(Received for publication, March 29, 1995; and in revised form, May 18, 1995)
From the
Thyroid hormone (T
Thyroid hormone influences biological processes by regulating
gene expression through its nuclear receptors, the thyroid hormone
receptors (TRs). ( We are studying the molecular regulation of amphibian metamorphosis,
a process that systematically transforms every single tissue of a
tadpole (5, 6) . Although different tissues undergo
very different changes, such as the complete resorption of the tail and
intestinal remodeling, the entire process is controlled by thyroid
hormone. The hormone presumably regulates a cascade of gene expression
in each metamorphosing tissues through TRs. Indeed, numerous genes that
are regulated directly or indirectly by thyroid hormone have been
identified in different tissues of metamorphosing Xenopuslaevis tadpoles(7, 8) . Among these are
the two TR We provide here evidence that TRs and RXRs function together to
mediate the regulatory effect of thyroid hormone on metamorphosis.
First of all, we demonstrate that the Xenopus RXR genes are
also expressed during metamorphosis. Furthermore, expression of both TR
and RXR genes is highly tissue specific and its regulation in different
tissues correlates strongly with tissue-specific transformation. In
addition, we show that Xenopus TR-RXR heterodimers can bind to
the TRE present in Xenopus TR
The pSP64(A)
plasmids containing TR and RXR cDNAs were linearized and transcribed in vitro using a SP6 kit (Ambion). The resulting capped mRNAs
were purified and resuspended in water. Each mRNA was injected at a
concentration of 100 ng/µl into the cytoplasm of about 30 stage VI Xenopus oocytes (27.6 nl/oocyte) as described (14) .
After overnight incubation at 18 °C, the oocytes were collected,
rinsed once with modified Barth's medium(14) , and then
homogenized in 600 µl of 20 mM Hepes, pH 7.5, 60 mM KCl, 5 mM MgCl To determine the
relative levels of TRs and RXRs in the oocytes, mRNA for each receptor
was coinjected with [ To determine the
DNA binding activity of TR-RXR heterodimers, 0.5 µl of TR and RXR
extracts were first mixed on ice for 10 min. The mixture or individual
extracts were then used in the DNA binding and competition experiments
as described previously(15) .
Figure 1:
Northern blots showing that Xenopus RXR
Although
metamorphosis is initiated by the rising concentrations of endogenous
thyroid hormone around stage 54, different tissues undergo their
specific transformations at very different stages(17) . In
particular, hindlimb development begins around stage 54 while tail
resorption takes place mostly around stage 62 and later. Reflecting
this stage dependence, the levels of at least the TR
Figure 2:
Northern blots showing tissue-dependent
regulation of RXR
Similarly, both RXR
Figure 3:
Coordinated regulation of TR and RXR genes
during tissue remodeling. The mRNA levels for TRs and RXRs in tail (A), hindlimb (B), and intestine (C) were
determined using a PhosphorImager to quantify the signals from Northern
blots similar to those shown in Fig. 2and normalized against
rpL8 signals. The mRNA levels for TR
Figure 4:
TRs and RXRs are efficiently synthesized
when their mRNAs are injected into oocytes. A, equal amounts
of TR or RXR mRNA were coinjected with
[
Figure 5:
TRE binding requires the presence of both
TRs and RXRs. Extracts from oocytes preinjected with mRNAs for the
indicated receptors were tested for their ability to bind the TRE
(xTRE) present in the TR
Figure 6:
TRE made of two direct repeats of AGGTCA
separated by 4 bp (xTRE) is the preferred binding site by Xenopus TR
When the extracts from
the oocytes injected with individual receptor mRNAs were tested for
binding to xTRE, no complexes were detected (Fig. 5). However,
when the extracts containing TR To test the DNA-binding
specificity by TR-RXR heterodimers, competition was performed with
various known TREs. All complexes formed between xTRE and different
combinations of TR-RXR heterodimers showed identical competition
profiles (Fig. 6). The unlabeled xTRE itself competed very
efficiently while its mutated version (mTRE), the mutations in which
abolish the T
Figure 7:
Transcriptional repression by unliganded
TR/RXR heterodimers and activation in the presence of T
As a control, our Western blot and
[
Using a tissue culture
transfection assay, we have shown recently that the TRE in the TR
Amphibian metamorphosis involves systematic
transformation of every single organ of a tadpole. Different tissues
metamorphose at very different developmental stages. For example,
hindlimb morphogenesis takes place very early, around stages
54-56, and tail resorption takes place very late, after stage 60,
while intestinal remodeling occurs between stages 58 and 66. Such
stage-dependence can be controlled at several levels. First of all, the
plasma T Finally the
coordinated, tissue-specific temporal regulation of TR and RXR genes
are likely to be an important factor in the timing of organ specific
metamorphosis. Thus, in the hindlimb around stages 54-56, even
though the plasma T Currently, it is unknown how these
receptor genes are regulated. Clearly, factors other than the receptors
themselves or T In conclusion, biochemical and
tissue culture transfection studies have suggested that TR/RXR
heterodimers mediate the effects of T
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
) plays a causative role in
amphibian metamorphosis. This regulation is thought to be mediated by
heterodimers of T
receptors (TRs) and retinoid X receptors
(RXRs). We report here that Xenopus TRs can indeed form strong
heterodimers with Xenopus RXRs on the T
response
element (TRE) present in Xenopus TR
genes. Using a
T
-responsive in vivo transcription system
established by introducing TRs and RXRs into Xenopus oocytes,
we demonstrated that TR-RXR heterodimers repressed TR
gene
promoter in the absence of T
and activated the promoter in
the presence of the hormone. Furthermore, by analyzing the expression
of TR and RXR genes, we showed that TR and RXR genes were coordinately
regulated in different tissues during metamorphosis. Thus high levels
of their mRNAs are present in the limb during early stages of limb
development when morphogenesis occurs and in the tail toward the end of
metamorphosis when it is being resorbed. Such correlations coupled with
our TRE-binding and in vivo transcriptional activation
experiments provide strong evidence that TRs and RXRs function together
to mediate the effects of T
during metamorphosis. These
results further suggest a possible molecular basis for the temporal
regulation of tissue-specific metamorphosis.
)TRs belong to the superfamily of steroid
hormone receptors(1, 2, 3) . These receptors
contain a highly conserved DNA binding domain consisting of two
Zn-fingers that mediate the specific recognition of their respective
hormone response elements (e.g. TREs for TRs) located in their
target genes. The hormone binding domain is located at the
carboxyl-half of the proteins and is unique to each hormone. In the
presence of their cognate hormones, these receptors can either activate
or repress the transcription of their target genes, depending on the
promoter context. Studies in mammals and birds have shown that while
TRs can bind to TREs as homodimers and more weakly as monomers, they
form strong heterodimers with several other members of the receptor
superfamily(3, 4) . In particular, the strongest
heterodimers formed are those with receptors for 9-cis-retinoic acid,
or retinoid X receptors (RXRs). Furthermore, the heterodimers between
TRs and RXRs confer specificity for gene regulation by thyroid hormone
in cotransfection experiments in tissue culture cells. However, it
remains to be determined whether TR-RXR heterodimers are the active
complexes mediating the effect of thyroid hormone in vivo.
and two TR
genes themselves. In addition, both
TR
and TR
genes are highly expressed during metamorphosis,
suggesting that they participate in the regulation of the process.
genes with high specificity
and affinity and activate the TR
promoter in a hormone-dependent
manner.
RNA Isolation and Analysis
RNA was isolated from
whole tadpoles or individually dissected tadpole organs at different
developmental stages as described(9) . Total RNA was
electrophoresed on a 1% agarose formaldehyde gel and analyzed by
Northern blot hybridization. To show that equivalent amounts of total
RNA were present, the blots were stained with methylene blue (10) and the same or duplicated blots were hybridized with a
cDNA probe for ribosomal protein L8 (rpL8), a gene whose mRNA is
maintained at relatively constant levels during
development(11) .Overproduction of TRs and RXRs in Oocytes and Gel
Mobility Shift Assays
Although there are two TR (
A and
B) and two TR
(
A and
B) genes in Xenopus and each of the TR
genes gives rise to two receptor isoforms,
the members in each subfamily are highly homologous(13) .
Therefore, we chose only TR
A and TR
A II for the analysis
below. pSP64(A)-TR
A and pSP64(A)-TR
A II for in vitro synthesis of TR mRNAs were kind gifts from Dr. A. Kanamori
(Carnegie Institution of Washington, also see (13) ). The cDNAs
for the entire coding regions of RXR
and RXR
(12) were polymerase chain reaction-amplified and cloned into a
modified pSP64poly(A) vector (Promega) (
)using the
restriction sites introduced in the polymerase chain reaction primers
to generate pSP64(A)-RXR
and pSP64(A)-RXR
.
, 5 mM dithiothreitol,
10% glycerol, 0.1% Nonidet P-40, and 1 mM phenylmethylsulfonyl
fluoride. The extracts were centrifuged twice at 12,000 rpm at 4 °C
for 15 min to remove yolk proteins and debris.
S]methionine (1
µCi/µl mRNA) into oocytes. The protein extracts were analyzed
by 12% SDS-polyacrylamide gel electrophoresis. The relative levels of
unlabeled receptors were analyzed by Western blotting using a
chemiluminescence kit from Amersham and polyclonal antibodies against
recombinant TR
A II and RXR
(15) .
Transcriptional Activation of the TR
A 1.9-kilobase EcoRI fragment from
the EcoRI site at -1.6 kilobase of TR Promoter by TRs
and RXRs in Oocytes
A promoter to
the EcoRI site at +300 of the CAT reporter gene in
pCAT-WT (15) was cloned into pBluescript KS(-) to
generate pKS-TR
Ae. The single-stranded pKS-TR
Ae was then
prepared as described(16) . The single-stranded DNA was
injected into the nuclei of the stage VI oocytes at a concentration of
50 ng/µl (23 nl/oocyte). When mRNAs for TRs and/or RXRs were used,
they were injected into the cytoplasm (23 nl/oocyte at a total
concentration of 10 ng/µl) 2-3 h before the injection of DNA,
and the oocytes were incubated overnight in modified Barth's
medium with 100 units of ampicilin and streptomycin in the presence or
absence of 50 nM thyroid hormone T
(3,5,3`-L-triiodothyronine). The injected
oocytes(15, 16, 17, 18, 19, 20) were
homogenized in 0.25 M Tris-HCl, pH 7.5 (10 µl/oocyte).
Half of the extract was transferred into a tube containing 500 µl
of RNAzol TM reagent (TEL-TEST, Inc., Friendswood, TX) for RNA
isolation and the remaining half was used to isolate DNA(14) .
Primer extension with a primer located in the CAT gene was used to
analyze the transcripts from the TR
A promoter as
described(14) . The DNA isolated from the other half of the
oocytes was analyzed by slot-blot hybridization to verify the amounts
of injected DNA.
Both RXR and TR Genes Are Regulated in a
Tissue-dependent Manner during Metamorphosis
The expression of Xenopus RXR and RXR
genes was initially examined
using RNA from whole animals at various embryonic and metamorphosing
stages. The mRNA level for RXR
gradually increased as zygotic
transcription began after midblastula transition (stage 9) and reached
highest values by stage 40, shortly after tadpole hatching at stages
35/36 (Fig. 1). The RXR
mRNA was then maintained at
relatively constant levels throughout tadpole development and
metamorphosis, resembling that of TR
mRNA(18) . In
contrast, the RXR
mRNA levels, which were relatively high in early
embryos due to maternal storage(12) , gradually decreased to a
minimum around neurula stages (stages 16/17) and was elevated slightly
in pre- as well as metamorphosing tadpoles (Fig. 1).
and RXR
are differentially regulated during
development. Each lane contained 10 µg of total RNA from whole
animals at different stages. Equal loading was confirmed by staining
the membrane for total RNA with methylene blue (not shown). The
positions of 28 S and 18 S rRNA are
indicated.
mRNA
correlate with metamorphosis in these different
tissues(18, 19, 20, 21) . To
investigate whether such regulation exist for other receptors, mRNA
levels for RXR
, RXR
, and TR
genes were determined in the
tail, hindlimb, and intestine at different stages ( Fig. 2and
3). Like the TR
genes, the RXR
gene was found to be highly
expressed in the tail around stages 62 to 64 when rapid resorption
occurred, and in the hindlimb around stages 54 to 56 when limb
morphogenesis took place. Very low levels of its mRNA were present in
either the tail or the hindlimb at other stages. In the intestine, the
RXR
mRNA was maintained at moderate levels throughout
metamorphosis, again resembling the expression pattern of the TR
genes.
gene during metamorphosis. Two µg of total
RNA from different organs at the indicated stages per lane were
analyzed. The probe rpL8 served as a loading
control.
and TR
genes were expressed at
relatively high levels during tail resorption (stages 62-64, Fig. 3). Unlike the other receptor genes, TR
mRNA levels in
the hindlimb were low even during morphogenesis (around stage 56). In
the intestine, both TR
and RXR
expression was elevated during
remodeling, in contrast to that of the TR
and RXR
genes. On
the other hand, it is worth pointing out that the absolute levels of
TR
and RXR
mRNAs were higher than the corresponding values
for TR
and RXR
mRNAs (data not shown; also see (20) ). However, the overall correlation of the mRNA levels of
these genes with tissue-specific transformations strongly suggest that
they all participate in metamorphosis.
and RXR
were determined
using 2 µg of total RNA. Due to the lower levels of their
expression, 10 µg of total RNA per sample was used for the analysis
of TR
and RXR
mRNAs. This made it difficult to determine
their expression in the hindlimb at stage 54 when the organ is very
small. The plasma T
levels were from Leloup and
Buscaglia(28) . Low levels of T
are likely present
around stage 54, as measurable amounts of T
, the precursor
for T
, is present by this
stage(28) .
TRs and RXRs Form Strong Heterodimeric Complexes on the
TRE in the TR
We have recently identified a TRE
(xTRE) consisting of two near perfect direct repeat of AGGTCA separated
by 4 base pairs in both TR Genes
A and TR
B genes of X. laevis(15) . While little binding of the TRE by
TR
or RXR
produced in E. coli was observed, strong
specific complexes were formed in the presence of both TR
and
RXR
. However, most of the proteins made in E. coli were
insoluble, making it difficult to determine the relative binding
affinity of different heterodimers. We, therefore, turned to Xenopus oocytes to generate functional receptors. When the
receptor mRNAs were injected into oocytes, they were efficiently
translated. By a combination of in vivo labeling with
[
S]methionine and Western blotting with
anti-Xenopus TR and RXR antibodies, we concluded that within a
few folds, the amounts of receptors made were the same when equal
amounts of mRNAs were injected into oocytes (Fig. 4). These
experiments also showed that there were little endogenous TRs and RXRs
in the oocytes even though their mRNAs appeared to be present during
oogenesis(12, 19) . Such a conclusion is in agreement
with recent results on TR levels in oocytes by Eliceiri and Brown (22) and is also consistent with the DNA binding and
transcription experiments described below.
S]methionine into oocytes and the resulting
labeled proteins were analyzed by SDS-polyacrylamide gel
electrophoresis. The arrowheads point to the positions of the
receptors. B and C, Western blot analysis of
S-labeled or unlabeled TR (B) and RXR (C) produced in oocytes. The anti-TR
antibody also
recognize TR
and the anti-RXR
antibody does not recognize
RXR
. The stars in B and C indicate the
same samples as used in A. By comparing the
S
signals and the signals from Western blots, it was concluded that
roughly equal amounts of TRs were present in the unlabeled extracts,
which were used in the DNA binding experiments ( Fig. 5and Fig. 6).
A promoter by the gel mobility shift
assay. The binding was carried with individual extracts or mixtures of
different combinations of TR and RXR
extracts.
(upper) or TR
(lower) heterodimers
with RXRs. Mixtures of oocyte extracts containing the indicated
receptors were analyzed for TRE binding. The xTRE were end-labeled and
binding was performed in the presence of the indicated amounts (ng) of
unlabeled competitors. The unlabeled xTRE itself competed effectively
while TREp and TREgh were weaker competitors. The mTRE failed to
compete.
or TR
were mixed with those
containing RXR
or RXR
, the resulting extracts formed strong
complexes with xTRE. Different combinations of receptors produced
roughly equal binding. The addition of thyroid hormone T
had little effect on the amount of complexes formed (not shown).
These results indicate that heterodimers formed between any TR and RXR
can bind to xTRE with similar affinities.
dependence of the TR
promoter in Xenopus tissue culture cells(15) , failed to compete.
In addition, a palindromic TRE made of two inverted repeat of AGGTCA
(TREp) and the TRE present in the human growth hormone gene (TREgh),
which differs from both TREp and xTRE, competed less efficiently for
the complex formation. This same TRE sequence preference has also been
found for mammalian TRs(23, 24) , consistent with the
high sequence conservation among the mammalian and frog receptors.
Transcriptional Regulation by TRs in Xenopus
Oocytes
The fact that there is little TRs and RXRs in oocytes
also made it possible to study the function of TRs. We took the
advantage that when single-stranded plasmid DNA containing a reporter
promoter is injected into oocytes, they undergo one round of
replication and the resulting double-stranded DNA is quickly
chromatinized to form templates with low levels of basal
transcription(14) . Thus, mRNAs of TRs or RXRs were
individually or co-injected into oocytes first. Following a few hours
of incubation to allow the synthesis of the receptors, single-stranded
plasmid DNA containing the T-inducible promoter of TR
A
gene was injected and the promoter activity was determined by primer
extension analysis of the transcribed RNA. The TR
A promoter
exhibited a weak activity in oocytes in the absence of T
and the addition of T
had little effect on this
activity (Fig. 7A). Identical results were found with oocytes
that were preinjected with only RXR
and RXR
mRNAs. Similarly,
when the promoter DNA was injected into oocytes preinjected with only
TR
or TR
mRNA, little or very low levels of activation was
observed in the presence of T
(Fig. 7A). In
contrast, preinjection of various combinations of TR and RXR mRNAs
resulted in suppression of the basal promoter expression and the
addition of T
relieved the suppression and led to strong
activation. These results indicate that TR alone is either insufficient
to activate the TRE-containing promoter or can only weakly activate the
promoter. On the other hand TR-RXR heterodimers are strong activators
of the TRE-containing promoter, in agreement with the TRE binding
results discussed above.
. A, oocytes were preinjected with mRNAs for the individual
receptors or combinations of TRs and RXRs. Subsequently, they were
injected with a single-stranded plasmid containing the
T
-inducible promoter of TR
A gene and the activity of
the promoter in the presence or absence of T
was determined
by primer extension. B, equal amounts of plasmid DNA were
injected into each oocyte sample. DNA was recovered from the same batch
of oocytes used for RNA analysis and subjected to slot-blot analysis
using
P-labeled plasmid probe.
S]methionine labeling showed that the amounts
of TRs and RXRs synthesized were similar (above). Furthermore, when the
injected DNA from the same batch of oocytes used for RNA analysis were
extracted and analyzed by hybridization, equal amounts of the plasmid
DNA were present in all samples (Fig. 7B). As only the
single-stranded plasmid injected into the nucleus would be replicated
and protected from degradation, these results indicate that equal
amounts of the promoter plasmid were present in all samples. Thus, the
results above reflect the true functional difference between the TR-RXR
heterodimers and TR or RXR homodimers.
Xenopus TRs Form Strong Heterodimers with RXRs and
Regulate Transcription in Oocytes
Four TR (two TR and two
TR
) and two RXR (RXR
and RXR
) genes have been cloned in X. laevis. We and others have shown that the TR
genes are themselves directly regulated by
T
(15, 25, 26, 29) . Our
current work clearly demonstrate that both TR
and TR
can form
strong TRE-binding heterodimers with either RXR
or RXR
.
Furthermore, the TRE sequence preference is identical among different
heterodimers and is the same as observed in mammals and birds, i.e. the direct repeats of AGGTCA separated by 4 base pairs being the
stronger TRE than either TREp or TREgh.
A
gene appears to confer the repression of the promoter in the absence of
T
and the addition of T
relieves this
repression(15) . Our results here demonstrate that such
repression is mediated by TR/RXR heterodimers. Furthermore, the
addition of T
leads to a strong activation of the promoter.
In contrast, TRs alone have little or very small effects on TR
promoter activity. This appears to differ from previous reports where
cotransfection of a reporter plasmid together with a plasmid to
overproduce only TR leads to T
-dependent transcription (e.g. see (30) ). However, RXRs and/or other
heterodimerization partners for TR seem to be present in tissue culture
cells(15, 30) . In addition, our transcription results
are consistent with the TRE-binding property of TR homo- and
heterodimers. However, it is worth mentioning that although we failed
to detect DNA binding by TR monomers or homodimers, such weaker binding
might be detectable under different conditions (e.g. see (30) ). The low level of transcriptional activity when TR mRNAs
alone were injected into oocytes may be due to either this weaker
binding by TRs alone or heterodimers formed by the TRs with the low
levels of endogenous RXRs that eluded our Western blot and DNA binding
assays. In any case, our results suggest that in developing tadpoles,
unliganded TRs can repress TRE-bearing promoters in the absence of
T
and activate them in the presence of T
.
Coordinated Regulation of TR and RXR Genes as a Means to
Control Temporal Regulation of Tissue-specific Metamorphosis
The
interesting regulation of amphibian metamorphosis by thyroid hormone
has led to the cloning and analysis of the expression of TR genes in
both X. laevis and Rana catesbeiana in
several
laboratories(18, 19, 20, 21, 31, 32) .
We have demonstrated here that the regulation of not only TR but also
RXR genes is highly tissue specific. In general, TR and RXR genes are
coordinately regulated in different tissues. Currently it is unknown
whether mRNA levels reflect protein levels. A recent quantification of
TR and TR
levels in Xenopus suggest that in general,
higher levels of mRNAs result in higher levels of the
receptors(22) . The exception is that TR
protein levels
did not correlate well with the mRNA levels during development in the
tail and head, suggesting the existence of mechanisms to regulate
translation and/or protein stability. Alternatively, TR
could
undergo post-translational modifications during development which
altered their ability to be immunoprecipitated and detected by Western
blot. Thus, it is likely that TR and RXR protein levels are also
coordinately regulated.
concentration peaks around stages 60 to 62 when
drastic tissue transformation takes place. Second, we have shown
recently that the expression a cytosolic T
-binding protein
is inversely correlated with tissue metamorphosis, i.e. low
levels in the hindlimb during morphogenesis and in the tail during
resorption but high levels at other stages(21) . This suggests
that the levels of free intracellular T
could be regulated
in a tissue dependent manner. Related to this, the Xenopus type III iodothyronine 5-deiodinase, which converts T
into an inactive form, has also been suggested to play a role in
regulating T
levels(27) .
levels are low, the high levels of TR
and RXR expression could allow the activation of genes important for
limb morphogenesis. Subsequently, as the limb undergoes growth and the
expression of these genes are no longer required, the reduced
expression of TRs and RXRs will lead to the down-regulation of these
genes although the plasma T
concentrations are even higher.
On the other hand, intestinal remodeling takes place from stage 58,
when its length begins to shorten, to stage 66, when secondary
epithelial cell differentiation is complete. This is the period when
high levels of T
are present. Furthermore, TR
and
RXR
expression are elevated even though the mRNA levels for
TR
and RXR
do not change appreciably. Finally, in the tail
before stage 50, TR and RXR expression is low, this coupled with
factors that could reduce free intracellular T
concentration, can effectively prevent metamorphosis to occur.
After stage 60, the elevated expression of TRs and RXRs then activate
the tail resorption process.
levels are involved. This is especially
true in premetamorphic tadpoles when there is little T
. The
high levels of TR and RXR mRNAs in the limb but not the tail or
intestine in those tadpoles strongly suggest the importance of
tissue-specific expression of yet unknown genes in receptor gene
regulation. Whatever the underlying mechanism is, the tissue-specific
regulation of the receptors and free cellular T
levels also
provides a molecular explanation for TR
gene expression, which is
directly regulated by T
. As TR
and RXR
are likely
the predominant forms of TRs and RXRs at least in premetamorphic
tadpoles based on absolute mRNA levels and protein quantification ( (20) and (22) and data not shown), the activation of
TR
genes are probably mediated mostly by TR
-RXR
heterodimers. In the intestine, TR
and RXR
levels appear to
be similar at all stages, thus TR
expression would be expected to
follow the levels of endogenous T
as we have observed. In
the tail, for the reasons discussed above, the activation of TR
genes would be limited to the later period of metamorphosis. Finally,
the relatively low levels of TR
expression in the hindlimb is due
first to insufficient amounts of T
and later the
down-regulation of TR and RXR genes.
in vivo.
However, it has been difficult to prove such a hypothesis as RXRs are
likely involved in gene regulation by several different types of
hormone receptors. On the other hand, thyroid hormone is the single
most important hormone that controls the drastic organ remodeling
during amphibian metamorphosis. Our demonstration of coordinated
regulation of TR and RXR genes coupled with the functional studies,
therefore, provide strong evidence for the importance of TR/RXR
heterodimers in vivo.
,
3,5,3`-L-triiodothyronine or thyroid hormone; CAT,
chloramphenicol acetyltransferase.
We thank Drs. E. De Robertis for the gifts of Xenopus RXR clones and A. Kanamori and D. D. Brown for the
gifts of TR cDNA clones. We are also grateful to Dr. A. Wolffe for
helpful comments on the manuscript and T. Vo for its preparation.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.