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AUTHOR COPY 91
Developmental expression and hormonal regulation of
glucocorticoid and thyroid hormone receptors during
metamorphosis in Xenopus laevis
L P Krain and R J Denver
Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, Michigan 48109, USA
(Requests for offprints should be addressed to R J Denver, Department of Molecular, Cellular and Developmental Biology, 3065C Natural Science Building,
The University of Michigan, Ann Arbor, Michigan 48109-1048, USA; Email: [email protected])
(L P Krain is currently at University of Michigan Hospital, Psychiatry, 9D UH 9812, Ann Arbor, Michigan 48109-0120, USA)
Abstract
Corticosteroids, the primary circulating vertebrate stress brain but increased significantly during prometamor-
hormones, are known to potentiate the actions of thyroid phosis, remained at a constant level throughout metamor-
hormone in amphibian metamorphosis. Environmental phosis, and increased to its highest level in the juvenile
modulation of the production of stress hormones may be frog. GR mRNA level in the intestine remained relatively
one way that tadpoles respond to variation in their larval constant, but increased in the tail throughout metamor-
habitat, and thus control the timing of metamorphosis. phosis, reaching a maximum at metamorphic climax. The
Thyroid hormone and corticosteroids act through struc- level of GR mRNA was increased by treatment with
turally similar nuclear receptors, and interactions at CORT in the intestine but not in the brain or tail. TR
the transcriptional level could lead to regulation of com- mRNA level increased in the brain, intestine and tail
mon pathways controlling metamorphosis. To better during metamorphosis and was induced by treatment with
understand the roles of corticosteroids in amphibian meta- T3. Analysis of possible crossregulatory relationships be-
morphosis we analyzed the developmental and hormone- tween GRs and TRs showed that GR mRNA was
dependent expression of glucocorticoid receptor (GR) upregulated by exogenous T3 (50 nM) in the tail but
mRNA in the brain (diencephalon), intestine and tail of downregulated in the brain of premetamorphic tadpoles.
Xenopus laevis tadpoles. We compared the expression Exogenous CORT (100 nM) upregulated TR mRNA
patterns of GR with expression of thyroid hormone in the intestine. Our findings provide evidence for tissue-
receptor beta (TR ). In an effort to determine the specific positive, negative and crossregulation of nuclear
relationship between nuclear hormone receptor expression hormone receptors during metamorphosis of X. laevis. The
and levels of ligand, we also analyzed changes in whole- synergy of CORT with T3 on tadpole tail resorption may
body content of 3,5,3 -triiodothyronine (T3), thyroxine, depend on the accelerated accumulation of GR transcripts
and corticosterone (CORT). GR transcripts of 8, 4 and in this tissue during metamorphosis, which may be driven
2 kb were detected in all tadpole tissues, but only the 4 and by rising plasma thyroid hormone titers.
2 kb transcripts could be detected in embryos. The level Journal of Endocrinology (2004) 181, 91–104
of GR mRNA was low during premetamorphosis in the
Introduction before the acceleration of thyroid activity, but then
increases dramatically throughout prometamorphosis,
The primary morphogen controlling amphibian metamor- reaching a maximum at metamorphic climax (Yaoita &
phosis is thyroid hormone, which regulates gene transcrip- Brown 1990). The expression of TR in X. laevis is
tion by binding to proteins that are members of the nuclear induced by thyroid hormone (i.e. it is autoinduced; Tata
receptor superfamily (Shi 2000). Two genes designated 1994) and the X. laevis TR gene contains at least
alpha and beta code for thyroid hormone receptors (TRs) one functional thyroid hormone response element in the
in vertebrates (Forrest 1994, Mangelsdorf et al. 1995). In proximal promoter region (reviewed by Shi 2000).
Xenopus laevis, TR mRNA increases shortly after hatch- Corticosteroids produced by the interrenal glands
ing and remains relatively constant throughout the larval (amphibian homologue of the mammalian adrenal cortex)
period and metamorphosis (Yaoita & Brown 1990). By are implicated in the positive control of metamorphosis
contrast, TR expression is low during premetamorphosis (Kikuyama et al. 1993, Hayes 1997). Corticosteroids, like
Journal of Endocrinology (2004) 181, 91–104 Online version via http://www.endocrinology.org
0022–0795/04/0181–91  2004 Society for Endocrinology Printed in Great Britain
AUTHOR COPY
92 L P KRAIN and R J DENVER · Hormone receptor gene expression during metamorphosis
thyroid hormone, regulate gene transcription by binding concentrate T3 from the environment, resulting in tissue
to nuclear receptors. Plasma corticosteroid concentrations concentrations of the hormone that are 4–6 times the
rise markedly during metamorphic climax in several environmental concentration. We discuss the implications
anuran species (Jaffe 1981, Krug et al. 1983, Jolivet-Jaudet of these findings for doses of T3 commonly used to induce
& Leloup-Hatey 1984, Kikuyama et al. 1986) and in the X. laevis metamorphosis.
urodele Ambystoma tigrinum (Carr & Norris 1988). This
rise in plasma corticosteroids is largely synchronous with
the rise in thyroid hormone production (Kikuyama et al. Materials and Methods
1993, Denver 1998). Several investigators have shown that
corticosteroids can synergize with thyroid hormone to Animals and hormone treatments
accelerate tadpole metamorphosis (see Kikuyama et al.
1993, Hayes 1997) and at least two mechanisms have been We obtained adult X. laevis from Xenopus I (Dexter, MI,
proposed for this effect. For example, treatment with USA) and spawned them in the laboratory by injecting
corticosteroids increases nuclear 3,5,3 -triiodothyronine into the dorsal lymph sac 1 µg gonadotropin-releasing
(T3) binding capacity in tailfin of two anuran species (Niki hormone agonist ([des-Gly10,-His(Bzl)6]-LH-RH ethyl-
et al. 1981, Suzuki & Kikuyama 1983b). Another mode of amide; Sigma Chemical Co., St Louis, MO, USA). We
action may be through the regulation of tissue deiodinases, reared tadpoles at 21–23 C on a 12 h light:12 h dark-
where corticosteroids can enhance conversion of thyroxine ness photoperiod in charcoal-purified, pH-adjusted water
(T4) to T3, the more biologically active hormone, and and fed them a suspension of pulverized rabbit chow.
decrease the degradation of T3 (Galton 1990). Hormones were added directly to the aquarium water. T3
There is biochemical evidence for the presence of was dissolved in a small volume of 0·01 M NaOH and
corticosteroid receptors in tadpole tissues (reviewed by diluted 100-fold in 0·6% saline. Hormone stocks were
Kikuyama et al. 1993) but there are currently no data on then diluted 300 000-fold in the aquarium water to give
the expression or hormonal regulation of corticosteroid final concentrations of 5 or 50 nM. CORT was dissolved
receptor genes during metamorphosis. Gao et al. (1994) in 100% ethanol and this stock was then diluted in the
isolated a cDNA for the X. laevis glucocorticoid receptor aquarium water to give a final concentration of 100 or
(GR) and showed, using Northern blot analysis, that the 500 nM (the final concentration of ethanol was 0·0001%).
gene is first expressed during late embryogenesis; how- Tadpoles were staged based on the system of
ever, they did not analyze GR expression during meta- Nieuwkoop & Faber (1956). Tadpoles were killed in 0·1%
morphosis. Csikos et al. (1995) isolated partial cDNA benzocaine before tissue collection. Animal rearing and
clones for a putative X. laevis mineralocorticoid receptor dissection procedures were done in accordance with the
(MR). They detected a low level of MR mRNA in rules and regulations of the University Committee on the
whole tadpoles at Nieuwkoop and Faber (NF) stage 60 Use and Care of Animals at the University of Michigan.
(Nieuwkoop & Faber 1956) by an RNase protection assay,
but were unable to detect MR transcripts by Northern
Hormone extraction and RIA
blotting (we are also unable to detect MR mRNA in
X. laevis tadpoles by Northern blotting, R J Denver, Tadpoles were snap frozen and stored at 80 C before
unpublished observations). hormone extraction. Thyroid hormones were extracted
In the present study we analyzed the developmental and from whole tadpoles following methods described by
hormone-dependent expression of mRNA for the X. laevis Denver (1993, 1998a). CORT was extracted from
GR in three tissues (brain, intestine and tail), and com- whole tadpoles following the method of Hayes & Wu
pared this with expression patterns of TR . The level (1995) with modifications described by Denver (1998a).
of GR mRNA in tadpole brain (the region of the Briefly, tadpoles were homogenized in 3–4 volumes of
diencephalon) was low during premetamorphosis, rose methanol containing 1 mM propylthiouracil for thyroid
during prometamorphosis and then again in the juvenile hormone extraction. For CORT extraction, tadpoles
frog. GR gene expression was strongly increased in tail were homogenized in 3–4 volumes of ethyl acetate. For
throughout prometamorphosis and metamorphic climax. estimation of recoveries, 1000 c.p.m. [125I]T3 or 3000
We found that positive or negative regulation by the c.p.m. [3H]CORT were added to each extract.
respective ligand, or crossregulation of nuclear hormone Recoveries ranged from 35 to 60% for [125I]T3 and 30 to
receptor gene expression was tissue-specific. While TR 45% for [3H]CORT. The validation of these methods for
was upregulated by its ligand in all three tissues examined, the extraction and recovery of hormones from tadpole
corticosterone (CORT) increased GR mRNA only in the tissues is described by Denver (1998a).
intestine. Crossregulation studies showed that GR mRNA The hormones T3, T4 and CORT were measured by
is negatively (brain) or positively (tail) regulated by RIA in extracts of whole X. laevis tadpoles. The T3 and T4
exogenous T3, and TR is upregulated by CORT in RIAs were as described (Mackenzie et al. 1978, Denver &
tadpole intestine. Our results also show that tadpoles Licht 1988, Denver 1998a) and the RIA for CORT was
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Hormone receptor gene expression during metamorphosis · L P KRAIN and R J DENVER 93
described by Licht et al. (1983). Primary antisera for T3
and CORT were purchased from Endocrine Sciences (San
Diego, CA, USA). Primary antiserum for T4 was obtained
from Dr Viggo Kruse (Denmark). All samples were
analyzed in single assays for each hormone.
RNA extraction and Northern blot analysis
Total RNA was extracted from pooled tadpole tissues
(eight to ten brains (diencephalon only), five or six
intestines or one to three tails) using Trizol reagent
(Invitrogen). For Northern blot analysis total RNA (10 µg)
was separated by electrophoresis in a 1% formaldehyde–
agarose gel, hydrolyzed in 0·05 M NaOH, 0·01 M NaCl,
and transferred to nylon membrane using 20 SSC as the
transfer buffer. Northern blots were stained with methyl-
ene blue to verify the integrity of the RNA and to assess
RNA loading. Blots were prehybridized in Hybrisol I
(Chemicon, Temecula, CA, USA) for 2–4 h and hybrid-
ized for 16 h at 42 C with 32P-labeled probes prepared
from cDNAs provided by Yun-Bo Shi (X. laevis; National
Institues of Health, USA) TR and ribosomal protein L8;
rpL8) and Olivier Destree (X. laevis GR; Utrecht, The
Netherlands). cDNAs were labeled with [32P]dCTP
by random priming (Roche). Blots were washed with
2 SSC, 0·5% SDS at room temperature for 10 min, then
0·25 SSC, 0·1% SDS at 65 C for 1 h before exposure to
X-ray film for 1–14 days. Blots were stripped by boiling in
1 TE (10 mM Tris, 1mM EDTA;pH 8.0), 1% SDS for
5 min and hybridized with a probe for the rpL8 gene (Shi
& Liang 1994) to control for RNA loading.
Data analysis
Tadpole wet weight (body weight; BW) was measured
using a digital balance (accurate to 0·01 g). Whole-body
hormone contents are expressed as ng hormone/g BW. Figure 1 Developmental changes in BW, whole-body T3, T4 and
Previous studies showed that correction of hormone con- CORT content in X. laevis during metamorphosis. Thyroid
hormone contents were determined by RIA following organic
tent using either DNA content or wet BW produced extraction as described in Materials and Methods. Data in the
identical results (Denver 1993). Following autoradiogra- CORT Figure are modified from Glennemeier & Denver (2002).
phy, Northern blot data were analyzed using a flatbed Each point represents the mean S.E.M. (n=6–8/stage). The
scanner and band densities were determined using asterisks designate significant differences from the preceding
developmental stage (P,0·05; Scheffe’s test).
Scion Image software (version 4·05; Scion Corporation
Frederick, MD, USA). Each mRNA data point shown on
the graphs represents three or four determinations in
which tissues were isolated from tadpoles from different analyzed by one-way ANOVA followed by a post-hoc test
spawns, RNA isolated, and Northern blots prepared. After (Scheffe’s multiple contrast test; P,0·05).
subtracting background on each Northern blot the densi-
tometric values for GR or TR were normalized to the
rpL8 band. Northern blot data were log10-transformed to Results
achieve homogeneity of variance before analysis by one-
way ANOVA. Expression data are presented in the graphs Developmental profiles of whole-body T3, T4 and CORT
as the mean percentage of maximum mRNA level (for We measured whole-body content of T3 and T4 in X.
developmental expression analyses) or the mean percent- laevis tadpoles from premetamorphosis through to the end
age of zero time (for the hormone treatment time course of metamorphosis (Fig. 1). This analysis showed the
analyses). Hormone data were log10-transformed and expected activation of the thyroid system characteristic of
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94 L P KRAIN and R J DENVER · Hormone receptor gene expression during metamorphosis
anuran metamorphosis. Whole-body T3 and T4 content embryos and is expressed only after hatching where it
remained low and relatively constant during premetamor- becomes the predominant transcript (data not shown for
phosis and early prometamorphosis. Whole-body T4 embryonic expression; see also Gao et al. 1994). Similarly,
showed a strong increase (4-fold) from NF stage 58 to we found that several X. laevis cell lines (XTC-2, XL-58,
stage 60 and remained elevated throughout metamor- A6, XL-177) expressed only the two smaller GR tran-
phosis (P,0·001; ANOVA). Mean whole-body T4 con- scripts (data not shown; see also Spindler & Verrey (1999)
tent declined after metamorphic climax, and although the for A6 cells).
mean was approximately twice that of premetamorphic Expression of GR mRNA in brain showed significant
values, this difference was not statistically significant. Peak changes during metamorphosis, approximately doubling
whole-body T4 content (6·5 ng/g BW) equals roughly between premetamorphic and early prometamorphic
7 nM, which compares with a peak plasma concentration stages (compare stages 49 and 52; P,0·0001; ANOVA).
of 10 nM as reported by Leloup & Buscaglia (1977) for Brain GR mRNA was then expressed at a constant level
X. laevis. For comparison, peak whole-body T4 content until after metamorphosis when it increased approximately
was 7 and 10 nM in larvae of Bufo japonicus (Niinuma et al. 40% above levels seen at metamorphic climax. In the
1991) and B. marinus (Weber et al. 1994) respectively. intestine, GR mRNA was expressed at a constant level
By contrast, the increase in whole-body T3 content during metamorphosis. There was a trend towards an
(3·3-fold) lagged behind changes in T4; whole-body T3 increase (20%) in the mean GR mRNA level at stage
did not show a statistically significant elevation until stage 60, which corresponds to the stage when TR mRNA is
62. Whole-body T3 content remained elevated at stage 64 strongly upregulated and the intestine begins to remodel
and declined after metamorphosis to levels seen in pre- (reviewed by Shi 2000). The level of GR mRNA in
and prometamorphic tadpoles (P,0·001; ANOVA). Peak the juvenile intestine then declined approximately 20%
whole-body T3 content (12 ng/g BW) equals roughly relative to metamorphic levels. The strongest develop-
18 nM, which compares with a peak (NF stage 62) plasma mental regulation of GR mRNA was seen in the tail
T3 concentration of 8 nM as reported by Leloup & where levels increased throughout prometamorphosis and
Buscaglia (1977) for X. laevis. For comparison, peak peaked at metamorphic climax (3-fold above early
whole-body T3 content was 6 and 8 nM in larvae of B. prometamorphic levels; P,0·05) when the tail is actively
japonicus (Niinuma et al. 1991) and B. marinus (Weber et al. resorbing.
1994) respectively.
Data for developmental changes in whole-body CORT
content are derived from Glennemeier & Denver (2002). Effects of exogenous T3 on nuclear hormone receptor mRNA
levels
By contrast to whole-body thyroid hormone content,
whole-body CORT content was highest during premeta- Addition of T3 (50 nM) to the rearing water caused
morphosis, declined significantly at the onset of prometa- statistically significant increases in TR mRNA levels in
morphosis, but then rose slightly during metamorphic all tissues (P,0·0001 for all; ANOVA; Fig. 4). The
climax (stage 62; P,0·0001; ANOVA). kinetics of upregulation was similar among the different
tissues; statistically significant increases in TR transcripts
were detected at 16 h in all tissues. It is likely that the
Developmental expression of TR and GR mRNAs in brain, upregulation occurred earlier than 16 h, since we and
intestine and tail during metamorphosis others have detected TR upregulation by 8–12 h (L P
We compared the developmental expression of TR and Krain & R J Denver, unpublished observations; Yaoita &
GR mRNAs in tadpole brain, intestine and tail by Brown 1990, Kanamori & Brown 1992, Eliceiri & Brown
Northern blotting (Figs 2 and 3). TR mRNA levels were 1994, Furlow & Brown 1999).
low and unchanging during premetamorphosis and early While the kinetics of TR upregulation was similar in
prometamorphosis but increased significantly in all tissues the three tissues analyzed, the dose sensitivity differed.
during late prometamorphosis (P,0·001; ANOVA). The Data for the 50 nM dose of T3 are shown in Fig. 4.
earliest elevation in brain TR mRNA level was detected Treatment with 5 nM T3 produced a similar increase
at stage 58. By contrast, TR mRNA levels increased later in TR mRNA in brain and intestine, but did not influ-
in the intestine and tail (stage 60). Maximal expression ence TR gene expression in the tail (data not shown).
occurred in the brain at stage 60, in the intestine at stage Others have documented the relative insensitivity of gene
62 and in the tail at stage 64. The TR mRNA levels in expression in the tadpole tail to exogenous T3, which is the
the brain of the juvenile frog remained at a late prometa- last tissue to transform (reviewed by Shi 2000).
morphic level (compared with stage 58) but returned to By contrast to TR , T3 treatment downregulated GR
premetamorphic levels in the intestine. mRNA in the brain (P,0·001; ANOVA; Fig. 5). A
Three GR transcripts of 8, 5 and 3 kb were detected by significant reduction in brain GR mRNA was detected by
Northern blot in the brain, intestine and tail (blot shown in 24 h after exposure to T3 (both 5 and 50 nM T3 – only the
Fig. 3 for brain only). The 8 kb transcript is not present in 50 nM dose is shown in Fig. 5). The effect of exogenous
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Hormone receptor gene expression during metamorphosis · L P KRAIN and R J DENVER 95
Figure 2 Developmental expression of TR mRNA in brain, intestine and tail during
metamorphosis. TR expression was analyzed by Northern blotting as described in
Materials and Methods. A representative Northern blot in brain is shown at the top (A).
The graphs (B) show the quantitation of mRNA levels based on densitometric analysis of
scanned autoradiograms. TR mRNA was normalized to the rpL8 band. Data are
expressed as percentage of maximum. Each bar represents the mean of three independent
experiments.
T3 (5 or 50 nM) on GR mRNA levels in the intestine was decreases at 48 and 72 h were (P,0·05). In the tail,
biphasic, with a transient upregulation followed by a 50 nM T3 upregulated GR mRNA, with a maximum
downregulation. The increases in intestinal GR mRNA level of expression at 72 h (3-fold above the 0 time
levels (8–24 h) were not statistically significant but the value; P,0·01). Note that this magnitude of change in
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96 L P KRAIN and R J DENVER · Hormone receptor gene expression during metamorphosis
Figure 3 Developmental expression of GR mRNA in brain, intestine and tail during
metamorphosis. GR expression was analyzed by Northern blotting as described in
Materials and Methods. A representative Northern blot in brain is shown at the top (A).
The graphs (B) show the quantitation of mRNA levels based on densitometric analysis of
scanned autoradiograms. GR mRNA was normalized to the rpL8 band. Data are expressed
as percentage of maximum. Each bar represents the mean of three independent
experiments.
gene expression is comparable with that seen in the tail of hormone to the tadpole-rearing water equals the
during spontaneous metamorphosis (Fig. 3). nominal concentration in the aquarium water i.e. tadpoles
equilibrate with their environment. The choice of a 5 nM
dose of T3 in many studies in X. laevis is based upon this
Changes in whole-body T3 content following exposure to assumption (Shi & Brown 1993), and the report by Leloup
exogenous T3
& Buscaglia (1977) that peak plasma T3 concentration
It is generally assumed that the tissue content/plasma during metamorphosis in this species reaches 8 nM. To
concentration of thyroid hormones achieved by addition test this assumption we analyzed whole-body T3 content
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Hormone receptor gene expression during metamorphosis · L P KRAIN and R J DENVER 97
Figure 4 Effects of in vivo T3 treatment on TR mRNA in brain, tail and intestine. Tadpoles were exposed to T3 (50 nM in the aquarium
water) for various times before harvest for tissue isolation, RNA extraction and Northern blot analysis. Water was changed and the T3 was
replenished every 24 h. A representative Northern blot from brain is shown at the top (A). The graphs (B) show the quantitation of mRNA
levels based on densitometric analysis of scanned autoradiograms. TR mRNA was normalized to the rpL8 band. Data are expressed as
percentage of the zero time value. Each bar represents the mean of four independent experiments.
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98 L P KRAIN and R J DENVER · Hormone receptor gene expression during metamorphosis
Figure 5 Effects of in vivo T3 treatment on GR mRNA in brain, tail and intestine. Tadpoles were exposed to T3 (50 nM in the aquarium
water) for various times before harvest for tissue isolation, RNA extraction and Northern blot analysis. Water was changed and the T3 was
replenished every 24 h. A representative Northern blot from brain is shown at the top (A). The graphs (B) show the quantitation of mRNA
levels based on densitometric analysis of scanned autoradiograms. GR mRNA was normalized to the rpL8 band. Data are expressed as
percentage of the zero time value. Each bar represents the mean of four independent experiments.
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Hormone receptor gene expression during metamorphosis · L P KRAIN and R J DENVER 99
Figure 6 Time-dependent changes in whole-body T3 content following exposure of
premetamorphic tadpoles to two doses of T3 in the aquarium water. T3 was added to the
aquarium water at time=0 and tadpoles were collected at various times and analyzed for
whole-body T3 content by RIA following organic extraction as described in Materials and
Methods. Each point represents the mean (n=8/time point) and vertical bars the S.E.M.
*Significantly different from zero time (P,0·001); **significantly different from zero time
and from the ambient T3 concentration (P,0·0001).
in NF stage 52 tadpoles reared in aquarium water with was not replenished). No effect of CORT treatment on
two different doses of T3 for various times. The nominal nuclear receptor mRNA was observed in the brain or tail
hormone concentrations in the aquarium water achieved (data not shown). In the intestine, CORT treatment
were 6 nM (for the intended 5 nM dose) and 70 nM resulted in a rapid, but transient upregulation of GR and
(for the intended 50 nM dose; Fig. 6). Our analysis TR mRNAs (Fig. 7).
showed that tadpole tissue hormone content reached
environmental concentrations within 4 h of immersion for
Changes in whole-body CORT content following exposure to
both doses (Fig. 6; compare line graphs with bars which
exogenous CORT
show the concentration of T3 in the water.) However, the
T3 tissue content continued to increase and reached a level Analysis of whole-body CORT content following
that was 4–6 times greater than the beginning environ- addition of CORT to a final concentration of 100 nM
mental concentration. The final whole-body T3 concen- (nominal concentration achieved 125 nM) or 500 nM
tration achieved was 24 nM for the low dose and (nominal concentration achieved 550 nM) to the
480 nM for the high dose. Thus, treatment of tadpoles aquarium water showed that while tissue CORT con-
with T3 by addition to the aquarium water results in tent increased rapidly, tissue content never reached the
pharmacological levels of the hormone in tadpole tissues. environmental concentration (Fig. 8). Furthermore,
whole-body CORT content declined at 24 and 48 h
following addition to the aquarium water. For the 100 nM
Effects of exogenous CORT on nuclear hormone receptor
dose, whole-body CORT content was significantly
mRNA levels elevated at 2 h after addition of hormone to the aquarium
In this experiment CORT was added to the rearing water water (P,0·001; ANOVA), continued to rise up to 8 h,
to a final concentration of 100 nM and tadpoles were but then declined at 24 and 48 h (but remained elevated
killed at different times thereafter up to 72 h. The water above the 0 time value). For the 500 nM dose, tissue
was not changed during the experiment (i.e. the CORT CORT content was significantly elevated at 2 h after
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100 L P KRAIN and R J DENVER · Hormone receptor gene expression during metamorphosis
Figure 7 Effects of in vivo CORT treatment on GR and TR mRNA
levels in premetamorphic tadpole intestine. CORT was added to
the aquarium water to a final concentration of 100 nM at time
zero and tadpoles harvested at various times for tissue isolation, Figure 8 Changes in whole-body CORT content following
RNA extraction and Northern blot analysis. TR and GR mRNAs exposure of premetamorphic tadpoles to two different doses of
were analyzed by Northern blotting as described in Materials and CORT in the aquarium water. The CORT was added to the
Methods. aquarium water at time=0 and tadpoles were collected at various
times and analyzed for whole-body CORT content by RIA
following organic extraction as described in Materials and
Methods. Each point represents the mean (n=8/time point) and
vertical bars the S.E.M.
addition of hormone to the aquarium water (P,0·0001),
but then gradually declined to 48 h (but remained elevated
above the 0 time value).
tions about tissue hormone content achieved by such
administrations.
Discussion
Developmental patterns of hormone production and nuclear
receptor expression
Corticosteroids have long been implicated in the positive
control of amphibian metamorphosis, but little is known Thyroid hormones and TR Our analyses of whole-
about their mechanism of action. This is the first study body T3 and T4 showed that both hormones exhibited the
to analyze the developmental expression and hormonal expected increases during prometamorphosis and meta-
regulation of GR mRNA, and to compare GR with TR morphic climax in X. laevis as has been described for
gene expression in X. laevis during metamorphosis. Our whole-body thyroid hormone content in other species
results show that the expression of GR mRNA increases in (Niinuma et al. 1991, Weber et al. 1994, Denver 1998a)
the brain and tail during metamorphosis. The strong and for plasma thyroid hormone concentrations in X. laevis
upregulation of GR in the tail leading up to metamorphic (Leloup & Buscaglia 1977, Tata et al. 1993) and other
climax may provide the molecular basis for corticosteroid anurans (Regard et al. 1978, Mondou & Kaltenbach 1979,
enhancement of T3-dependent tail regression (reviewed Weil 1986). We found that peak tissue hormone contents
by Kikuyama et al. 1993). This accumulation of GR in X. laevis were comparable with those reported for other
transcripts in tail may be mediated by rising plasma thyroid anurans (Niinuma et al. 1991, Weber et al. 1994, Denver
hormone concentrations, since we also found that exogen- 1998a). The whole-body T3/T4 ratio was highest at stage
ous T3 increased GR mRNA in this tissue. Our data 62, which is consistent with the upregulation of deiodinase
support the findings of others that show that there are type II (D2) at this stage of development in tadpoles
tissue-specific differences in the timing of the onset of (shown for D2 activity and mRNA in various tissues of
TR expression and its peak, which may underlie the tadpoles of Rana catesbeiana (Becker et al. 1997), also D2
asynchronous tissue morphogenesis that occurs during mRNA in X. laevis pituitary (R G Manzon & R J Denver,
spontaneous metamorphosis. Our studies also highlight unpublished observations)).
important methodological issues relating to the admin- It is worth noting that Leloup & Buscaglia (1977) did
istration of hormones in the aquarium water, and assump- not report a significant rise in plasma T4 or T3 until NF
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Hormone receptor gene expression during metamorphosis · L P KRAIN and R J DENVER 101
stages 59–60. Similarly, Tata (1993) was also unable to more sensitive to T3 than most other tadpole tissues (not
detect an increase in plasma T3 until these late prometa- including the hind limb, which may show the earliest
morphic stages. Findings in X. laevis are also consistent responses to T3; see Kawahara et al. 1991, Denver 1998b).
with measures of plasma T4 and T3 in the bullfrog, TR mRNA levels in the intestine and tail increased at
R. catesbeiana, where significant increases in plasma the time when T4 content was first elevated (stage 60) and
hormone concentration were not observed until late continued to increase through metamorphic climax.
prometamorphosis/early climax (Regard et al. 1978). Maximal TR mRNA level in the intestine (stage 62) was
Thus, the published plasma T3 and T4 concentrations in coincident with the peak in whole-body T3 content, while
tadpoles during metamorphosis are consistent with our maximal TR mRNA level in the tail (stage 64) occurred
measures of whole-body hormone content, which showed when T3 was beginning to decline.
significant increases only during late prometamorphosis.
However, failure to detect significant elevations in thyroid CORT and GR In X. laevis, CORT was found to be the
hormones in tadpo


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