Congenital Hypothyroid Pax8-/- Mutant Mice Can Be Rescued by Inactivating the TR
Gene
Frédéric Flamant1,
Anne-Lise Poguet1,
Michelina Plateroti,
Olivier Chassande,
Karine Gauthier,
Nathalie Streichenberger,
Ahmed Mansouri and
Jacques Samarut
Laboratoire de Biologie Moléculaire et Cellulaire de lEcole
Normale Supérieure de Lyon (F.F., A.-L.P., M.P., O.C.,
K.G., J.S.), Unité Mixte de Recherche Centre National de la
Recherche Scientifique 5665 LA INRA913, 69364 Lyon Cedex 07, France;
Laboratoire dHistologie et dEmbryologie Moléculaires
(N.S.),
Faculté de Médecine Laennec, Université Claude
Bernard Lyon I, 69372 Lyon Cedex 08; Max-Planck Institute for
Biophysical Chemistry (A.M.), Department of Molecular Cell Biology,
D-37077 Gottingen, Germany
Address all correspondence and requests for reprints to: Frédéric Flamant, Laboratoire de Biologie Moléculaire et Cellulaire de lEcole Normale Supérieure de Lyon, Unité Mixte de Recherche Centre National de la Recherche Scientifique 5665 LA INRA913, 46 Allée dItalie, 69364 Lyon Cedex 07, France. E-mail:
Frederic.Flamant{at}ens-lyon.fr
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ABSTRACT
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Mice devoid of all TRs are viable, whereas
Pax8-/- mice, which lack the
follicular cells producing T4 and T3 in
the thyroid gland, die during the first weeks of postnatal life. A
precise comparison between the two types of mutants reveals that their
phenotypes are similar, but the defects in spleen, bone, and small
intestine are more pronounced in
Pax8-/- mice. This is interpreted as
the result of a negative effect of the unliganded TR on thyroid hormone
target genes expression in the
Pax8-/- mutants.
Pax8/TR
compound mutants can survive to adulthood,
and the expression of target genes is partially restored. This
demonstrates the importance of TR
aporeceptor activity in
several aspects of postnatal development.
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INTRODUCTION
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THE TR GENES TR
and
TRß [NR1A1 and NR1A2 (Nuclear Receptors Nomenclature
Committee, 1999)] belong to the superfamily of nuclear hormone
receptor genes and encode several isoforms. Among these, TR
1,
TRß1, TRß2, and TRß3 (1) bind
T3 and activate transcription in the
nucleus. Like RAR, PPAR, VDR, farnesoid-X-activated receptor, liver X
receptor, and Nurr1, the TRs form heterodimers with RXR and bind
to DNA response elements located in transcription promoters (TRE).
T3 directly activates gene transcription by
binding to the C-terminal domain of TR, inducing a conformation change
and the recruitment of transcription coactivators (2, 3).
For a number of genes, T3 does not induce
activation but, rather, induces transcriptional repression
(4). Although the underlying mechanism is still unclear,
it is usually assumed that it results from the negative action of
liganded TR bound to specific TRE (5, 6).
The fact that DNA binding of TR is not hormone dependent also raises
the possibility for a biological activity of the unliganded receptors,
or aporeceptors (7). In vitro experiments show
that unliganded TR/RXR heterodimers bound to DNA recruit transcription
corepressors with histone deacetylase activity and exert a negative
effect on TRE (8). However, the physiological relevance of
this phenomenon has not been clearly evaluated. The recent availability
of several mutant mice provides new possibilities to address this
question in living animals.
Pax8 is a gene encoding a paired-box-containing protein with
a highly restricted expression pattern, which is necessary for the
differentiation of thyroid cells (9). The only known
primary defect in Pax8-/- knockout mice
is the absence of thyroid follicular cells (10). This
defect results in the almost complete absence of
T4 in postnatal life, when autonomous hormone
production normally replaces maternal supply.
Pax8-/- mice die around weaning time but
can be rescued by T4 treatment. Several
TR-knockout mice have also been produced (11, 12). Surprisingly, all single and compound TR-knockout mutants
are viable, with one exception (13, 14), which is
discussed elsewhere (15, 16). Thus, there is now ample
evidence that mice devoid of all TR are viable (17)
although very poorly fertile. Therefore, it seems that the absence of
TR is less deleterious than the deficiency in T4
and T3 observed in
Pax8-/- mice.
We combined the TR
0 mutation, which
is viable and deletes all the TR
isoforms
(15), and the lethal Pax8 mutation and found that animals
homozygous for both knockout mutations can survive to adulthood. The
lethality observed in Pax8-/- mice is
thus a consequence of the TR
1 aporeceptor activity.
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RESULTS
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Pax8-/- Mice Have Similar But
More Severe Defects Than
TR
0/0ß-/-
Mice
The postnatal consequences of the absence of
T4 and T3 synthesis in
Pax8-/--knockout mice have not been
studied before. However, the fact that these mice do not survive beyond
weaning time indicates that they are different from mice lacking TR
(15, 17). We thus decided to perform a systematic
comparison between the Pax8-/- mice and
TR
0/0ß-/-
mice devoid of all TR isoforms, obtained by intercrossing
TR
+/0TRß-/-
animals (15).
Statistical analysis reveals that
Pax8-/- mice are present in Mandelian
ratio at birth (9 of 35 newborns) but unlike
TR
0/0ß-/-
mice, they are under-represented in offspring 2 wk after birth (Table 1
). T4 is almost
undetectable in Pax8-/- blood at this
age, confirming that maternal supply is minimal after birth (data not
shown). Early growth appeared quite variable but clearly slower in
Pax8-/- mice than in
TR
0/0ß-/-
mice. We did not observe the spontaneous survival of
Pax8-/- mice beyond 30 d, but we
were able to rescue these mutants by injecting T4
daily between d 15 and 21, as reported previously (10).
This confirms that the lethality of
Pax8-/- mice before or during
weaning time is a direct consequence of hypothyroidism.
TSH activates T4 secretion and
T3 synthesis, which exerts in turn a negative
feedback regulation on pituitary TSH secretion. The number of
TSH-secreting cells in pituitary is highly elevated in both
TR
0/0ß-/-
and Pax8-/- mice (Fig. 1A
), and the circulating level of TSH is
always superior to 40 mU/liter (data not shown). TSH
gene
expression was also found to be similar in these two genotypes by
quantitative RT-PCR analysis (data not shown). Thus, although
T4 and T3 levels are very
low in Pax8-/- mice and very high in
TR
0/0ß-/-
mice, the two situations are functionally equivalent.
Anatomical and histological analyses were thus performed 1416 d
after birth, with a particular attention for the organs that have been
previously shown to be affected in various TR-knockout mice.
Spleen hematopoiesis is impaired in TR
-knockout mice,
mainly reflecting a defect in B cell development (18).
TR
0/0ß-/-
animals display a visible splenic hypotrophy. This feature is much more
pronounced in Pax8-/- mice (Fig. 1B
),
and histological examination confirms a dramatic reduction in the size
and number of follicles (data not shown). Bone morphology was studied
by x-ray, whole mount alizarine staining of skeleton, and on
histological sections (Fig. 1
, C and D). Both types of mutant
display the defects reported previously for 3- to 6-wk-old
TR-knockout mice (13, 17), i.e.
retarded ossification and mineralization of long bones, which is
clearly visible in epiphysis. These defects are likely to result from a
defect in growth plate cell differentiation (19). Again,
this phenotype was always more pronounced in
Pax8-/- mice than in
TR
0/0ß-/-
mice.
Small intestine epithelium is another important target for
T3 during the profound remodeling occurring at
weaning time (20). Epithelial cell proliferation and
differentiation in the distal small intestine, assessed by
immunostaining of the Ki67 proliferation marker and by measuring
lactase activity, respectively, were more
severely reduced in
Pax8-/- than in
TR
0/0ß-/-
mutants (Fig. 2
and Table 2
). Cells were
also much more vacuolized in Pax8-/-
epithelium. When T4 and T3
were injected to Pax8-/- mice, a
recovery of crypt cell proliferation was observed after 48 h.
Morphological examination showed that the highly vacuolized epithelial
cells, typical of immature stages (21), were replaced by
apparently normal cells in the proximal portion of the villi (Fig. 2
, lower panel). The only modest increase in epithelial lactase
activity probably reflects the fact that the complete renewal of
intestinal epithelium would take more than 48 h of hormonal
treatment.

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Figure 2. Comparative Analysis of Small Intestine Distal
Ileum of Pax8-/- and
TR 0/0ß-/-
Mice
Mice were analyzed at d 15. A, Haematoxylin-eosin staining. Villi
epithelial cells are highly vacuolized in
Pax8-/- animals. This feature
disappears in the proximal portion of the villi after 48 h of
thyroid hormones treatment. B, Ki67 immunostaining of proliferating
cells. Average number of Ki67+ per crypt is indicated.
Bars, 100 µm.
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The overall conclusion of the above observations is that
Pax8-/- and
TR
0/0ß-/-
mutant mice display similar phenotypic traits, but that these defects
are always more pronounced in the
Pax8-/- animals.
Survival Without Thyroid Gland and TR
At this point, three main hypotheses could account for our
observations: 1) A hypothetical third TR gene would encode a
receptor, which would transduce T3 signal in some
TR
0/0ß-/-
tissues and introduce a partial functional compensation. It has been
shown that knocking out both the TR
and TRß
genes was sufficient to abrogate any high-affinity
T3 binding in adult liver (17).
Furthermore, there is at this time no indication for the presence of an
uncharacterized TR-related sequence in the GenBank database
(Robinson-Richevi, M., and V. Laudet, unpublished
observations). The existence of a third unknown TR
gene, expressed in other tissues, is therefore highly unlikely. 2)
Nongenomic effects of thyroid hormones might persist in
TR
0/0ß-/-
but not in Pax8-/- animals to ensure
survival. The physiological relevance of such effects are controversial
and the underlying mechanism is poorly understood, but
T4 is able to act directly on cell membranes and
cytoplasmic components to activate an alternative signaling pathway
(22, 23, 24). 3) TR aporeceptors present in the
Pax8-/- mice may have a
detrimental physiological activity. According to in
vitro data, TR/RXR heterodimers and TR/TR homodimers
(25) act as transcription repressors in the absence of
T3. Thus, TR target genes expression would be
more sensitive by the absence of T3 than by the
absence of receptors.
This third hypothesis leads to a testable prediction: compound mutant
mice, which would lack both the thyroid hormones and the receptors,
should be viable with a phenotype similar to the one observed in
TR
0/0ß-/-
mice. To test this prediction, we crossed the TR and
Pax8 mutants to generate
Pax8-/-TR
0/0
and
Pax8-/-TRß-/-
mice (Table 1
).
The
Pax8-/-TRß-/-
combination was lethal before weaning (Table 1
and Fig. 3
). Anatomical and histological
examinations failed to reveal a significant difference between these
mice and Pax8-/- mice, except in one
case (not shown), in which femural ossification was slightly more
advanced in the compound mutant. In particular, the distal small
intestine, bones, and spleen were always deeply affected (Table 2
and
Fig. 4
). This feature is not surprising,
considering that previous knockout analysis failed to identify any
function of TRß in these organs (14, 26).
Histological analysis of pituitary revealed the presence of a high
number of TSH-secreting cells, similar to the one already observed for
in Pax8-/- and
TR
0/0ß-/-
(data not shown). This observation argues against any major influence
of the TRß aporeceptor on TSH
and
TSHß genes expression.

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Figure 3. Early Growth Curve of Compound and Single
Mutants
One representative male is presented for each genotype.
Pax8-/- and
Pax8-/-TRß-/-
mice died ( ) before weaning. Statistical variations are omitted for
clarity, but variability at d 15 is reported in Table 1 .
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Figure 4. Histological Analysis of
Pax8-/-/TR Compound
Mutants
Mice were analyzed at d 15. A, Spleen; B, forelimb and hindlimb whole
mount alizarine staining. Secondary ossification of epiphysis is
visible only for
Pax8-/-TR 0/0
mice mainly in femur and tibia. C, Distal ileum morphology. Vacuoles
similar to the one observed in
Pax8-/- epithelium are found only in
Pax8-/-TRß-/-
mice.
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The offspring of
Pax8+/-TR
+/0
and Pax8+/-TR
0/0 mice contained both
Pax8-/-TR
+/0
and
Pax8-/-TR
0/0
pups. The majority of
Pax8-/-TR
0/0
mice survived to adulthood (Table 1
), although growth was delayed
compared with mice lacking only TR (Fig. 3
). Anatomical and
histological examinations confirmed a partial to complete
phenotypic recovery in bone, spleen, and small intestine (Table 2
and
Fig. 4
). Surprisingly, some of the
Pax8-/-TR
+/0
mice (three of eight) also spontaneously survived to adulthood,
indicating that the absence of a single TR
gene copy limits the
aporeceptor detrimental effect.
Attempts to obtain triple
Pax8-/-TR
0/0TRß-/-
mutant mice were hampered by the very low fertility of double mutants.
Only one
Pax8-/-TR
0/0TRß-/-
male was obtained, which survived to adulthood and was not studied
further.
From these observations, we can conclude that the lethality observed in
Pax8-/- mutant mice is due to the
negative effect of the TR
1 aporeceptor.
Direct in Vivo Evidence for TR Aporeceptors-Mediated
Gene Regulation
T3 target genes in the small intestines,
bone, and spleen are not known. Establishing a direct link between the
presence of TR aporeceptors and the repression of
T3 target genes expression in these tissues is
therefore problematic. By contrast, although knockout of TR
genes does not induce any obvious histological defect in liver and
heart, their function is known to be regulated by
T3. Several T3 target genes
expressed in these two organs are now well characterized, and we
decided to measure the expression of two of these. Liver cells express
both TR
and TRß genes, but TRß
mRNA is more abundant than TR
mRNA. The opposite
situation is found in the heart. This is consistent with the results of
knockout mice analysis, which revealed that TRß has a
predominant function in liver, whereas TR
is the main
regulator of heart rate (27, 28, 29, 30).
Among the best characterized T3 targets in liver
is the gene encoding type I deiodinase gene (D1),
which is very tightly regulated (31, 32) and which, in
humans, contains several TRE in its promoter (33). As
predicted, expression of D1 is greatly reduced in
TR
0/0ß-/-
mice, undetectable in Pax8-/-
mice, and highly induced after
T4/T3 treatment (Fig. 5A
). The persistence of D1
expression in
TR
0/0ß-/-
livers is a good indication that unliganded TR can repress
D1 transcription by binding to positive TRE in
Pax8-/- livers. The fact that
D1 expression is not detectable in either
Pax8-/-TRß-/-
mice or
Pax8-/-TR
0/0
mice is also an indication that both TR
and TRß aporeceptors can
contribute to this repression.

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Figure 5. Type I Deiodinase and HCN2
Expression in Compound Mutants
A, Liver RNA from 15-d-old mice was analyzed by Northern blotting.
Equal loading was confirmed by using a probe for the hypoxyanthine
phosphoribosyl transferase mRNA and ethidium bromide (Eth.Br.) staining
of the gel. B, Heart RNA was analyzed by RNase protection with a
mixture of probes for the HCN2 (lower
band) and hypoxyanthine phosphoribosyl transferase (HPRT,
upper band) mRNA. The
Pax8-/-TRß-/-
is from a different experiment. C, Quantification of
HNC2 mRNA level in three independent samples for each
genotype. Ratios compared to wild-type average level, are
represented.
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HCN2 is a gene encoding one of the pacemaker current
components, which has been recently identified as a
T3 target in cardiac cells, although the binding
of TR to the promoter has not been demonstrated
(34). HCN2 expression followed the expected
expression pattern in heart with a mRNA level that is very low in
Pax8-/-, higher in
TR
0/0ß-/-
hearts, and inducible by
T4/T3 treatment.
HCN2 expression was clearly restored in
Pax8-/-TR
0/0
mice but not in
Pax8-/-TRß-/-
mice. Variations within mice sharing the same phenotype were very
limited (Fig. 5C
). These data are consistent with a repression of
HCN2 transcription by the TR
1, but not the
TRß, aporeceptor.
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DISCUSSION
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The purpose of this work was to address the possible biological
activity of unliganded TR in vivo. A key observation is that
mice lacking a thyroid gland can survive only if they do not express
the TR
gene. Thus, the TR
1 aporeceptor is
able to compromise postnatal survival.
Congenital Hypothyroidism Is Lethal in Mice
Severe congenital hypothyroidism, as observed in
Pax8-/- mice, is lethal at weaning time.
T4 treatment during the second week of postnatal
life is only sufficient to rescue
Pax8-/- mice, showing that
T4 and T3 are dispensable
for survival after weaning. A similar lethality has been observed in
other transgenic mice expressing a mutated cAMP response element
binding protein gene in thyroid follicular cells in which
T4 production is abrogated by a less direct
mechanism (35). By contrast, drug treatment
(36) and TSH receptor mutations (37, 38)
induce a hypothyroid status that is not lethal. These discrepancies are
likely to result from a moderate reduction of T3
levels in these last cases. The exact causes for the lethality linked
to the Pax8-knockout remain hypothetical. In agreement with
previous observations that showed that only TR
is
important in this process (16), we found that the delay in
small intestine development is much more pronounced in the lethal
Pax8-/- and
Pax8-/-TRß-/-
mutants than in all other genotypes. This delay is certainly
sufficient to explain why Pax8-/- and
Pax8-/-TRß-/-
mice do not survive beyond the weaning period, which normally occurs
during the third postnatal week. These mutants are, however, already
under-represented in offspring and underweight at d 1015.
T3 may therefore fulfill other unidentified vital
functions during the first days of postnatal life in bone, spleen,
heart, or other organs that were not studied here. Finally, because
Pax8-/- embryos receive hormones through
the placenta of their Pax8+/- mothers, we
can not rule out another vital function for T3
during embryonic and fetal life.
TR Mutations Recapitulate Hypothyroidism
The resemblance between Pax8-/--
and
TR
0/0ß-/--knockout
mice suggests that the defects observed in bone, small intestine
epithelium, and spleen directly reflect congenital hypothyroidism. This
confirms that none of the phenotypic traits that we have observed are
side effects of the Pax8 mutation per se.
Meanwhile, it argues against a possible involvement of the nonreceptor
isoforms encoded by the TR genes in the determination of
these phenotypic traits (39, 40).
TR
1 and TRß Aporeceptor Activity
The new observations presented here establish the first in
vivo evidence that the repressor activity of the TR
1
aporeceptor is of physiological relevance. We were able to directly
verify this assumption only for the HCN2 gene
expression in heart. Unfortunately, the TRE responsible for the
activation of this gene by T3 are presently
unknown and other known T3 target genes usually
do not display the same high induction rate. We were thus unable to
address the possibility that T3 target genes may
differ in their sensitivity to TR
1 aporeceptor. Based on
histological and functional analysis, we can, however, predict that
genes activated by T3 are actively repressed in
Pax8-/- bone, spleen, and intestinal
epithelium and that this repression is abrogated in
Pax8-/-TR
0/0
mice. The mutant collection that we produced may then serve to identify
some of these genes.
The large similarities observed between the phenotypes of
Pax8-/- and
Pax8-/-TRß-/-
mice does not provide any firm indication for TRß
aporeceptors activity. However, the persistence of
D1 expression in
TR
0/0ß-/-
but not Pax8-/- mice suggests that this
gene is sensitive to both TR
1 and TRß aporeceptors. D1
expression has recently been reported to be absent in mice devoid of
TR, but this discrepancy might result from differences in the detection
sensitivity achieved by Northern blotting (41). According
to published data (29), other T3
target genes in the liver do not follow the same expression pattern,
and T3 regulation in this organ seems to be
very complex (4). The expression of D1 in
TR
0/0ß-/-
liver could thus be interpreted in several different manners. According
to recent data, TRß aporeceptors exert a physiological
function mainly in brain (42). This conclusion was reached
after the introduction of a point mutation in the activation
function-2 domain of TRß performed to mimic the
human syndrome of resistance to thyroid hormone in mice. Even mice
heterozygous for this mutation display cerebellar and hippocampal
abnormalities presumably absent in knockout mice and therefore
attributed to TRß aporeceptors activity. However, this
interpretation remains questionable, as a slightly different mutation
provides different results (43). Therefore, the
possibility remains than TR
and TRß
aporeceptors activities are intrinsically different
(44).
Possible Physiological Implication of Aporeceptor-Mediated Gene
Regulation
The detrimental effects of TR aporeceptors on mouse postnatal
development mimics human pathological situations such as thyroid
disgenesis (45), agenesis (46), and perhaps
resistance to thyroid hormone (47). A remaining issue is
whether the TR aporeceptors also regulate transcription in a normal
situation. There is no firm indication that T3
can be completely absent in any tissue expressing TR
or
TRß in healthy animals, but this question should be
examined more precisely in the future. For example,
T3 is not transported in brain but is produced
locally by T4 deiodination. Deiodination in brain
is performed by type 2 deiodinase, an enzyme with a restricted
distribution (48) and under tight regulation (49, 50). T3 could therefore be absent in some
brain areas during the fetal or postnatal life.
T3 may also be unable to access to some embryonic
tissues at a time when embryos already express TR
.
Do Other Aporeceptors Regulate Gene Transcription in
Vivo?
The TR/T3 pathway offers a favorable
opportunity to verify the physiological relevance of aporeceptors
activity by genetic means. First, there is a well-defined source of
ligand in the organism, and the Pax8 mutation provides an
extremely precise way to perform a genetic ablation. Second, all the
receptors isoforms are encoded by only two genes. It is likely,
however, that aporeceptor function also exists for other related
receptors. For example, it might explain why VDR knockout is
not equivalent to vitamin D deficiency (51) or why
retinaldehyde dehydrogenase type 2 knockout, which abrogates RAR
ligand synthesis, entails early embryonic lethality (52),
whereas RAR compound mutants usually survive beyond this
stage (53). The recent knockout of nuclear
corepressor 1 also suggests that many nuclear aporeceptors have
important developmental functions (54), although this
corepressor interacts at least in vitro with a broad range
of transcription factors.
In conclusion, this work demonstrates that TR
1 aporeceptor have
physiological effects in vivo and that
T3 is required to relieve these effects during
the first weeks of postnatal development.
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MATERIALS AND METHODS
|
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Mice
All pups were kept with their mother for at least 5 wk to ensure
natural weaning. Genotypes were determined on toe lysates at d 1015
by PCR DNA analysis. The TR
0 allele
(15) is a complete deletion of all the known
TR
isoforms. It differs from the
TR
- allele used in previous studies
(13) by the fact that it is viable and fertile.
TR
0/0 animals were analyzed with a
mixture of 4 oligonucleotides (5'-ATCGCCTTCTATCGCCTTCTTGACG,
5'-TTCAGGAGGATGATCTGGTCTTCGCAAG, 5'-GAGGAGGCGAAAGGAGGAG, and
5'-TGCCCTGGGCGTTAGTGCTG) and the following temperature schedule:
95 C for 5 min (94 C for 20 sec, 60 C for 20 sec, 72 C for 60 sec) 5
times; (94 C for 20 sec, 56 C for 20 sec, 72 C for 60 sec) 27
times to amplify both the wild type (115 bp) and the mutant (660
bp) allele. The PCR protocol to detect the TRß mutation
was reported previously (14). The Pax8-knockout
mutation was described before (10). The genotype at this
locus was determined using 3 oligonucleotides
(5'-GGATGTGGAATGTGTGCGAGG, 5'-GCTAAGAGAAGGTGGATGAGAG, and
5'-GATGCTGCCAGTCTCGTAG) and the following temperature schedule: 94 C
for 5 min (94 C for 15 sec, 60 C for 15 sec, 72 C for 30 sec, 10 times;
94 C for 15 sec, 57 C for 15 sec, 72 C for 30 sec, 25 times), 72 C for
5 min. This amplifies a 390-bp fragment for the wild-type allele and a
370-bp fragment for the knockout allele. All genetic background were
initially a combination of C57/BL and 129Sv later backcrossed to 129Sv.
Wild-type controls were littermates of Pax8 mutants. Experiments were
all performed with young animals kept with their mother. When
indicated, thyroid hormones [1 µg T3 and 10
µg T4 (Sigma, St. Louis, MO) in
100 µl PBS] were injected ip at d 13 and 14, and animals were
sacrificed at d 15.
Histology and Enzymatic Activities
Standard histological techniques were used (55).
Distal small intestine (56) and bone (13)
were analyzed as described. Values of SD were calculated
from at least 3 animals. For TSH immunocytochemistry, pituitary glands
were fixed in Bouin-Hollande-Sublimate for 4 d and then embedded
in parrafin. Serial sections were treated by the indirect
immunoperoxidase method with streptavidin-biotin-complex (DAKO Corp. A/S, Copenhagen, Denmark) using a 1/20000 dilution of an
anti-ßrTSH antibody (a kind gift from Dr. A. F. Parlow, NIDDK)
as described previously (57).
RNA Analysis
RNA were purified using column purification
(QIAGEN, Valnecia, CA). Liver RNA was analyzed by Northern
blotting using Hybond N+ membrane for transfer
(Amersham Pharmacia Biotech, Piscataway, NJ) and an
antisense RNA probe synthesized from a cloned D1 cDNA
with 32P-labeled UTP (Amersham Pharmacia Biotech) for hybridization. HCN2 mRNA in heart was
measured using a RNase protection assay (Ambion, Inc.,
Austin, TX) including a hypoxanthine phosphoribosyltransferase
antisense probe for calibration. Quantitation was performed using a
PhosphorImager and the ImageQuant software (Molecular Dynamics, Inc., Sunnyvale, CA).
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ACKNOWLEDGMENTS
|
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We thank Jean-Paul Roux for bone sections; Samuel Refetoff for
helpful discussion; Bernd Gloss for the HNC2 probe gift; and
Nadine Aguilera, Djamel Belgarbi, and Christelle Morin for animal
breeding.
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FOOTNOTES
|
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This work was supported by the Max Planck Society, the Association pour
la Recherche contre le Cancer, the Comité Départemental de
la Loire de la Ligue Nationale contre le Cancer, and the Human Frontier
scientific Program (RGO347/1999.M).
1 These authors contributed equally to this work. 
Abbreviations: D1, Type I deiodinase gene; TRE,
transcription promoter.
Received for publication April 25, 2001.
Accepted for publication September 27, 2001.
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