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ARTICLES |
Molecular Endocrinology Laboratory, University of Massachusetts Medical School, Worcester, Massachusetts 01655
Address all correspondence and requests for reprints to: Alan P. Farwell, M.D., Division of Endocrinology and Metabolism, Department of Medicine, University of Massachusetts Medical School, 55 Lake Avenue North, Worcester, Massachusetts 01655. E-mail alan.farwell{at}bangate.ummed.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Fundamental to the morphological abnormalities in the hypothyroid brain are disordered neuronal migration and blunted axonal projection (5, 6, 7, 8, 9, 10, 11, 12). Neurons require multiple guidance cues to migrate down specific paths to their target destinations (13, 14, 15, 16). Key guidance signals are produced by the extracellular matrix protein (ECM)1, laminin (17, 18, 19, 20, 21, 22, 23). Laminin is secreted into the ECM by the astrocytes and fixed to the astrocyte surface by binding to specific transmembrane receptors known as integrins (17, 24, 25, 26). The integrins are clustered together into structures known as focal contacts by binding to the F-actin microfilaments (27, 28, 29), and this clustering results in the presentation of the laminin bound to the integrins in a very specific pattern. The migrating neurons recognize these laminin-derived guidance signals through integrins located in the migrating neurite. In this fashion, the neuron is able to follow the laminin-derived path to its target destination. Once neuronal migration is complete, laminin disappears from the brain paranchyma and is restricted to the vasculature (19, 20, 30, 31). Any factor that disrupts the formation of the laminin derived pathways would disrupt neuronal migration.
The postnatal development of the rat cerebellum is an excellent model to study the effects of thyroid hormone on brain development (5, 8, 9, 10, 11, 12, 32, 33). There is extensive proliferation of granular neurons for the first few days after birth to form the external granular layer. This is followed by migration of the granular neurons from the external granular layer to the internal granular layer, where they form connections with interneurons in the inner layer and with the Purkinje cells. By postnatal day 10, extensive migration is occurring and the purkinje cells are elaborating processes. By postnatal day 21, the external granular layer is gone and the extensive wiring network of the cerebellum is established.
In this study, we examined the effects of thyroid hormone on the expression and distribution of laminin in the developing rat cerebellum.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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EHS laminin, rabbit polyclonal antilaminin IgG, and BSA were purchased from Sigma (St. Louis, MO). Antirabbit IgG-horseradish peroxidase conjugate was purchased from Promega Corp. (Madison, WI), rabbit polyclonal anti-GFAP IgG was purchased from Biomedical Technologies (Stoughton, MA) and Hybond ECL nitrocellulose was obtained from Amersham Pharmacia Biotech (Arlington Heights, IL). The Lumiglo chemiluminescent kit, True Blue Peroxidase Substrate kit and HRP Blocking Solution was obtained from Kirkegaard & Perry Laboratories (Gaithersburg, MD). All other reagents were of the highest grade available.
Tissue harvest and hormone assays
Animals were killed every other day from birth to postnatal day
20. In all experiments, animals were weighed, killed by decapitation,
and their blood collected for hormone assays. The cerebellum was
rapidly isolated, and the vermis fixed by quick freezing the tissue in
OTC compound with liquid nitrogen. The tissue blocks were kept at -70
C until sectioning. The remaining cerebellar hemispheres were
homogenized in 10 volumes of 50 mM Tris (pH 8.0), 0.4
M NaCl, 5 mM EDTA and 1% Nonidet-40. The
homogenates were then sonicated and kept at -70 C until use.
Serum TSH was measured in duplicate by RIA using materials obtained from the National Pituitary Agency, National Institutes of Health (Bethesda, MD). Serum T4, T3 and rT3 were determined in duplicate by species-adapted specific RIAs.
Immunocytochemistry
Tissue blocks were cut in 6-µm sections from similar
orientations focusing on the midline of the vermis and three sections
were mounted per slide. Sections were thaw-mounted and then fixed at
-20 C in iced acetone for 15 min, followed by iced methanol for 5 min.
Sections were rehydrated in 150 mM NaCl, 10 nM
Na2HPO4, pH 7.4 (PBS), endogenous peroxidases
were blocked by incubation with HRP Blocking Solution and nonspecific
binding sites were blocked by incubation with BSA blocker (1 mg/ml BSA
in PBS). Laminin was identified by incubating sections with
antilaminin IgG (1:100 dilution in BSA blocker) or BSA blocker alone
(nonspecific binding control) for 1 h and immune complexes were
visualized by incubation with an antirabbit IgG conjugated with HRP
(1:2500 dilution in BSA blocker) followed by development with True Blue
Peroxidase Substrate (antilaminin sections) or an antirabbit IgG.
Sections were then dehydrated through alcohols to xylene and mounted
with Permount (Fisher, Fairlawn, NJ). Astrocytes
were identified by incubating sections with antibodies to the
astrocyte-specific marker, glial fibrillary acidic protein (GFAP)
(1:500 dilution in BSA blocker) for 1 h. Immune complexes were
visualized by immunofluorescence after incubation with an antirabbit
IgG conjugated with Texas Red (1:50 dilution in BSA blocker). A
Carl Zeiss Axioskop microscope equipped with an
Olympus Corp. OM4 camera and Kodak TMAX ASA
100 (laminin) or 400 (GFAP) film (Eastman Kodak Co.,
Rochester, NY) was used for image acquisition.
Slot-blot immunoanalysis
Cerebellar homogenates were thawed, 10 µl aliquots were
obtained for DNA analysis, and the remaining samples spun in a
microfuge for 5 min at room temperature. DNA content was determined by
measuring the binding of DNA to 33258 Hoechst dye (34, 35) with a TKO
100 Mini Fluorometer (Hoefer Scientific Instruments, San
Francisco, CA), and sample aliquots were normalized as to their DNA
content to ensure analysis of equivalent samples of cells. Cerebellar
homogenates containing 200 ng DNA were diluted in 20 mM
Tris-HCl, 137 mM NaCl, pH 7.5, and proteins were denatured
in a boiling water bath for 5 min then applied to nitrocellulose via
slotblot under vacuum. Blots were blocked for 1 h with milk
blocker (20 mM Tris-HCl, 0.1%(vol/vol) Tween-20, 5 g/ml
powdered milk, 137 mM NaCl, pH 7.5) then probed for 1
h at room temperature with anti-laminin IgG (1:1000 dilution in milk
blocker). Nonspecific signal was determined by incubating parallel
blots with antilaminin IgG preabsorbed with excess laminin and was
subtracted from the signal obtained with the antisera alone. Immune
complexes were then visualized by incubation with an antirabbit IgG
conjugated to horseradish peroxidase (1:2500 in blocker) and developed
with the Lumiglo chemiluminescent kit. Blots were analyzed by scanning
densitometry and laminin was quantified by comparison with results
obtained with a standard curve of EHS laminin (0–5 ng) that was run on
each blot.
Statistical methods
Where indicated, results are reported as mean ±
SE. Statistical analysis was performed by single-factor
ANOVA. When necessary, justification for performance of single-factor
ANOVA was determined by two-factor ANOVA with replication. Statistical
significance was determined to be achieved at the P <
0.05 level.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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are the hormone levels
achieved in the euthyroid and PTU-treated neonatal rats. Serum
T4 and TSH concentrations showed the most marked changes
between the treatment groups. In the euthyroid animals, serum
T4 concentrations steadily increased from birth to a
plateau at postnatal day 14–16, while remaining undetectable in the
PTU-treated animals throughout the treatment period. Euthyroid serum
T4 concentrations became significantly different from those
of the PTU-treated animals by postnatal day 2 and remained so for the
entire treatment period. Serum TSH concentrations remained stable in
both sets of animals, with the TSH concentrations in the PTU-treated
rats 2- to 3-fold higher than those in the euthyroid animals. Serum
T3 concentrations steadily increased after birth in the
euthyroid animals and were higher at all time points than in the
PTU-treated animals, reaching statistical significance at postnatal day
4. Serum T3 concentrations in the PTU-treated animals
remained stable throughout the treatment period. For the first few days
after birth, serum rT3 concentrations were 2- to 3-fold
greater in the PTU-treated rats than in the euthyroid animals. From
postnatal day 6 and throughout the remainder of the treatment period,
serum rT3 concentrations remained similar in both treatment
groups. These results are similar to those reported by others (4, 10, 11, 32, 36).
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). Body weight also
increased in the PTU-treated rats, but at a slower rate and reached a
significant plateau after postnatal day 12. Again, these observations
are similar to those made by others (10, 11, 32) and provide further
evidence that the PTU-treated animals were indeed hypothyroid.
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is the biochemical
analysis of the time course of laminin expression in the neonatal rat
cerebellum. At birth, laminin content in the cerebellum was similar in
both the euthyroid and PTU-treated animals (Fig. 3, Table 1). In the euthyroid animal, the
cerebellar content of laminin steadily increased after birth, becoming
significantly greater than that observed in the PTU-treated rats by
postnatal day 4 and reaching peak levels by postnatal day 8. Cerebellar
laminin content then decreased to plateau levels after postnatal day 10
and remained stable throught the rest of the treatment period. In
contrast, there was little change in cerebellar laminin content from
birth through postnatal day 12 in the PTU-treated animal (Fig. 3).
Cerebellar laminin content then increased to levels equal to the
euthyroid rats by postnatal day 14 and remained stable throughout the
rest of the treatment period. The maximal laminin content in the
cerebellum of the PTU-treated rats was approximately 35% less than
that found in the euthyroid cerebellum (Table 1). Importantly, the
pattern of laminin expression in the cerebellum of the PTU-treated rat
did not show a peak, as was observed in the euthyroid rats; rather,
levels steadily increased from low levels to plateau levels. These data
represent changes in the total laminin content of the cerebellum, which
includes laminin deposited in the vasculature as well as that deposited
in the cerebellum paranchyma. The regional expression of laminin in the
neonatal rat cerebellum was examined next by immunocytochemistry.
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are representative
sections of the euthyroid rat cerebellum stained for laminin. Control
sections incubated with either nonimmune serum or laminin antisera
preincubated with excess laminin showed no significant staining at any
time point (data not shown). At birth, the specific cerebellar layers
have not yet formed (11, 32, 33). Because laminin is a major component
of the basement membrane of the blood vessels (37), the only structure
stained for laminin at this time point is the vasculature. By postnatal
day 5, definite layers of cerebellar cells have formed. Laminin
staining is readily apparent in the molecular layer and is distinct
from the vasculature. By postnatal day 10, there is intense laminin
staining diffusely throughout the molecular layer in punctate clusters
and clumps (Fig. 4). This punctate pattern of laminin has been
previously described in the neonatal cerebellum (18, 19, 20, 30, 38). By
postnatal day 15, laminin staining in the euthyroid cerebellum has
disappeared from the molecular layer and is again restricted to the
vasculature (Fig. 4). In addition, there are few. If any, cells
remaining in the EGL, indicating that granular neuron migration has
been completed (8, 9, 10, 11, 32, 39).
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. Similar to
what was observed in the euthyroid animal, laminin is restricted to the
vasculature at birth in the PTU-treated cerebellum. However, in
contrast to the euthyroid cerebellum, no significant staining for
laminin is observed in the molecular layer until postnatal day 10 in
the PTU-treated cerebellum, and the most extensive laminin staining
observed in these animals is found at postnatal day 15 (Fig. 5).
Importantly, this pattern of laminin staining in the molecular layer of
the PTU-treated cerebellum at postnatal day 15 is clearly less diffuse,
with smaller punctate collections of stain, than the pattern of laminin
staining in the molecular layer of the euthyroid animal observed at
postnatal day 10 (Fig. 4
). By postnatal day 18, laminin staining has
disappeared from the molecular layer in the PTU-treated cerebellum. The
EGL still contains several rows of cells on postnatal day 18,
consistent with the observation by others that granular neuron
migration continues up until postnatal day 21 in the hypothyroid rat
(8, 9, 10, 11, 32, 39).
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are representative sections from the
cerebellum obtained from euthyroid and PTU-treated rats on postnatal
day 10. The top sections are stained for laminin, again showing
extensive staining in the molecular layer in the euthyroid cerebellum
and minimal staining in the molecular layer of the PTU-treated
cerebellum. The bottom sections show extensive GFAP staining of cells
in the molecular layer of both the euthyroid and PTU-treated animals,
consistent with the presence of the radial Bergman glial fibers (14, 40). If anything, GFAP staining in the PTU-treated cerebellum is more
extensive, extending into the EGL. Similarly, there is extensive GFAP
staining in the cerebellum of both animals at postnatal days 14–15
(Fig. 7), indicating the presence of
abundant astrocytes. By postnatal days 14–15, laminin has disappeared
from the molecular layer of the euthyroid cerebellum while still being
expressed in the molecular layer of the PTU-treated cerebellum (Fig. 7). These data show that the differential appearance and disappearance
of immunoreactive laminin in the molecular layer of the cerebellum
between the euthyroid and PTU-treated developing rats is not due to
differential astrocyte formation in these animals.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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In this paper, we show that the punctate forms of laminin are
transiently expressed in the molecular layer of the rat cerebellum at
times coinciding with migration of the granular neurons from the
external granular layer to the inner granular layer. In the euthyroid
rat, small and large punctates of laminin first appear around postnatal
day 3–5 when the molecular layer is formed (Fig. 4). Maximum
expression of laminin in the cerebellum is found between postnatal days
8–10, and the punctate laminin disappears from the molecular layer of
the cerebellum by postnatal day 15 (Fig. 4). This pattern is markedly
different from that observed in the hypothyroid rat, where the punctate
laminin does not begin to appear in the molecular layer until postnatal
day 10, peaks on postnatal days 14–15, and disappears by postnatal day
18 (Fig. 5). The only previous study that examined laminin expression
in the developing cerebellum identified punctate laminin in the
molecular layer of the cerebellum at postnatal day 10, which then was
not present on postnatal day 24 (20). This the first study to document,
in detail, the developmental expression of laminin in the rat
cerebellum.
The delayed pattern of laminin expression in the hypothyroid brain is
likely to represent a pathological process rather than one due to the
general delay in brain development that occurs in the cretinous brain.
Essentially, all of the granular neurons have left the external
granular layer by the time punctate laminin disappears in the euthyroid
rat (Fig. 4), indicating that granular cell migration is complete. In
the hypothyroid rat cerebellum, many granular neurons remain in the
external granular layer when the punctate laminin disappears,
suggesting that granular neuron migration through the molecular layer
is still ongoing when punctate laminin disappears in the hypothyroid
cerebellum (Fig. 5). Further, maximal expression of laminin in the
euthyroid cerebellum is significantly greater than that observed in the
hypothyroid cerebellum (Table 1). Thus, the expression of laminin is
both delayed and diminished in the hypothyroid cerebellum. These data
suggest that the regulation of laminin expression in the cerebellum by
thyroid hormone may play a major role in the profound derangements of
neuronal migration observed in the cretinous brain.
The mechanism whereby thyroid hormone regulates the expression and
distribution of laminin in the developing rat cerebellum remains to be
established. Many actions of thyroid hormone are mediated by specific
nuclear receptor proteins that act as ligand-activated
transcription factors (50, 51, 52). Nuclear receptors for thyroid hormone
are developmentally expressed in the brain (53, 54, 55), and
T3-regulated genes have been identified in the cerebellum
(for review see Refs. 2, 56). Interestingly, many of these
T3-regulated genes eventually achieve euthyroid expression
levels even in the absence of T3 (57) or in the absence of
specific thyroid hormone receptor isoforms in transgenic mice (58, 59).
In contrast to the steady increase in expression observed with these
previously identified T3-regulated genes, peak laminin
expression in the euthyroid rat occurs early in cerebellar development,
followed by a fall to a lower plateau level in the euthyroid rat.
Laminin content in the PTU-treated cerebellum, which eventually
achieves the lower plateau levels observed in the euthyroid rat, never
reaches the peak levels observed in the euthyroid animal (Table 1).
Finally, few of the genes that have been identified to be altered in
the hypothyroid cerebellum have shown to also play a major role in
neuronal migration. If laminin gene expression in the cerebellum is
discovered to be under direct regulation by thyroid hormone, it would
provide the first evidence of thyroid hormone-dependent transcriptional
regulation of a protein directly involved with neuronal migration.
Laminin is synthesized and secreted by astrocytes (24, 25, 26, 42, 60);
thus, any action of thyroid hormone on laminin gene expression would
have to be targeted to the astrocyte. We (61) and others (62) have
shown that astrocytes lack significant numbers of functional nuclear
thyroid hormone receptors (TRs). The predominant (>95%) thyroid
hormone receptor in astrocytes is the nonT3-binding isoform
c-erbA
2 (61). Small quantities of TR1 (61) and TRß2
(63) have been identified in cultured astrocytes, although the dominant
negative activity of the c-erbA2 (64, 65, 66) is likely to
render these TR isoforms transcriptionally inert. These studies suggest
that actions of thyroid hormone in astrocytes are not mediated by
thyroid hormone receptors (62).
How might thyroid hormone influence laminin expression in astrocytes, if not by nuclear receptor-mediated transcriptional regulation? We have shown that thyroid hormone, primarily T4, regulates microfilament organization (67, 68, 69) and vesicle recycling (70, 71) in cultured astrocytes via an extranuclear mechanism (26). T4-dependent alterations in the microfilament organization in astrocytes also results in altered integrin-laminin interactions (72) and altered organization of laminin bound to the astrocyte surface (26). If similar events occur in vivo, abnormal deposition of laminin into the extracellular matrix in the absence of thyroid hormone would likely activate extracellular matrix-degrading proteases that function to maintain the integrity of the extracellular matrix (73) and alter laminin protein expression without alterations in gene expression. Studies are ongoing in our lab to examine this potential paradox.
In summary, we have shown that the appearance of laminin in the molecular layer of the hypothyroid cerebellum is markedly delayed and less abundant than in the euthyroid cerebellum. These data indicate that the disruption of the timing of appearance and regional distribution of laminin in the absence of thyroid hormone may play a major role in the profound derangements of neuronal migration observed in the cretinous brain.
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Acknowledgments |
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Footnotes |
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Received February 5, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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2 in selected tissues:
cerebellar Purkinje cells as a model for ß 1 receptor-mediated
developmental effects of thyroid hormone in brain. Proc Natl Acad Sci
USA 88:3887–3891 2) is due to
competition for binding sites on target genes. J Biol Chem 268:20904–209102 does not require binding to a thyroid
hormone response element. Mol Endocrinol 9:86–95This article has been cited by other articles:
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