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Gene Expression in the Perinatal Rat Cerebellum: Ontogeny and Thyroid Hormone Regulation1
Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, Boston, Massachusetts 02115
Address all correspondence and requests for reprints to: Dr. Noriyuki Koibuchi, Division of Genetics, Department of Medicine, Brigham and Women’s Hospital and Harvard Medical School, 75 Francis Street, Thorn 1004, Boston, Massachusetts 02115. E-mail: koibuchi{at}rascal.med.harvard.edu
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Abstract |
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gene is disrupted, exhibits cerebellar abnormalities similar to
those seen in the hypothyroid animals, despite normal thyroid function.
We, therefore, speculated that TH (tetraiodo-L-thyronine;
T4) may regulate ROR gene expression, which then may
regulate genes essential for normal brain development. To test this
hypothesis, we studied the changes in ROR gene expression in
perinatal hypothyroid rat cerebellum and the effect of TH replacement
using Northern blot analysis, ribonuclease protection assay and
in situ hybridization histochemistry. During cerebellar
development, an approximately 3-fold increase in the cerebellar content
of ROR messenger RNA (mRNA) was seen in both
propylthiouracil-treated, and propylthiouracil-treated and
T4-replaced animals. However, the increase was accelerated
when T4 was injected, although the ROR mRNA content was
identical, with or without T4, by 30 days after birth
(P30). In contrast, T4 treatment suppressed the TH receptor
1 and c-erbA2 mRNA content by P30; retinoic acid X
receptor-ß mRNA content was not influenced by thyroid status. A
significant hybridization signal for ROR mRNA was seen only over
Purkinje cells in the cerebellar cortex by in situ
hybridization histochemistry. These results indicate that TH alters the
timing of expression of the ROR gene in the Purkinje cells of the
cerebellar cortex, which may, in turn, influence Purkinje cell
differentiation. |
Introduction |
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Studies in the rodent show that TH plays an important role in neurogenesis in the cerebellum. As neuronal development of the rodent cerebellum is largely postnatal (3), perinatal hypothyroidism dramatically affects the morphogenesis of neurons (4, 5, 6, 7). In particular, dendritic arborization of the Purkinje cell and synaptic formation between Purkinje cells and granule cells are dramatically affected (4, 5, 6, 7). Based on these facts and the finding that TRs are expressed in Purkinje cells during development (8, 9), the Purkinje cell is considered to be a critical target of TH. Unfortunately, the molecular mechanisms mediating abnormal Purkinje cell neurogenesis seen in the hypothyroid animal are not clear. To date, the only gene that is specifically expressed in the Purkinje cell and is directly regulated by TR is the Purkinje cell protein-2 gene (pcp-2) (10), whose function is not yet known.
An animal model that exhibits morphological and neurological
abnormalities of the cerebellum similar to those seen in the
hypothyroid animals is the homozygous staggerer
(sg) mouse (11). In sg mice, Purkinje cells have
atrophic dendrites, and granule cell axons exhibit disturbed synaptic
connections (12, 13). Similar abnormalities are seen in the hypothyroid
animal. The abnormal neurogenesis seen in the sg mouse has
been considered to be the result of abnormal Purkinje cells that fail
to form synaptic connections with the axons of the granule cells, thus
leading to granule cell death (12, 14). Recently, it has been shown
that disruption of the orphan nuclear hormone receptor ROR gene
results in significant cerebellar abnormalities in sg mice
(15). ROR is a novel member of the steroid hormone nuclear receptor
superfamily and is related to the retinoic acid receptors. Three
isoforms (ROR1, ROR2, and ROR3) that share common DNA- and
putative ligand-binding domains, but possess distinct amino-terminal
domains, are generated by alternative RNA processing in humans (16),
and two isoforms (ROR1 and ROR4) have been isolated from mouse
brain (15, 17). Although its ligand has not been identified, and its
physiological function is not clear, ROR, as manifest by its
messenger RNA (mRNA), is widely expressed, i.e. heart, lung,
liver, muscle, spleen, ovary, and peripheral blood leukocytes (16, 18).
ROR transcripts are expressed in several brain regions; in
particular, there is a high level of expression in Purkinje cells of
the mouse cerebellar cortex (15, 17). ROR1 and ROR2 bind as a
monomer to a hormone-response element composed of a 6-bp AT-rich
sequence 5' to a half-site core motif, PuGGTCA (ROR response element),
to activate transcription (16). In the sg mouse, on the
other hand, expression of the pcp-2 gene, which is directly
regulated by TH in wild-type animals (10), is suppressed, although TR
expression (15) and serum TH levels are within the normal range (19).
Also, TH treatment does not induce thymidine kinase activity in
sg mice, which is normally induced in proliferating granule
cells by interaction with Purkinje cells (20). These results suggest
that ROR may be involved in the regulation of gene expression by TRs
in Purkinje cells.
To examine whether TH regulates the expression of the ROR gene,
which may then regulate the gene expression essential for normal
cerebellar development, we have studied the ontogenic change in ROR
mRNA content in the cerebellum of hypothyroid animals and the effect of
TH replacement using Northern blot analysis, ribonuclease (RNase)
protection assay, and in situ hybridization histochemistry
(ISH) for ROR mRNA. During this study, we also cloned a partial rat
ROR1 complementary DNA (cDNA).
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Materials and Methods |
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Total RNA from pooled cerebella (7–10 brains for P2, 5–7 brains for P7, 2–3 brains for P15, 1–2 brains for P30, and 1 brain for adults) was extracted using the acid guanidinium thiocyanate-phenol-chloroform method (23). Some brains of P15 and P30 pups were used to cut 10-µm thick frozen sections on a cryostat and were fixed with paraformaldehyde according to a procedure described previously (21, 22).
Plasmids and oligonucleotides
The protein-coding region of mouse ROR1 cDNA was provided by
Dr. B. A. Hamilton, Whitehead Institute (Cambridge, MA) (15). A
part of the ligand-binding domain (LBD) of the cDNA was amplified by
PCR using the oligonucleotide, 5'-GCAGCTTCTACCTGGACATCCAG-3' (sense nt
661–683), as the forward primer and 5'-GTCGCACAATGTCTGGGTATATTG-3'
(antisense nt 1506–1483) as the reverse primer and was cloned into the
pGEM-T vector (Promega, Madison, WI). The LBD of rat TR1 cDNA (bases
371-1256), cloned into the pSG5 vector (Stratagene, La Jolla, CA), was
prepared as previously described (24). This fragment contains a region
with sequences identical to those of c-erbA2 (bases
371-1125). Mouse retinoic acid X receptor-ß (RXRß) cDNA cloned into
pcDNAI/Amp was provided by Dr. D. J. Mangelsdorf (University of
Texas Southwestern Medical Center, Dallas, TX).
RT-PCR amplification of rat ROR fragment
Polyadenylated RNA of adult rat cerebella (mothers of the pups)
was isolated as described previously (25) and was reverse transcribed
with mouse mammary leukemia virus reverse transcriptase and
oligo(deoxythymidine) primers. The resulting cDNA was then amplified by
PCR with sense and antisense primers for mouse ROR cDNA as described
above, resulting in an 846-bp DNA fragment, which was then cloned into
the pGEM-T vector. The fragment was partially sequenced from the
5'-region using version 2.0 of the sequencing kit from Amersham
(Arlington Heights, IL). To exclude the possibility of a mutation that
might have occurred during the PCR procedure, two sets of samples were
prepared separately. We confirmed that the sequence was identical
between the two samples. Then, the fragment was digested with
PstI restriction endonuclease, which retains a 259-nt
section of the fragment, and ligated.
Northern blot analysis
Northern blot analysis was performed using procedures described
previously (21). Thirty micrograms of total RNA obtained from pooled
cerebella from animals on each postnatal day were subjected to
electrophoresis and transferred to a nylon membrane (Nytran, Schleicher
and Schuell, Keene, NH). For normalization, 30 µg RNA (n = 6)
obtained from adult animals were always analyzed with other RNA
samples. cDNA fragments for PCR-amplified mouse ROR1 fragment, rat
TR1 LBD, and full-length mouse RXRß, prepared as described above,
were excised from each vector and labeled with 32P, using
the random priming method. The blots were hybridized first with ROR
probe. Hybridization was carried out at 42 C overnight. Then the blots
were washed with a graded series of sodium chloride-sodium
phosphate-EDTA buffer (SSPE) and exposed to Kodak X-Omat film (Eastman
Kodak, Rochester, NY) for 3–7 days at -80 C. After the exposure, the
blots were stripped with 0.5% SDS at 100 C. Then blots were hybridized
sequentially with probes for TR1 LBD and RXRß. The blots were also
hybridized with 32P-labeled rat cyclophilin cDNA probe (26)
for normalization. The band densities were determined by laser
densitometry (Molecular Dynamics, Sunnyvale, CA). The amount of total
RNA in each sample was internally standardized within each blot by
correcting each mRNA level according to the level of the cyclophilin
mRNA. Blot to blot variations were corrected according to the level of
each mRNA from six adult samples subjected to electrophoresis.
Statistical analysis was performed using ANOVA. Post-hoc
comparison was performed using Duncan’s new multiple range test.
RNase protection assay
A RNase protection assay was performed using the RPA II kit
(Ambion, Austin, TX). A rat ROR cDNA fragment in pGEM-T vector was
linearized with SphI restriction endonuclease, and a
32P-labeled antisense riboprobe containing 259 nt of ROR
cDNA fragment and 85 nt of vector DNA was transcribed using SP6 RNA
polymerase. 32P-Labeled riboprobe for rat cyclophilin was
also transcribed with T7 RNA polymerase using the cDNA provided with
the kit. Twenty-microgram samples were then hybridized with both probes
(ROR, 300,000 cpm/sample; cyclophilin, 20,000 cpm/sample) overnight
at 45 C, followed by RNase A/RNase T1 digestion. The protected
fragments were separated on a 5% polyacrylamide-8 M urea
gel. Then the gel was transferred to chromatography paper and exposed
to x-ray film at -80 C overnight. The amount of RNA was internally
standardized using cyclophilin mRNA levels, and the difference in each
blot was standardized with ROR mRNA levels of the same adult RNA
samples, as described for the Northern blot analysis. Statistical
analysis was determined as described for Northern blot analysis.
ISH
An 35S-labeled antisense riboprobe for rat ROR
mRNA was prepared as described for the probe for the RNase protection
assay. A 35S-labeled sense riboprobe was also prepared by
linearizing the fragment with PstI followed by transcription
with T7 RNA polymerase. After prehybridization with 120 µl/slide of
prehybridization buffer (21), 1.5 x 106 cpm/slide of
sense or antisense probe, dissolved in hybridization buffer (21), were
pipetted onto the section. Then a 24 x 50-mm glass coverslip was
placed over the section. Hybridization was carried out for 24 h at
50 C. The next day, sections were rinsed with 2 x SSC (standard
saline citrate) containing 50% formamide for 1 h at 65 C,
followed by RNase A (20 µg/ml) treatment for 30 min at 37 C. Sections
were rinsed with solution containing 2 x SSC and 50% formamide
for 45 min at 65 C, dehydrated through the graded series of ethanol,
dipped in Kodak NTB-3 autoradiography emulsion, and exposed for 2
weeks. Then the sections were developed, counterstained with cresyl
violet, and overlaid with a glass coverslip.
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Results |
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shows the change in body weight in PTU-treated (hypothyroid) animals
and PTU-treated animals with daily single injections of T4
(T4-replaced). The changes in body weight were identical to
those seen in our previous study (21, 22).
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and other mRNAs in the cerebellum was
performed. When LBD of the TR1 cDNA was used as a probe, two bands
corresponding to TR1 [
5.0 kilobases (kb)] and
c-erbA2 (2.6 kb) mRNA were detected (Fig. 2). The TR1 mRNA level was
significantly higher in the hypothyroid rat on P15 and P30, and the
c-erbA2 mRNA level was slightly higher in the hypothyroid
rat on P30 (Fig. 3). An intense 2.4-kb
band and a weak 3.0-kb band, corresponding to RXRß cDNA, were
observed (Fig. 2). As the 3.0-kb band was too weak for quantitative
analysis, only the 2.4-kb band was used for analysis. No significant
changes in RXRß mRNA were detected throughout the experiment (Fig. 3). We also tried to detect TRß1 mRNA using the same blots; the
signal was too weak for quantitative analysis (data not shown). A
single band (10 kb in size) was seen after hybridization with the
mouse ROR cDNA. The intensity of the band appeared to increase with
age. Quantitative analysis showed about a 3-fold increase by P30
compared with that on P2. On P15, a more intense band was observed in
the T4-replaced animal. Such a tendency, however, was not
seen on P2, P7, or P30. When the ratio of the cyclophilin-normalized
intensity between hypothyroid and T4-replaced animals on
P15 in each blot was calculated, the level was 1.4 ± 0.09-fold
higher in T4-replaced animals (n = 5). However, the
ratio of each band with adult ROR mRNA signal greatly varied among
the blots, and the data could not be properly normalized. As a
consequence, we did not see any significant difference in levels of
ROR mRNA between hypothyroid and T4-replaced animals
using this approach (data not shown).
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mRNA between hypothyroid and T4-replaced animals on P15, we
could not fully standardize the levels of the mRNA. Therefore, to study
further the change in ROR mRNA levels, we applied a RNase protection
assay to increase the sensitivity of the quantitative analysis.
First, we cloned a cDNA encoding part of the LBD of rat ROR using a
RT-PCR method. As ROR1 through ROR4 share common DNA-binding
domains and LBDs (15, 16, 17), this probe may detect all ROR
transcripts. However, because no ROR cDNA has been cloned from the
rat, we decided to produce cDNA encoding a region of the LBD of ROR.
The nt sequence of the partial rat ROR cDNA and a comparison with
mouse (15) and human (16) sequences are shown in Fig. 4. The amino acid sequence is completely
identical with other ROR sequences. The sequence similarity of the
nt sequences is 98% with mouse and 89% with human. Using part of this
cDNA fragment, we then performed a RNase protection assay. An example
of the autoradiogram and the result of quantitative analysis are shown
in Fig. 5. As in the Northern blot
analysis, an approximately 3-fold increase in ROR mRNA level from P2
to P30 was observed in both groups of animals. However, T4
treatment clearly accelerated the increase in ROR mRNA levels.
Statistical significance of the difference was achieved on P15. The
ROR mRNA content was approximately 85% greater in
T4-replaced animals. By P30, with or without T4
treatment, the ROR mRNA levels were equivalent.
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gene is expressed in a specific subset
of cells in the cerebellum, we applied ISH. As shown in Fig. 6, A and B, TH treatment dramatically
changed the morphology of the cerebellar cortex, as reported previously
(4, 5, 6, 7), on P15. T4 treatment reduced the size of the
external granule cell layer and increased that of the molecular layer.
Such a difference was not seen on P30 (Fig. 6, E and F). When sections
were hybridized with antisense probe, a significant concentration of
silver grains was seen over specific cells (Fig. 6). When sections were
hybridized with a sense probe, a significant hybridization signal was
not seen (data not shown). These results suggest that a specific
hybridization signal was detected. In the cerebellum, a specific
hybridization signal was noted only over Purkinje cells (Fig. 6). No
other significant signal was detected over other groups of cerebellar
cells, such as granule cells and cells in the deep cerebellar nuclei.
These results, showing ROR gene expression only in Purkinje cells,
are consistent with previous findings in the mouse cerebellum (15, 17).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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mRNA content in the developing cerebellum of the
perinatal hypothyroid rat. The ROR mRNA level, however, becomes
identical by P30, with or without T4 treatment. In
contrast, the TR1 level was lower in T4-replaced animals
on P15 and P30, and the c-erbA2 level was lower on P30,
confirming previous findings (27). In contrast, RXRß mRNA levels were
not affected by altered thyroid status during cerebellar development.
The difference in ROR mRNA content between hypothyroid and
T4-replaced animals was most evident on P15. The results of
the ISH study suggest that the ROR gene is expressed only in
Purkinje cells in the rat cerebellum. As generation of Purkinje cells
occurs prenatally, and altered thyroid status does not change the
number of Purkinje cells (6), these results indicate that TH modulates
the expression of the ROR gene within Purkinje cells in the rat
cerebellum during a specific period of development.
Outgrowth of primary dendrites of Purkinje cells begins around P12, and
differentiation of these dendrites is initiated with the formation of
synapses with the parallel fibers from granule cells by P15. These
events are associated with the outgrowth of dendritic spines in the
Purkinje cells (28). TH deficiency during this period results in
significant decreases in dendritic arborization and synaptogenesis in
Purkinje cell. TH replacement started after this critical period cannot
restore these abnormal neurogenesis (29). On the other hand, dendritic
arborization and synaptogenesis are greatly disturbed in the
sg mouse (12, 13), in which the ROR gene is disrupted,
suggesting that ROR is essential for normal dendritic arborization
and synaptogenesis. As timely and coordinated gene expression is
essential for normal cerebellar development, the reduced expression of
the ROR gene during this critical period seen in hypothyroid animals
in the present study may contribute to the abnormal neurogenesis of
Purkinje cell. Although a specific gene that is expressed in the
Purkinje cell and is directly regulated by ROR has not yet been
identified, our results indicate that TH in part exerts its effect on
neurogenesis by regulating the gene expression of ROR, which then
regulates the expression of genes essential for normal differentiation
of Purkinje cells.
Whether the ROR gene is directly regulated by TH is not known. Two
genes that are known to be specifically expressed in brain and directly
regulated by TR are pcp-2 and myelin basic protein (10, 30).
Although distinct TREs have been identified within the promoter regions
of both genes, TH appears to regulate their gene expression in the rat
cerebellum only during the perinatal period. During cerebellar
development, there is a delayed increase in these genes in the
hypothyroid state. However, the mRNA content of these two genes between
hypothyroid and euthyroid animals becomes identical after the critical
period (31, 32). Furthermore, the expression of many other cerebellar
genes, which are altered by thyroid status, achieves the same level
after the critical period with or without TH regardless of
morphological differences (22, 31, 32). In the present study, ROR
mRNA content also became identical in hypothyroid and
T4-replaced animals by P30 despite the fact that TH
increases its content on P15. This indicates that ROR gene
expression may also be regulated by TH with the same timing mechanisms
as other TH-regulated brain genes. Further study is necessary to
determine whether expression of the ROR gene is directly regulated
by TR.
In addition to the possibility that TH may regulate the expression of
the ROR gene, which then regulates gene expression essential for
normal development of Purkinje cells, there is another issue to be
considered. ROR1 binds as a monomer to a palindromic TRE and various
direct repeat hormone-response elements, providing that an AT-rich
sequence precedes one of two core motifs (AGGTCA) (16). This suggests
that a subset of natural TREs containing the proper AT-rich sequence
could serve as dual response elements for TR and ROR. Therefore, the
change in the ratio of TR to ROR concentrations in Purkinje cell by
thyroid status could change the rate of transcription of specific genes
containing such a dual hormone response element and result in abnormal
neurogenesis. Hence, it would be interesting to investigate the
possible interactions of TR and ROR in the control of gene
expression in Purkinje cells.
In summary, during cerebellar development, about a 3-fold increase in
the cerebellar content of ROR mRNA was seen in both PTU-treated and
PTU-treated and T4-replaced animals. However, the increase
was accelerated when T4 was injected, although the ROR
mRNA content was identical, with or without T4, by P30.
These results indicate that TH alters the timing of expression of the
ROR gene in Purkinje cells of the cerebellar cortex, which may, in
turn, influence Purkinje cell differentiation.
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Acknowledgments |
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Footnotes |
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Received November 12, 1997.
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References |
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2 in selected tissues:
cerebellar Purkinje cells as a model for ß1 receptor-mediated
developmental effects of thyroid hormone in brain. Proc Natl Acad Sci
USA 88:3887–3891- and ß-thyroid hormone receptor mRNAs,
including the ß2-subtype, in the developing mammalian nervous system.
J Neurosci 12:2288–2302[Abstract]
in staggerer mice. Nature 379:736–739[CrossRef][Medline]
, a novel family of orphan hormone
nuclear receptors. Genes Dev 8:538–553
isoforms during neonatal brain development. Mol Endocrinol 5:1109–1119This article has been cited by other articles:
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; hypothyroid) and T4-replaced (•) animals. Mean body
weights ± SEM of the animals killed at the indicated
age are shown. The numbers of animals are 38 (P2, hypothyroid), 33 (P2,
T4-replaced), 24 (P7, hypothyroid), 25 (P7,
T4-replaced), 18 (P15, hypothyroid), 18 (P15,
T4-replaced), 11 (P30, hypothyroid) and 10 (P30,
T4-replaced). **, P < 0.01
vs. hypothyroid animals of the same age.
