 h before the rats were killed. Levels of specific mRNA species were measured by real-time RT-PCR and expressed relative to an internal control. B, Effects of acute TH delivery on rat brain MBP mRNA levels at PN10. PN10 hypothyroid rat pups were acutely treated with 2 µg/mg body weight T3 and T4. Levels of specific mRNA species were measured by RT-PCR and expressed relative to an internal control. Brains were all harvested at 0900 h on PN10. Bars indicate the relative MBP mRNA level mean ± SEM (n = 6). ANOVA analysis was performed to assess statistically significant changes in mRNA levels.

 
A rapid, T3-dependent up-regulation of another TH-responsive brain gene, basic transcription element binding protein (BTEB), was observed using these RNAs (data not shown). BTEB is expressed in the developing neurons and not developing oligodendrocytes (35). Up-regulation of BTEB mRNA levels demonstrates that the ip administered TH activated expression of TH-responsive genes expressed in the brain.

TH regulates AC area
We next assessed the effects of TH on AC development. We stained sections of PN5, 10, and 50 euthyroid and hypothyroid rat brains with H&E. A distinct staining pattern and paucity of cells identifies the AC during development. We observed that the hypothyroid anterior commissure is smaller in area at both PN10 and 50. These gross observations were quantified.

Quantification revealed no significant differences between the hypothyroid and euthyroid AC area at PN5 (Fig. 3BGo). However, we noted a statistically significant, 22% decrease in the relative cross-sectional area of the hypothyroid AC when compared with euthyroid controls at PN10. This difference persisted through PN50 with the hypothyroid AC cross-sectional area measuring 30% less than euthyroid controls. Thus, TH regulates AC area in the developing rat brain.



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  		FIG. 3. TH regulates development of the AC. A, The AC is identified by a distinct eosin staining pattern and is outlined in black. B, TH regulates AC area. Rat dams drank either methimazole-treated water (0.025%) or normal water from midgestation until the time that rats were killed. Hypothyroid or control rat pups were harvested at the noted postnatal day of development. Digital images of H&E-stained sagittal brain sections were obtained. Area calculations for the AC were obtained using NIH Image 1.62. Bars indicate the area mean ± SEM (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN10 (P < 0.0001) and PN50 (P < 0.0001). C, TH regulates total AC cell number. AC nuclei were identified by their dark blue hematoxylin staining pattern and were enumerated by direct counting. Bars indicate the cell number mean ± SEM (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN10 (P = 0.001) and PN50 (P < 0.0001). D, In situ hybridization using a PLP probe was performed on sagittal brain sections. PLP-positive cells were identified in the AC by their dark purple cytoplasmic staining and were enumerated by direct counting of Photoshop preserved images (PN50 section shown here). The AC is outlined by the black oval. Each arrow points to a single PLP-positive cell; however, most PLP-positive cells are not marked by arrows. The AC is easily identified as the PLP-positive cell-rich, oval-shaped region. E, TH regulates accumulation of mature oligodendrocytes in the developing AC. Bars indicate the PLP-positive cell number mean ± SEM number (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN50 (P < 0.0001). *, Statistical significance.

 
TH regulates the number of cells within the AC
In keeping with the area observations, we observed no significant change in the number of H&E-stained nuclei in the hypothyroid PN5 AC (Fig. 3CGo). However, differences were observed by PN10. We observed a 33% reduction in cells counted per hypothyroid AC cross-section compared with euthyroid controls. This difference persisted through PN50, where we observed a 29% reduction in cells counted per hypothyroid AC cross-section compared with euthyroid controls. Thus, TH regulates the number of cells within the developing PN10 and 50 AC.

TH regulates the total number of oligodendrocytes populating the AC
We next used in situ hybridization to identify and quantitate mature oligodendrocytes within the AC. PLP is an oligodendrocyte-specific protein integral to the myelin sheath. PLP mRNA is exclusively expressed by mature oligodendrocytes and is localized in the oligodendrocyte cell body. In situ hybridization using a PLP riboprobe was performed on sagitally cut rat brain sections taken from hypothyroid and euthyroid PN5, 10, and 50 rat brains. Oligodendrocytes are identified within the AC by virtue of their deep purple staining after in situ hybridization (Fig 3DGo). Quantitation of the oligodendrocytes revealed a significant difference in PLP-positive cells on PN5 (Fig. 3EGo). At PN10, the number of PLP-positive cells within the hypothyroid AC was reduced by 36%. Even greater changes were observed at PN50 where the number of PLP-positive cells was reduced by 47% in the hypothyroid animals. Thus, TH regulates the number of oligodendrocytes within developing rat brain AC.

TH regulates CC area
TH may exert differential effects on oligodendrocyte proliferation, survival, and maturation within separate intrahemispheric commissures. Thus, a second brain white matter tract, the CC, was examined.

Gross analysis suggested that the PN5 hypothyroid CC is larger in area than the euthyroid control, similar in area to the euthyroid CC at PN10, and smaller than the euthyroid CC at PN50. Quantitation supports these observations, revealing that the PN5 hypothyroid CC is increased in area by 63% compared with the euthyroid control (Fig. 4AGo). There was no statistically significant difference in area observed between the two treatment groups at PN10. However, by PN50 the hypothyroid CC is significantly reduced in area by 64%. Thus, TH regulates the CC area during brain development.



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  		FIG. 4. TH regulates development of the CC. A, TH regulates CC area. Hypothyroid or control rat pups were harvested at the noted postnatal day of development. Digital images of H&E-stained sagittal brain sections were obtained. Area calculations for the CC were obtained using NIH Image 1.62. Bars indicate the area mean ± SEM (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN 5 (P < 0.0001) and PN 50 (P < 0.0001). B, TH regulates total CC cell number. CC nuclei were identified by their dark blue staining pattern. The number of nuclei within each CC was estimated by multiplying the known CC area by the average CC cell density. Bars indicate the cell number mean ± SEM (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN 5 (P < 0.0001) and PN 50 (P < 0.0001). C, TH regulates oligodendrocyte accumulation in the CC. In situ hybridization using a PLP probe was performed on sagittal brain sections. PLP-positive cells were identified in the CC by their dark purple cytoplasmic staining and were enumerated by automated cell counting using NIH Image 1.62 on Photoshop preserved images. Bars indicate the PLP-positive cell number mean ± SEM (n = 6). ANOVA shows a statistically significant difference between hypothyroid and euthyroid animals at PN10 (P = 0.0002) and PN50 (P < 0.0001). *, Statistical significance is noted.

 
TH regulates the number of cells within the CC
As was observed in the AC, the total number cells populating the CC correlated with changes in the measured CC area. The PN5 hypothyroid CC exhibited a 40% increase in the number of nucleated cells compared with the euthyroid controls (Fig. 4BGo). This difference was not observed in the PN10 CC. In contrast to PN5, the PN50 hypothyroid CC exhibited a 66% decrease in the number of nucleated cells compared with euthyroid controls. Thus, TH effects developing CC cellularity during brain development.

These findings support the hypothesis that TH regulates the population of supporting cells surrounding the CC axons. We next examined the effects of TH deprivation on the CC oligodendrocyte population.

TH regulates the total number of oligodendrocytes populating the CC
In situ hybridization was performed on sagitally cut rat brain sections at the noted postnatal days. The PN5 hypothyroid CC showed a 26% reduction in the number of PLP-positive cells compared with euthyroid controls (Fig. 4CGo), although this difference was not statistically significant. By PN10, the hypothyroid CC contained 30% fewer PLP-positive cells compared with the euthyroid CC. Finally, PN50 hypothyroid CC PLP-positive cells were reduced by 32% compared with euthyroid controls. Thus, TH regulates the number of mature oligodendrocytes in the developing rat CC.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
TH regulates brain MBP mRNA levels
Our findings demonstrate that TH controls the accumulation of mature oligodendrocytes in brain white matter tracts. We hypothesize that reduced accumulation of MBP-expressing mature oligodendrocytes during hypothyroidism leads to concomitant reductions in MBP mRNA levels.

The effects of TH on myelination have been known for several decades (21, 22, 23, 24, 25, 26). Consequently, MBP was one of the first brain genes identified as a target of TH-dependent gene regulation during brain development (27, 28, 29, 30, 31). Identification of a thyroid hormone response element in the MBP proximal promoter region (36, 37) supported the hypothesis that MBP mRNA levels are regulated by TH in a direct fashion. Several in vitro transient transfection studies have demonstrated that MBP proximal promoter regions containing the identified MBP thyroid hormone response element are TH responsive (37, 38, 39, 40, 41). Finally, a nuclear run-off analysis using 18-d-old hypothyroid rat brain nuclei revealed a 2-fold decrease in the MBP transcription rate (37). Together, these data suggest that the MBP gene is a direct target of TH in the developing brain.

Numerous in vivo studies demonstrate reduced MBP mRNA levels in hypothyroid animals (27, 28, 29, 30, 31) (see also Fig. 2Go). However, none of these in vivo studies assessed the effects of acute TH administration on regulation of MBP mRNA levels. If the MBP gene was directly up-regulated by TH, we would expect a rapid increase in MBP mRNA levels after TH stimulation. We now report that MBP mRNA levels are not altered by acute TH administration during rat brain development (Fig. 2Go). Two in vitro studies lend further support to these findings. In the first study, 72-h T3 treatment of primary oligodendrocyte cultures did not result in altered MBP mRNA levels (42). The second study assessed the effects of T3 on the rate of MBP transcription using a nuclear run-off approach. T3-dependent stimulation of the MBP transcription rate was not observed (43). Together, these studies suggest that TH may not directly regulate the rate of MBP transcription. Rather, TH may indirectly alter MBP mRNA levels by controlling the number of MBP-expressing oligodendrocytes populating the developing brain.

TH regulates commissure development
Commissures are predominantly composed of intrahemispheric axons and myelinating oligodendrocytes. Our data demonstrate that the sizes of the AC (Fig. 3BGo) and the CC (Fig. 4AGo) are altered in the hypothyroid neonate. These results are consistent with previously published data (9, 12).

Several hypotheses could explain these observed TH-dependent effects on CA. Firstly, postnatal development in the absence of TH may result in a decreased number of axons contained within a commissure. Gravel et al. (10), however, demonstrated no effect of hypothyroidism on the total number of axons per CC. Additionally, Ferraz et al. (12) showed no effect of hypothyroidism on the total number of axons located within the AC. Secondly, hypothyroidism may decrease the number of myelinated axons per commissure resulting in reduced commissure area. Previously published studies support this hypothesis. These data demonstrate that the hypothyroid PN25 and 60 rat CC contains a greater than 20-fold reduction in myelinated axons compared with euthyroid controls (10). This finding is supported and extended by Berbel et al. (11). They observed a significant reduction in myelinated axons from PN17 through adulthood within the CC of rats rendered hypothyroid from midgestation. In addition, they determined that myelinated axons were significantly reduced in the AC from PN17 through adulthood in the hypothyroid rat. Unmyelinated axons are reduced in diameter and are more tightly packed. Thus, the decreased number of myelinated axons observed in hypothyroid commissures contributes to the reduced cross-sectional hypothyroid commissure area.

TH regulates oligodendrocyte number within developing white matter tracts
A reduction in myelinated axons in hypothyroid commissures may be explained by either reduced myelin production from myelinating oligodendrocytes or normal myelin production from a reduced number of myelinating oligodendrocytes. To test the latter hypothesis, we assessed the total number of nuclei and oligodendrocytes per cross-sectional euthyroid and hypothyroid commissure area.

We determined that the observed TH-dependent changes in commissure area are accompanied by concomitant changes in total cell number (Figs. 3CGo and 4BGo). This change is consistent with the results of Bass and Young (44), demonstrating reduced DNA content in hypothyroid brain white matter.

We next assessed the effects of sustained neonatal hypothyroidism on the number of oligodendrocytes populating the AC and CC. We determined that the total number of oligodendrocytes populating the PN10 and adult AC and CC were significantly reduced when animals were rendered hypothyroid from midgestation (Figs. 3EGo and 4CGo). This reduction is most consistent with the commissure area calculations obtained from PN50 rats. This is not surprising because the number of myelinated axons within commissures are near maximal at this time point (11). The reduction in PN10 commissure area observed in hypothyroid animals is also consistent with the area calculations. The only discrepancy is noted in the PN10 CC where the CC hypothyroid area is the same as the euthyroid, but a significant increase in oligodendrocyte numbers is noted.

We hypothesize that TH-dependent regulation of oligodendrocyte accumulation determines the size of developing commissures. The commissure size may be reflective of the contribution of populating cell body volume to the overall volume occupied by the commissure. Additionally, the commissure volume may be reflective of the numbers of myelinated axons populating the commissure because a myelinated axon is substantially thicker in cross-sectional area compared with an unmyelinated axon (9). Therefore, reductions in oligodendrocyte numbers may lead to the myelination of fewer axons in the hypothyroid animal. Thus, we further hypothesize that TH-dependent regulation of oligodendrocyte accumulation determines the extent of axonal myelination within the developing commissure.

Mechanisms regulating oligodendrocyte accumulation in developing white matter tracts
TH regulates the proliferation of oligodendrocyte precursor O-2A cells (14, 15). In vitro studies demonstrate that in the absence of TH, oligodendrocyte precursor cells proliferate indefinitely in response to growth factors. The precursor cells do not differentiate into maturing oligodendrocytes unless the cell cycling is stopped. TH is thought to provide the signal required for cessation of O-2A cell cycling. Thus, in the absence of TH the developing brain will be populated with increased numbers of oligodendrocyte precursor cells but paradoxically, decreased numbers of developing oligodendrocytes.

TH also regulates the survival of developing oligodendrocytes (18). We have shown that TH protects developing oligodendrocytes from apoptosis during a precise stage of development (18). Specifically, we found that developing oligodendrocytes are able to survive for at least 10 d in vitro when cultured in the presence of TH. However, oligodendrocytes cultured in the absence of TH die between developmental d 3 and 4. These findings suggest a protective role for TH during oligodendrocyte development. Thus, reduced levels of TH in the hypothyroid neonate may lead to decreased survival of developing oligodendrocytes and concomitant reductions in oligodendrocytes populating brain white matter tracts.

TH likely regulates oligodendrocyte accumulation via multiple molecular mechanisms. Recent data suggest that TH may inhibit O-2A cell proliferation by repressing expression of the transcription factor E2F-1 (45). E2F family members regulate the expression of genes that promote entry into the cell cycle (46). TH also controls the expression of oligodendrocyte survival factors such as neurotropin-3 and IGF-I during late brain development (6, 17, 47, 48). Therefore, TH may regulate the accumulation of mature oligodendrocytes by controlling expression of these survival factors. Nonetheless, the mechanisms responsible for TH-dependent control over oligodendrocyte accumulation in vivo remain unclear.

Conclusions
TH regulates both O-2A cell proliferation and the survival of developing oligodendrocytes. O-2A cell proliferation and immature oligodendrocyte development occurs during late brain development. If TH levels are diminished during this period of time decreased numbers of mature oligodendrocytes will ultimately populate the mature brain. A reduction in oligodendrocyte numbers will lead to reduced axonal myelination and decreased expression levels of myelin-associated genes such as MBP. Reduced axonal myelination will interfere with efficient axonal transfer of neuronal signals. Thus, reduced oligodendrocyte accumulation in white matter tracts will contribute to the profound functional deficits observed when brain development occurs in the absence of TH.


   Acknowledgments
 
We would also like to thank Jennifer Metkowski for excellent technical assistance and Lucy Mittag for secretarial support.


   Footnotes
 
This work was supported by National Institutes of Health Grants 1F32-DK62577-01 and RO1 DK54060.

C.M.S. and M.M.S. contributed equally to this work.

Abbreviations: AC, Anterior commissure; BTEB, basic transcription element binding protein; CC, corpus callosum; H&E, hematoxylin and eosin; MBP, myelin basic protein; O-2A, oligodendrocyte-type II astrocyte; PLP, proteolipid protein; PN, postnatal; TH, thyroid hormone.

Received January 21, 2004.

Accepted for publication July 9, 2004.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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