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Endocrinology Vol. 145, No. 3 1386-1391
Copyright © 2004 by The Endocrine Society

Aberrant Maturation of Astrocytes in Thyroid Hormone Receptor {alpha}1 Knockout Mice Reveals an Interplay between Thyroid Hormone Receptor Isoforms

Beatriz Morte, Jimena Manzano, Thomas S. Scanlan, Björn Vennström and Juan Bernal

Instituto de Investigaciones Biomédicas Alberto Sols (B.M., J.M., J.B.), Consejo Superior de Investigaciones Científicas y Universidad Autónoma de Madrid, 28029 Madrid, Spain;. Departments of Pharmaceutical Chemistry and Cellular and Molecular Pharmacology (T.S.S.), University of California, San Francisco, California 94143-2280; and Laboratory of Developmental Biology (B.V.), CMB, Karolinska Institute, Stockholm S-17177, Sweden

Address all correspondence and requests for reprints to: Dr. Juan Bernal, Instituto de Investigaciones Biomédicas, Arturo Duperier 4, 28029 Madrid, Spain. E-mail: jbernal{at}iib.uam.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the effects of thyroid hormones on the development of neurons and oligodendrocytes are well documented, less is known about the hormonal effects on astrocytes. Our analyses of cerebellar slices from 2-month-old T3 receptor protein (TR){alpha}1-deficient mice show that mature astrocytes, Golgi epithelial cells, and their Bergmann processes had strongly reduced glial fibrillary acidic protein (GFAP) and nestin immunoreactivity, in contrast to wild-type mice. Furthermore, the Bergmann processes exhibited an irregular GFAP staining. A similar expression of nestin and GFAP was observed in 11-d-old (P11) mutant pups. Surprisingly, however, hypothyroidism normalized the appearance of these markers in the P11 mutants, suggesting that liganded TRß is detrimental to astroglial cell differentiation in the absence of TR{alpha}1. To test this hypothesis, hypothyroid mice were treated from birth until P11 with the TRß-selective ligand GC-1. This treatment was devastating in the TR{alpha}1-/- mice, causing little if any nestin or GFAP immunoreactivity, whereas the wild-type mice were normal. The results thus indicate an important interplay between thyroid hormone receptor isoforms in astroglial cell maturation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THYROID HORMONE EXERTS profound influences on brain development and function, and many of its effects are due to the control of cellular migration and differentiation (1, 2). The sites of action of thyroid hormone in tissues may include the plasma membrane (3) and the mitochondria (4), although the general consensus is that most actions of thyroid hormone, including those on neurodevelopment, are mediated by nuclear hormone receptors (5). There are several T3 receptor proteins (TRs), encoded by two distinct genes, TR{alpha} and TRß. The TR{alpha} gene encodes several proteins including TR{alpha}1, TR{alpha}2, and the truncated proteins {Delta}TR{alpha}1 and {Delta}TR{alpha}2, of which only TR{alpha}1 binds T3 and activates or represses target genes. The TRß gene produces several amino terminal protein variants, TRß1, TRß2, TRß3, and {Delta}TRß3, that all bind T3.

All receptor isoforms are expressed in brain, but given the high proportion of the TR{alpha}1 protein, which accounts for about 70–80% of total receptor content (6), it is likely that most actions of thyroid hormone on brain are mediated through this isoform. It was therefore very surprising to find that TR{alpha}1-/- mice do not display obvious signs of brain misdevelopment (7). Specifically, the cerebellum, a sensitive target of thyroid hormone during development, shows no histological signs of abnormal development as far as migration of granular cells and differentiation of Purkinje cells are concerned. Alterations of these processes are hallmarks of early hypothyroidism in developing animals. We also found that hypothyroidism in the absence of TR{alpha}1 did not result in the profound alterations of granule cell migration and Purkinje cell differentiation observed in hypothyroid wild-type animals. These data suggested that the repressing activity of unliganded TR{alpha}1 is responsible for the developmental insults of hypothyroidism, a conclusion also supported by results from Flamant et al. (8).

Glial cells are also targets of thyroid hormone, but the specific role of T3 receptors has been clarified only in some instances. For example, in addition to the well known effects of thyroid hormone on oligodendrocyte differentiation, gene expression and myelination, recent studies demonstrate that TR{alpha}1 is important in the timing of oligodendrocyte precursor cell maturation by controlling their exit from the cell cycle (9).

Another type of glial cell, the astrocyte, has very important functions in the central nervous system (for a review, see Ref. 10). In addition to their structural role, they influence cell migration; are an important source of matrix proteins, adhesion molecules, and growth factors; play a role in neurotransmission; regulate the ionic composition of the extracellular milieu; contribute to stability and function of the blood-brain barrier; are metabolically coupled to neurons; and are implicated in a variety of central nervous system disorders, including neurodegenerative diseases.

Early hypo- or hyperthyroidism affects the astroglial cell population of the cerebellum (11). More recent work has shown that, in vivo, thyroid hormone influences the expression of astroglial genes (12, 13, 14) and radial glia maturation (15). Some of the effects of thyroid hormone on neuronal proliferation and differentiation could be mediated by a primary action on astrocytes. In cultured astrocytes thyroid hormone influences actin polymerization and integrin–laminin interactions (16, 17, 18), but the role of T3 receptors in these processes is unclear.

We have therefore examined the astroglial population in developing mice harboring a selective deletion of TR{alpha}1 (19). Surprisingly, TR{alpha}1 deficiency yields a severely altered pattern of astrocyte maturation in the cerebellum, which is normalized after induction of hypothyroidism. This observation, combined with results of treatment with the TRß-specific ligand GC-1, indicates that the interplay between TR{alpha} and TRß is essential for normal astroglial cell maturation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal procedures
Animal care procedures were conducted in accordance with the guidelines set by the European Community Council Directives (86/609/EEC). Animals were under temperature- (22 ± 2 C) and light (12 h light:12 h-dark cycle; lights on at 0700 h)-controlled conditions and had free access to food and water. We used adult and developing TR{alpha}1-deficient mice (TR{alpha}1-/-) (19) and wild-type mice with the BALB/c genetic background. Chemical thyroidectomy from birth was induced by administration of 0.02% 2-mercapto-1-methylimidazole (Sigma Chemical Co., St. Louis, MO) and 1% sodium perchlorate in the drinking water throughout the experiment. Before weaning, the solution was administered to the lactating dams from the time of delivery. Hormonal treatments were started on P0 and consisted of daily single ip injections of 10 ng/g of the TRß-selective agonist GC-1(7). At least four male mice, from different litters and for each condition, were examined. The animals were killed as indicated for each experiment.

Immunohistochemistry
Animals were perfused transcardially with buffered 4% paraformaldehyde, and the brains were carefully dissected, cryoprotected in 30% sucrose at 4 C, and frozen in dry ice. Immunohistochemistry was performed on free-floating, 25-µm cryostat sections performed through the vermis. Sections from two mice for each condition were simultaneously analyzed, and the experiment was performed twice. The sections were incubated overnight with a monoclonal antiglial fibrillary acidic protein (GFAP) antibody (final dilution 1:2000) (Dako A/S, Glostrup, Denmark) or a rabbit polyclonal antinestin antibody (final dilution 1:7500) (20) at 4 C. They were then incubated with biotinylated horse antimouse or goat antirabbit secondary antibodies at a 1:200 dilution (Vector Laboratories, Burlingame, CA) followed by ABC (Vector). Peroxidase was then visualized with diaminobenzidine (0.05%) and H2O2. For immunofluorescence, the slices were incubated with Alexa Fluor 594 goat antirabbit and streptavidin Alexa Fluor 488 (Molecular Probes, Eugene, OR). For microscopy we used a Nikon Elite optical microscope equipped with a Nikon dn100 digital camera and a TCS SP2 confocal microscope (Leica Microsystems, Heidelberg, Germany). A qualitative assessment of the immunohistochemical patterns was performed by recording the relative intensity and homogeneity of the stain distribution throughout the sections. For comparisons of different groups, we used lobule VII in which the alterations observed were intermediate between lobules V-VI (strongest) and lobule X (mildest).

Western blotting
Cerebella from four P11 mice were pooled and homogenized in 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, 10% glycerol, 1 mM EDTA, 50 mM Tris-HCl (pH 8.0) containing 2 µg/ml leupeptin, 2 µg/ml aprotinin, and 100 µg/ml phenyl-methyl-sulfonyl fluoride. Aliquots of 50 µg of the extracts were electrophoresed in 12% polyacrylamide gels and transferred to nitrocellulose blotting membranes (Schleicher & Schuell, Keene, NH). The membranes were washed in 0.5 M NaCl and 20 mM Tris-HCl (pH 7.5) containing 0.1% Tween 20 and then in 5% skimmed milk. Finally the membranes were incubated with anti-GFAP (1:10,000), antinestin (1:10,000), and anti ß-actin (1:500) antibodies and developed with LUMINOL (Santa Cruz Biotechnology, Santa Cruz, CA). Thyroid hormone is known to affect actin polymerization but not total actin content in the cerebellum (21). For quantification, the films were scanned and densitometry was measured using the Scion Image program version 4.02 (http://www.scioncorp.com).

Primary cell cultures
Astrocyte cultures were established from P1 cerebella after mechanically disaggregating the tissue in Hanks’ balanced salt solution (Life Technologies, Inc., Grand Island, NY). The cells were washed in DMEM containing 20% fetal calf serum (FCS). Then 2.7 million cells were plated on polyornitin-treated, 100-cm diameter plates and incubated until confluence, usually 9 d, in DMEM containing 20% FCS. The cells were then washed and incubated for 24 h in DMEM containing 10% FCS. After trypsinization, 100,000 cells were seeded in each of six P60 plates in the same medium for 3 d. Immunohistochemistry revealed the absence of neurons in the culture, as shown by immunohistochemistry for ß3-tubulin. Cells were positive for nestin or nestin and GFAP. 5-Bromo-2'-deoxyuridine (BrdU) was added at a final concentration of 10 µm for 20 h. The cells were then washed in PBS and fixed before BrdU immunohistochemistry was performed. For quantifications four fields from each plate, containing an average of 135 cells (range 60–250) from the wild-type cultures and 119 cells (range 87–184) from the TR{alpha}1-/- cultures were analyzed.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Two main classes of astroglial cells are present in the cerebellum, astrocytes and Golgi epithelial cells (GECs). Astrocytes are present in the white matter and also throughout the internal granular layer (IGL) in which they serve a trophic function for the granular cells. GECs are located in the Purkinje cell layer and send radial processes (Bergmann processes) through the molecular layer to the surface of the cerebellum. These two classes of cells are readily characterized by immunostaining for the intermediate filament GFAP. Figure 1Go shows an immunohistochemistry experiment comparing the GFAP staining pattern between a wild-type mouse and a TR{alpha}1-/- mouse at 6 months of age. Strikingly, and despite lack of obvious alterations in cerebellar layering previously reported (7), the TR{alpha}1-deficient mice had a strongly reduced expression of GFAP, making it difficult to delineate single astrocytes. GECs and their processes also stained less and had a strongly altered distribution with a nonuniform, patchy appearance.



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  		FIG. 1. GFAP staining of cerebellar slices of P60, wild-type (WT), and TR{alpha}1-/- mice. ml, Molecular layer; igl, internal granular layer; pcl, Purkinje cell layer. The upper panel shows the entire lobule, at low magnification, and the lower panel shows a higher magnification of the ml and igl. The arrowheads indicate the position in which the GECs are located. Note the lack of staining of astrocytes in the TR{alpha}1-/- sample and the irregular staining of Bergmann processes in the ml. Bars, upper panel, 200 µm, lower panel, 50 µm.

 
To elucidate whether these alterations were the result of an altered astrocyte terminal differentiation, we analyzed the patterns of intermediate filament expression during the postnatal development. During this time, expression of the intermediate filaments, nestin, vimentin, and GFAP are sequentially expressed as astroglial maturation proceeds (22, 23, 24), causing fully mature astrocytes to express mainly GFAP. At P11 the Bergmann processes were positive for nestin, and some cells in the IGL were still positive for this marker in wild-type mice (Fig. 2AGo, WT). GFAP staining revealed numerous mature astrocytes well distributed throughout all cerebellar lobules. Astrocytes were present primarily in the IGL and the white matter, whereas Bergmann processes were observed in the molecular layer.



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  		FIG. 2. Expression of nestin and GFAP in the cerebellum in developing wild-type and TR{alpha}1-/- mice. A, Patterns of nestin and GFAP staining in P11 cerebellar slices in intact wild-type (WT) and TR{alpha}1-/- mice. For both nestin and GFAP, the entire lobules are shown at low magnification. In addition, for nestin staining higher magnification of the molecular layer (ml) and internal granular layer (igl) is also shown. B, Result of Western blots of cerebellar extracts using antinestin and anti-GFAP antibodies. For control of protein loading, anti-ßactin was also used. The numbers under the protein bands show their densitometric values. D, The pattern of nestin staining in P2 mice is shown. C, Nestin and GFAP staining in hypothyroid wild-type (WTH) and TR{alpha}1-/- (TR{alpha}1-/-H) mice. (Scale bars, 200 µm.)

 
Similar analysis in the TR{alpha}1-/- mice revealed an abnormal development of astrocytes. Nestin-positive cells were almost absent in the IGL, and there was much less staining of the Bergmann processes than in wild-type mice (Fig 2AGo, TR{alpha}1-/-). GFAP staining was also decreased. In addition, both nestin and GFAP staining were heterogeneous throughout the slices and displayed a patchy distribution. Mature astrocytes were observed primarily in the white matter. Furthermore, the amounts of nestin and GFAP proteins were strongly decreased at P11 in the TR{alpha}1-/- mice as measured by Western blotting (Fig. 2BGo). At earlier stages, i.e. P2, there was no difference in the patterns of nestin-expressing cells between TR{alpha}1-/- and wild-type mice (Fig. 2DGo).

That TR{alpha}1-/- mice showed an aberrant astroglial development was unexpected because migration of granular cells and differentiation of Purkinje cells are normal (7). Furthermore, hypothyroidism does not affect migration and differentiation in these mice, whereas these parameters are strongly perturbed in hypothyroid wild-type mice. We therefore studied the effect of hypothyroidism on astrocyte development. As described previously (7), the Purkinje cells of wild-type mice, but not of TR{alpha}1-/- mice, were affected by hypothyroidism (not shown). Concerning astrocyte maturation, Fig. 2CGo illustrates that hypothyroid wild-type mice showed the typical signs of delayed astrocyte development such as increased nestin staining and abnormal accumulation of GFAP+ astrocytes in the white matter. Surprisingly, hypothyroid TR{alpha}1-/- mice exhibited a normal pattern of nestin and GFAP staining and an overall normal morphology, similar to that seen in euthyroid wild-type mice.

One possible interpretation for the effects described above is that normal astrocyte maturation is dependent on a balance between opposing influences of TR{alpha}1 and TRß1. According to this hypothesis, removal of TR{alpha}1 in a euthyroid setting would allow liganded TRß1 to perturb astrocyte differentiation, whereas hypothyroidism would deprive TRß1 of ligand and therefore also neutralize its negative effects. To test this, hypothyroid wild-type and TR{alpha}1-/- mice were treated with the TRß-selective ligand GC-1. Although T3 treatment would have sufficed in the TR{alpha}1-deficient mice, selective activation of TRß in the wild-type mice necessitated the use of the selective ligand. Results of nestin and GFAP expression in intact and hypothyroid wild-type and TR{alpha}1-/- mice, with or without GC-1 treatment, are shown in Fig. 3Go. Again, the TR{alpha}1-/- mice showed little or no staining for nestin, whereas staining for GFAP in the IGL was of low intensity and was present mainly in the white matter. In contrast, the wild-type mice contained some nestin-positive astrocytes dispersed in the IGL, and the Bergmann processes were labeled. At this age, however, most astrocytes expressed GFAP. Remarkably, hypothyroidism in the TR{alpha}1-/- mice reverted the altered nestin and GFAP expression and produced a pattern that was indistinguishable from normal. In the wild-type mice, as expected, hypothyroidism induced changes, suggesting a delayed maturation of astrocytes, with more nestin-positive cells than in the euthyroid mice. Treatment of hypothyroid wild-type mice with GC-1 had no noticeable effect, whereas the same treatment of hypothyroid TR{alpha}1-/- mice abrogated the normalization caused by hypothyroidism and resulted in a reversal to decreased nestin and GFAP staining. These results thus suggest that liganded TRß1 actually inhibited astrocyte maturation in the absence of TR{alpha}1.



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  		FIG. 3. Immunostaining patterns for nestin and GFAP in the cerebellum of P11 mice. The groups analyzed were as follows: wild-type mice (WT); TR{alpha}1-/- mice (TR{alpha}1-/-); hypothyroid wild-type mice (WTH); hypothyroid TR{alpha}1-/- mice (TR{alpha}1-/-H); hypothyroid wild-type mice treated with GC-1 (WTH+GC-1); and hypothyroid TR{alpha}1-/- mice treated with GC-1 (TR{alpha}1-/-H+GC-1). For both nestin and GFAP, the entire lobules are shown at low magnification. For nestin stain, higher magnification of the molecular layer and the IGL is also shown. (Scale bars, 200 µm.)

 
Other strains of TR-mutant mice had no alterations of GFAP expression (not shown). These included TRß knockout mice (25), and knock-in mice expressing a dominant-negative TR{alpha}1 mutation (26).

To test whether astrocytes from TR{alpha}1-/- mice had an intrinsic defect in proliferation or differentiation, we determined the number of proliferating cells and differentiated astrocytes in confluent cultures of P1 cerebella. Neurons were not present in the culture, as shown by ß3-tubulin staining. Total number of cells was counted by nuclear staining with 4',6'-diamino-2-phenylindole, proliferating cells after incubation with BrdU, and differentiated cells after immunostaining with GFAP. Cultures from TR{alpha}1-/- mice contained a lower proportion of BrdU-positive cells, compared with cultures from wild-type mice (51 ± 0.05% vs. 60 ± 0.06%, six plates per culture, P < 0.05). The proportion of mature, GFAP-positive cells did not, however, differ significantly (23% for TR{alpha}1-/- and 25% for wild type).


   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Recently we described that neither euthyroid nor hypothyroid TR{alpha}1-/- mutant mice present obvious alterations of cerebellar structure, despite the well-known role of thyroid hormone in development and gene expression in this tissue (7). The two main cellular targets of thyroid hormone, the granular and the Purkinje cells, developed normally. Migration of granular cells from the external germinal layer to the internal granular layer was not delayed, and maturation of the rich dendritic arborization of the Purkinje cells was normal. Furthermore, hypothyroidism did not interfere with these processes in the mutant mice, in contrast to the dramatic effects seen in the wild-type mice. These findings led us to propose that cerebellar insults caused by hypothyroidism are a consequence of the repressing, or more generally, altered transcriptional activity of unliganded TR{alpha}1. These analyses of cerebellar layering focused on neuronal cells. In this work we extended our analysis to the glial cell population of TR{alpha}1-/- mice and, to our own surprise, found dramatic alterations in the patterns of cerebellar glial cell distribution during both postnatal development and adulthood.

The two types of astroglial cells, the GECs and the astrocytes, can be readily identified by studying intermediate filaments. Mature astroglial cells express GFAP, although GECs retain expression of intermediate filaments typical of immature cells such as nestin and vimentin. At the early stages of postnatal development, the nestin staining pattern was similar in wild-type and TR{alpha}1-/- mice but at P11 GEC did not express nestin in TR{alpha}1-/- mice, and GFAP-positive cells were almost absent from the granular layer, being present mainly in the white matter. The nestin gene contains a T3-responsive element in a critical region of the second intron required for proper nestin expression (27). This region also binds retinoic acid receptor; retinoid X receptor, and chicken ovalbumin upstream transcription factor. In the absence of TR{alpha}1, complex interactions among these receptors, in particular the repressor activity of chicken ovalbumin upstream transcription factor, might play a role in the altered nestin expression.

The altered expression of intermediate filaments in astroglial cells may have important physiological consequences. GFAP-null mice have been reported to undergo normal development and to show no gross alterations in behavior or brain morphology (28, 29, 30). Others have reported that GFAP is necessary for integrity of the blood-brain barrier and for long-term maintenance of myelination (31). Mice deficient in GFAP, like TR{alpha}1-/- animals, have normal migration of granule cells despite profound alterations of Bergmann fibers (32). Because astrocytes modulate synaptic function, GFAP-/- mice have a disturbance of long-term potentiation in the hippocampus (30). In the cerebellum, disturbance of GECs and Bergmann fibers may alter Purkinje cell function because they constitute the insulating elements of Purkinje cell processes in the molecular layer. Accordingly, mice deficient in GFAP display a defect in long-term depression in the cerebellar cortex (33). Of considerable interest is the recent observation that mice deficient in GFAP and vimentin have a reduced astrocytic scar in the spinal cord after injury and display better functional recovery than wild-type mice (34). Whether absence of TR{alpha}1, with its concomitant deficiency in GAFP, facilitates recovery from spinal cord injuries deserves further investigation.

Thyroid hormones influence differentiation of astrocytes in vivo and in vitro, increasing GFAP expression (35) and the maturation of GECs in the cerebellum (36). They also regulate expression of astrocyte target genes during development (12, 14). These effects might be mediated by astrocyte receptors, although the presence of T3 receptors in astrocytes has been somewhat controversial, mainly due to the technical difficulties of detecting the receptor in whole-brain preparations. Early reports failed to detect any significant T3 binding in neuron-free astrocyte cultures (37). However, other studies showed the presence of T3 receptors in the C6 astrocytoma cell line (38) and in purified glial nuclei from postnatal rat brain (39). Leonard et al. (40) described that cultured astrocytes from fetal brain express TR{alpha}1 and TR{alpha}2, whereas Lebel et al. (41) found that astrocytes isolated from postnatal brain express TR{alpha}1 and TRß1. In these later studies, T3 addition to the cultures further increased TRß1 expression. Carlson et al. (42) showed that, whereas cultured type 2 astrocytes express TR{alpha}1 and TRß1, type 1 astrocytes express only TR{alpha}1. However, on the basis of RT-PCR analysis, these authors suggested that most T3 binding in astrocytes is due to the TRß2 isoform.

Therefore, because most studies agree on the presence of TR{alpha}1 in astrocytes, we believe that the effects described in this work arise from a direct consequence of the lack of TR{alpha}1-/- in the affected cells. On the other hand, there are more discrepancies regarding the presence of TRß receptor subtypes in astrocytes. These discrepancies may be due to the experimental conditions such as the age of the animals used for cell preparations or the particular culture conditions, which may alter TRß expression. For these reasons we cannot discard that the effects of GC-1 are indirectly exerted through cells other than astrocytes. This possibility is not, however, against the major conclusion that signals from both TR{alpha}1 and TRß are needed for proper astrocyte maturation. It should also be pointed out that TR{alpha}1-/- mice have slightly elevated concentrations of T3 in brain (7, 43), thus contributing to the unbalanced stimulation of TRß in the absence of TR{alpha}1. On the other hand, it is at present not possible to speculate on the role that other products of the TR{alpha} gene, i.e. TR{alpha}2 and the truncated products {Delta}TR{alpha}1 and {Delta}TR{alpha}2 may play in the astroglial phenotype described here.

In vitro, astrocytes from TR{alpha}1-/- mice had a significantly lower rate of proliferation, but the percentage of cells expressing GFAP was not different from astrocytes derived from wild-type animals. In vivo, hypothyroidism normalized the altered staining pattern in TR{alpha}1-/- mice. This was completely unexpected, and two conclusions may be drawn from these observations. First, liganded TRß1 exerts negative influences on astroglial cell differentiation. This view was supported by the results of GC-1 treatment. This T3 analog binds to TRß isoforms with a 10-fold higher affinity than for TR{alpha}1 (44). GC-1 treatment in vivo reaches the brain and induces T3-sensitive genes (7, 45). This ligand reverted the effects of hypothyroidism on astrocytes in TR{alpha}1-/- but not in wild-type mice, causing a disappearance of nestin staining and a much reduced number of GFAP+ cells in the mutant mice. These results suggest that proper astrocyte differentiation requires a balance of TRß1 and TR{alpha}1 activity because the negative effects of liganded TRß1 are observed only in mice devoid of TR{alpha}1. The second conclusion is that the delayed maturational pattern observed in hypothyroid wild-type mice is a consequence of repression by unliganded TR{alpha}1. This is supported by the fact that hypothyroidism does not have the same effect on wild-type as on TR{alpha}1-/- mice; in addition, GC-1 had no effect on hypothyroid wild-type mice. The data therefore corroborate our previous findings that other histological alterations observed in developing hypothyroid cerebella are a consequence of repression, or altered regulation of transcription, by unliganded TR{alpha}1.


   Acknowledgments
 
We thank F. Nuñez, M. Marsá, A. Bordalla, and P. Señor for the care of animals and E. Moreno for technical help.


   Footnotes
 
This work was supported by Grant BFI2002-00489 from the Ministry of Science and Technology, FIS, Instituto de Salud Carlos III, Red de Centros RCMN (C03/08) (to J.B.), the National Institutes of Health (DK 52798) (to T.S.S.), and The Swedish Cancer Society and Karobio AB (to B.V.). B.M. is a postdoctoral fellow from the Community of Madrid, and J.M. is a predoctoral fellow from the Ministry of Science and Technology.

B.M. and J.M. contributed equally to this work.

Abbreviations: BrdU, 5-Bromo-2'-deoxyuridine; FCS, fetal calf serum; GEC, Golgi epithelial cell; GFAP, glial fibrillary acidic protein; IGL, internal granular layer; TR, T3 receptor protein..

Received August 27, 2003.

Accepted for publication November 14, 2003.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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