Stage-Sensitive Blockade of Pituitary Somatomammotrope Development by Targeted Expression of a Dominant Negative Epidermal Growth Factor Receptor in Transgenic Mice
Meejeon Roh,
Andrew J. Paterson,
Sylvia L. Asa,
Edward Chin and
Jeffrey E. Kudlow
Departments of Medicine/Endocrinology (A.J.P., E.C., J.E.K.) and
Cell Biology (M.R., J.E.K.) University of Alabama at Birmingham
Birmingham, Alabama 35294
Department of Pathology (S.A.)
University of Toronto Toronto, Ontario, Canada M5G 2M9
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ABSTRACT
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The epidermal growth factor receptor (EGFR) and
its ligands EGF and transforming growth factor-
(TGF
) are
expressed in the anterior pituitary, and overexpression of TGF
in
the lactotrope cells of the pituitary gland in transgenic mice results
in lactotrope hyperplasia and adenomata, suggesting a role for EGFR
signaling in pituitary cell proliferation. To address the role of EGFR
signaling in pituitary development in vivo, we blocked EGFR
signaling in transgenic mice using the dominant negative properties of
a mutant EGFR lacking an intracellular protein kinase domain (EGFR-tr).
We directed EGFR-tr expression to GH- and PRL- producing cells
using GH and PRL promoters, and a tetracycline-inducible gene
expression system, to allow temporal control of gene expression.
EGFR-tr overexpression in GH-producing cells during embryogenesis
resulted in dwarf mice with pituitary hypoplasia. Both somatotrope and
lactotrope development were blocked. However, when EGFR-tr
overexpression was delayed to the postnatal period either by directing
its expression with the PRL promoter or by delaying the onset of
induction with tetracycline in the GH cells, no specific phenotype was
observed. Lactotrope hyperplasia during pregnancy also occurred
normally in the PRL-EGFR-tr mice. These data suggest that EGFR
signaling is required for the differentiation and/or maintenance of
somatomammotropes early in pituitary organogenesis but not later in
life. (Molecular Endocrinology 15: 600613, 2001)
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INTRODUCTION
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The pituitary gland contains a mixed population of cells the
function of which is to secrete the six pituitary hormones in the
quantities and timing appropriate to maintain homeostasis for the
entire organism. The initial stages of development of the anterior
pituitary from Rathkes pouch to the fully differentiated gland have
been well characterized. The control of the spatial and temporal
emergence of these cells during embryogenesis appears to result from
counter-directional gradients in growth factors related to transforming
growth factor-ß (TGFß) [bone morphogenetic protein 2
(BMP2), ventral, and fibroblast growth factor 8 (FGF8)
(dorsal)], respectively (1, 2, 3). These growth factors, depending on the
ratio of concentration at a particular cell, differentially induce the
expression of the particular transcription factors that in turn control
the fate of the cell. Transcription factors that are expressed in
pituitary primordium include Six3 (4), Rpx/Hesx1 (5, 6), and PAX-6 (7).
Subsequently, concurrent with organ commitment, a Lim-homeodomain
factor, Lhx-3/P-Lim/mLim-3 (8, 9), and OTX-related factor Pitx1/P-OTX
(10, 11) are expressed throughout early pituitary development and in
the mature gland. The further specification of pituitary cell lineage
can be monitored by a tissue-specific POU domain factor Pit-1 and
orphan nuclear receptor SF-1 (for review see Refs. 12, 13, 14, 15). These
transcription factors provide the molecular memory in response to
transient signaling gradients by reciprocal interactions based on both
DNA binding-dependent and -independent actions and thus, the cells
commit to the lifelong expression of their specific pituitary hormone
(16).
Once the populations of cells have been established, the situation is
not static. Although the life span of pituitary cells is not known,
there is evidence that a constant turnover of these cells occurs (17),
with both a disposal and replacement mechanism present. This turnover
allows the selective expansion of pituitary cell subpopulations that
have been observed under various pathological and physiological
conditions (18, 19, 20). This selective growth of pituitary subpopulations
is an example of postnatal development that occurs adaptively in
response to environmental signals. We have previously proposed that the
epidermal growth factor receptor (EGFR) system plays a role in
pituitary adaptation. This receptor and its ligands, epidermal growth
factor (EGF) and transforming growth factor-
(TGF
), are expressed
in multiple cell types in the pituitary (21, 22, 23, 24), and TGF
, EGF,
EGFR, and erbB2/neu have been also shown to be expressed in human
pituitary adenomas (25, 26, 27, 28, 29, 30). EGF and TGF
can modulate hormone
production; EGF can stimulate the secretion of ACTH, GH, LH, and TSH
(31, 32, 33, 34, 35), and TGF
can stimulate LHRH (36). Conversely, hormones can
modulate the EGFR system; GH can enhance EGFR concentration in mice
(37, 38), and TGF
expression is up-regulated by estrogen treatment
in the uterus, mammary gland, and pituitary (38, 39). In MCF7 mammary
cancer cells, estrogen-stimulated growth can be blocked by anti-EGFR
and anti-TGF
antibodies (39), and the pubertal development of the
mammary ductal system requires EGFR signaling (40). In the pituitary,
the up-regulation of TGF
expression precedes the lactotrope
hyperplasia that is induced by estrogen in rats (38). Furthermore,
overexpression of TGF
in the lactotropes of transgenic mice results
in lactotrope hyperplasia and lactotrope adenomas, implying that the
signaling system downstream of TGF
is intact in lactotropes. These
studies suggest that the EGFR system plays a role in pituitary hormone
secretion and proliferation.
The pituitary lactotropes and somatotropes arise from a common
precursor (41), and both cell types have been shown to express TGF
and the EGFR in the mature pituitary gland (22, 23). To determine the
role of the EGFR system in the development of these cells, we made use
of a dominant negative form of the EGFR. This truncated receptor,
EGFR-tr, when expressed in molar excess to the wild-type receptor,
blocks EGF-dependent signaling in cultured cells (40). When the same
EGFR-tr construct was expressed in the mammary glands (40) or a similar
construct in the epidermis of transgenic mice (42), mammary duct or
epidermal development was markedly impaired, implying a role for EGFR
signaling in the development of these tissues. To determine the role of
the EGFR in somatotropes and lactotropes during and after development,
we expressed, at high levels, EGFR-tr in these respective cells of
transgenic mice. For lactotrope expression, the EGFR-tr was expressed
both under direct control of the PRL promoter and with the use of the
tetracycline-inducible system. For somatotrope expression, the GH
promoter with the use of the tetracycline-inducible system was used
(43, 44, 45). The tetracycline system was used to allow amplification of
gene expression to assure a large molar excess of EGFR-tr expression
over endogenous EGFR expression in this dominant negative strategy. In
this paper we show that the EGFR-tr was indeed highly expressed in the
respective pituitary cells. However, the phenotype differed greatly in
the two models. Whereas EGFR-tr expression in somatotropes blocked the
development of these cells, resulting in a dwarf phenotype, the
expression of this same construct in lactotropes resulted in no
discernable phenotype. These results raise interesting questions
concerning the cell- and stage-specific role of the EGFR in signal
transduction in the pituitary and perhaps elsewhere.
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RESULTS
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Pituitary-Specific Transgenic Models
Four lineages of transgenic mice were generated for this study and
the schematic diagrams are shown in Fig. 1
, AC. In one lineage, the PRL-EGFR-tr
transgene was designed to directly drive expression of the dominant
negative (40), cytoplasmic kinase domain-deleted EGFR in the
PRL-producing pituitary cells of female transgenic mice. Another
lineage contained a transgene consisting of the PRL promoter driving
expression of the tetracycline-controlled transactivator, tTA, in
lactotropes (PRL-tTA). In the absence of doxycycline, the tTA activator
protein constitutively drives high-level expression from promoters
containing the tetracycline-response element (TetRE), while
tetracycline turns transcription off, hence the "tet-off"
nomenclature. The tet-off system has been used in numerous transgenic
models (46, 47, 48, 49, 50). To express the dominant negative EGFR-tr in the mice
under tetracycline control, lines were developed with the TetRE-EGFR-tr
transgene. Finally, to drive expression of transgenes in the
somatotropes, a GH-rtTA lineage was developed using the 1.7-kb segment
of the rat GH promoter, which had been shown previously to drive high
level expression in the pituitary gland (51). The rtTA gene encodes the
reverse tetracycline activator. This activator requires the presence of
tetracycline to activate promoters containing the TetRE. We have
recently shown the utility of this tet-on system using a skin-specific
promoter (45). In each case, the B6x SJL mouse strain was used as the
genetic background for the development of the transgenic lines.

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Figure 1. Schematic Representation of the PRL-EGFR-tr
Transgene Construct and the Tet-Off and Tet-On Transgenic Mouse System
A, 3.0-kb PRL promoter, 2.23-kb cytoplasmic-deleted EGFR (EGFR-tr), and
0.8-kb SV40 polyadenylation signal are indicated. B and C, Schematic
outlines of the Prl-tTA/TetRE-EGFR-tr Tet-Off and GH-rtTA/TetRE-EGFR-tr
Tet-On systems. The Prl-tTA or GH-rtTA transgenes direct expression of
tTA or rtTA to the PRL- or GH-producing cells in the pituitary gland.
The binding of tTA or rtTA to TetRE and the induction of the EGFR-tr
transgene can occur only in the absence (Tet-Off) or presence (Tet-On)
of doxycycline (Dox).
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Transgene-positive founder mice were identified by PCR using primers
that span between the promoters and the coding sequence. The number of
founder mice for each of the transgenes was as follows:
PRL-EGFR-tr, 5; PRL-tTA, 8, GH-rtTA, 8; and TetRE-EGFR-tr, 5. The
integration of the transgene into chromosomal DNA and the relative gene
dose was confirmed by Southern blot and slot blot analysis,
respectively, using a transgene-specific probe derived from the tTA,
EGFR, or SV40 sequence (Fig. 2
, AD).
All transgenes were integrated intact into host chromosomal DNA as
evidenced by the single hybridizing band having a size that corresponds
to the respective transgene. In each case, the founders with the
highest or second highest copy number were used for subsequent
studies.

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Figure 2. Southern Blot Analysis of Prl-EGFR-tr, Prl-tTA,
GH-rtTA, and the TetRE-EGFR-tr Transgenic Mouse Genomic DNA
Genomic DNA, extracted from the liver of offspring of each transgenic
founder mouse [81, 39 lines for Prl-EGFR-tr (A), 65 for Prl-tTA
(B), 36 and 43 for GH-rtTA (C), and 29 for TetRE-EGFR-tr (D), or
their wild-type littermates (WT)] was digested with a unique
restriction enzyme within the transgene and subjected to Southern blot
analysis. Fifty picograms of each transgene (TG) fragment excised from
the original transgene-containing plasmids were loaded as a positive
control. The membranes were probed with 32P-labeled EGFR (A
and D), tTA (B), and SV 40 (C), respectively. Lines 24 and 35 in
panel A are nontransgenic mouse lines.
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EGFR-tr Expression in Pituitary Directed by the PRL and GH
Promoters
For lactotrope-specific expression, either the constitutive 39
or 81 PRL-EGFR-tr lines, or the 65 PRL-tTA and 29 TetRE -EGFR-tr
mouse lines, were used. These lines, with high gene dose, were
characterized for gene expression and phenotyping for most of our
studies. PRL-tTA and TetRE-EGFR-tr were used in cross-breeding
experiments to generate bitransgenic animals. To confirm the EGFR-tr
expression in the pituitary gland, F1 generation
mice from either direct PRL-EGFR-tr or bitransgenic mice were killed
for Northern blot analysis. EGFR-tr was overexpressed in the
PRL-EGFR-tr female mice (Fig. 3C
) and in
bitransgenic female mice having both the PRL-tTA and TetRE-EGFR-tr
transgenes but not in wild-type or single positive transgenic
littermates (Fig. 3A
). The expression of the EGFR-tr was much greater
than expression of the endogenous wild-type EGFR as indicated by the
observation that the transgene transcript was easily detectable while
the transcript from the endogenous gene was either undetectable or
barely detectable at the exposure used for these Northern blots. Since
the PRL model uses the tet-off system, EGFR-tr was constitutively
expressed unless doxycycyline was provided to the mice in the drinking
water (Fig. 3D
, lane 1). When doxycycline was given for 7 days to turn
off the transgene expression, the expression of EGFR-tr became
undetectable (Fig. 3D
, lane 2). However, in those PRL-tTA mice given
doxycycline for 7 days, the cessation of doxycycline for as much as 2
weeks was insufficient to induce expression of the TetRE-EGFR-tr
transgene in bitransgenic mice (Fig. 3D
, lanes 35). This result
indicates that the dose of doxycycline given for 1 week was sufficient
to allow the development of a tissue reservoir of the antibiotic
sufficient to sustain suppression of transgene expression even after 14
days of withdrawal in this tet-off model. This observation and our
excellent results with inducibility using the tet-on system in the K14
skin model (45) prompted us to use the tet-on system for somatotrope
transgene expression. Mice, bitransgenic with the GH-rtTA and TetRE
-EGFR-tr transgenes, also showed EGFR-tr overexpression in the
pituitary gland. To demonstrate that this expression was conditional,
these bitransgenic mice were raised in the absence of doxycycline until
the age of 4 weeks, at which time doxycycline was given for various
times up to 4 weeks. Pituitary gland total RNA was isolated and
analyzed by Northern blotting. The membrane was first probed with
32P-labeled rtTA cDNA and then was stripped and
rehybridized with the EGFR-tr cDNA. Northern blotting showed that
EGFR-tr was expressed only in bitransgenic mice, both in female (Fig. 3B
, lane 4) and male (data not shown), but not in wild-type or single
positive transgenic mice (Fig. 3B
, lanes 13, 5, and 6). At the
exposure used, the endogenous EGFR mRNA, with a predicted size of
4.74.9 kb in mice, was not detectable, indicating the EGFR-tr
transgene transcript was expressed at a much higher level under GH-
rtTA control than the endogenous EGFR. The expression of EGFR-tr
transgene in the pituitary was inducible with doxycyline. Two-month-old
bitransgenic mice having both GH-rtTA and TetRE-EGFR-tr transgenes were
treated without doxycycline (lane 1) or with the antibiotic for 1 day
(lane 2) or 3 days (lane 4). Transgene expression was observed
only when doxycyline was given to the bitransgenic mice (lanes 2 and 4)
for as little as 1 day. No expression was observed in single positive
mice (lane 3). The induction was reversible when the animals were
treated for 1 day with doxycycline followed by 3 days withdrawal of
treatment (Fig. 3E
, lane 5).

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Figure 3. Northern Blot Analysis of the Transgene Expression
The pituitary gland from each transgenic mouse line was removed and
processed for total RNA isolation. The RNA was separated on 1%
formaldehyde-agarose gel, transferred onto the Hybond-N+
membrane, and hybridized with 32P-labeled cDNA of tTA or
rtTA for the detection of tTA or rtTA expression-positive mice (A and
B, upper panel). The membrane was subsequently stripped
and rehybridized with the mouse EGFR-tr cDNA for the detection of
TetRE-EGFR-tr expression-positive mice (A and B, middle
panel). The expression of EGFR-tr transgene was observed only
in bitransgenic mice (panel A, lane 6, and panel B, lane 4). A,
Northern blot of Prl-tTA/TetRE-EGFR-tr transgenic mouse. Lane 1, male;
lanes 26, female mice from the 65 transgenic mouse line. Lanes
24, single positive for Prl-tTA; lane 5, single positive for
TetRE-EGFR-tr; lanes 1 and 6, double positive for both Prl-tTA and
TetRE-EGFR-tr, respectively. About 2-month-old mice were used, no Dox
treatment at any time. Notice that the transgene was expressed
only in bitransgenic female mice. B, Northern blot of
GH-rtTA/TetRE-EGFR-tr transgenic mouse. Lanes 14, female; lanes 5 and
6, male mice from the 36 transgenic mouse line. Lane 1, wild-type;
lanes 2 and 5, single positive for GH-rtTA; lanes 3 and 6, single
positive for TetRE-EGFR-tr; lane 4, double positive for both GH-rtTA
and TetRE-EGFR-tr, respectively. One-month-old mice were used, and Dox
was given for 1 week to induce transgene expression. The rtTA
transcripts were detected in the pituitary of all GH-rtTA-bearing
animals (lanes 2, 4, and 5), while the expression of EGFR-tr transgene
was detected only in the bitransgenic mice (lane 4). C, Northern blot
of Prl-EGFR-tr transgenic mice. Lane 1, wild-type; lane 2, 39 line;
lane 3, 81 line, respectively. Two-month-old female mice never
exposed to Dox were used. D, Induction of EGFR-tr expression by Dox
withdrawal in Prl-tTA/TetRE-EGFR-tr mice. Lane 1, no Dox at any time;
lane 2, Dox for 1 week; lanes 3 and 4, Dox for 1 week, then Dox
withdrawal for 1 week; lane 5, Dox for 1 week, then Dox withdrawal for 2 weeks. All are
2-month-old bitransgenic female mice. E, Induction of EGFR-tr
expression by Dox administration and the reversibility of transgene
expression in GH-rtTA/TetRE-EGFR-tr transgenic mouse. The expression of
EGFR-tr transgene was only observed when Dox was given in the
bitransgenic mouse (lanes 1 and 2, and 4), but not in single positive
mice (lane 3), and the induction was completely reversed after
withdrawal of Dox for 3 days after a 1-day Dox treatment (lane 5). In
all treatments, 2-month-old bitransgenic mice were used except in lane
3 (lane 3 used a single positive mouse). In each panel, 28S ribosomal
RNA was shown as a loading control.
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Transgene expression was tested to determine whether targeting was both
pituitary- and cell type-specific. To determine organ specificity, a
bitransgenic mouse with the PRL-tTA/TetRE-EGFR-tr transgenes was killed
at the age of 2 months without prior exposure to doxycycline. To test
the GH promoter, a 2-month-old GH-rtTA/TetRE-EGFR-tr bitransgenic mouse
was exposed to doxycycline 1 week before being killed. In both cases,
Northern blots showed high level transgene expression in the pituitary
but not the brain, liver, kidney, lung, or heart (Fig. 4A
). To determine in which pituitary
cells transgene expression was occurring, adjacent sections of
transgenic pituitary were studied by in situ hybridization
and immunohistochemistry as indicated in Fig. 4B
. For the GH promoter,
a 2-month-old GH-rtTA/TetRE-EGFR-tr bitransgenic was induced with
doxycycline for 1 week and adjacent sections were probed with
35S-labeled antisense GH or EGFR riboprobes,
respectively. Most, but not all, cells expressing GH also expressed the
EGFR-tr transgene (long arrows) (Fig. 4B
). We did not
observe cells that were not positive for GH expression that expressed
the transgene. Some GH-producing cells did not express the EGFR-tr
transgene as shown by short arrows. For characterization of
the PRL promoter, the PRL-producing cells were identified by PRL
immunohistochemistry, and transgene expression in an adjacent section
was identified by in situ hybridization with an
35S-labeled antisense EGFR riboprobe. For this
study, a bitransgenic mouse with the PRL-tTA/TetRE-EGFR-tr transgenes
was killed at the age of 2 months without prior exposure to
doxycycline. The EGFR-tr transgene was expressed in most, if not all,
PRL-containing cells (see arrows), and expression of the
transgene was not observed in other cell types. Together these data
demonstrate that both PRL and GH promoter drive both pituitary-specific
and cell type-specific transgene expression.

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Figure 4. Northern Blot and in Situ
Localization Showing Both Tissue- and Cell Type-Specific Expression of
EGFR-tr mRNA
A, A 2-month old female bitransgenic mouse having both PRL-tTA and
TetRE-EGFR-tr transgenes (left panel) or GH-rtTA and
TetRE-EGFR-tr transgenes (right panel) was killed, and
RNA from several tissues was processed for Northern blot analysis. The
PRL-tTA/TetRE-EGFR-tr mouse never received doxycycline so that
transgene expression was induced whereas the GH-rtTA/TetRE-EGFR-tr
mouse was treated with doxycycline for 1 week to induce transgene
expression. In both cases, EGFR-tr was expressed only in pituitary
gland. B, Adjacent sections of pituitary gland from the respective
2-month-old bitransgenic mice, induced for transgene expression as
described in panel A, were analyzed by in situ
hybridization using the EGFR-tr riboprobe in one section and either
immunostaining to identify PRL containing cells (upper
panel) or in situ hybridization using the GH
riboprobe to identify somatotropes (lower panel) in the
adjacent section. Arrows indicate the cells in which the
transgene was expressed in PRL-producing cells with the use of PRL
promoter, and GH-producing cells with the use of GH promoter,
respectively. The short arrows in lower panel indicates
GH-mRNA-containing cells that do not appear to express the EGFR-tr
transgene. BV, Blood vessel. Bar, 30 µm.
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No Phenotype in the PRL-EGFR-tr and PRL-tTA/TetRE -EGFR-tr
Transgenic Mice
Transgenic mice that overexpress EGFR-tr in PRL-producing cells,
whether under direct control of the PRL promoter or under indirect
control through the tet-off system, showed no phenotype. The rates of
linear growth and weight gain were identical to those of their
nontransgenic littermates. In addition, the suckling offspring of the
transgenic lactating mice showed normal weight gain and development
(Fig. 5A
), suggesting that the ability of
the transgenic mice to lactate was not impaired. Grossly and on a
histological level, pituitary size was identical in the control
wild-type and transgenic mice. Immunohistochemical staining for PRL,
GH, ACTH, TSH, ßFSH, and ßLH showed a normal hormonal distribution
pattern compared with control wild-type or single positive mice (data
not shown). PRL staining also showed a normal pattern both before and
during pregnancy, suggesting that these transgenic mice can adapt
normally to pregnancy (Fig. 5B
). Pituitary cell proliferation was also
determined in these mice using bromodeoxyuridine (BrdU) incorporation.
The animals were injected 1 h before examination of the pituitary
with BrdU, and incorporation was assessed by BrdU immunostaining in
pituitaries taken from both nonpregnant and pregnant female mice. The
transgenic mice showed no difference from control wild-type or single
positive mice (data not shown); that is, the labeling index of 23%
was similar in the transgenic and control mice. These results indicate
that expression of the dominant negative EGFR-tr in lactotropes, from
the earliest point in development when the PRL promoter drives
expression of this transgene, results in normal lactotrope development
and adaptation to pregnancy. This result suggests that EGFR
signaling is not required for terminal lactotrope differentiation,
maintenance, or adaptation.

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Figure 5. Growth Curve of Offspring of Prl-tTA/TetRE-EGFR-tr
Mice and Immunostaining for PRL (PRL) in the Pituitary of Prl-EGFR-tr
Mice
A, The eight suckling offspring of a lactating Prl-tTA/TetRE-EGFR-tr
bitransgenic mouse, never receiving Dox, were weighed at the indicated
times (TG, ) and compared with the eight offspring of a lactating
nontransgenic littermate (Con, ). Weaning occurred at approximately
day 25. B, The pituitary glands of Prl-EGFR-tr transgenic (TG) and wild
type (WT) littermates were compared before and during pregnancy. The
nonpregnant female littermates were 10 weeks old, while the pregnant
mice were 3 months old and at day 16 of gestation. Gestational timing
was confirmed by measurement of the fetal size. The pituitary glands
were stained for PRL content and are shown at a final magnification of
approximately 170x.
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Gross Phenotype in the GH-rtTA/TetRE -EGFR-tr Transgenic Mice
The lack of phenotype in the lactotropes did not exclude the
possibility that other pituitary cell types might use EGFR signaling
for development or adaptation. It has been shown that both somatotrope
and lactotrope cells originate from a common GH-producing precursor
cell (somatomammotroph) that must proliferate during organogenesis
(41). GH gene expression precedes PRL gene expression by about 2 weeks
(52); therefore, we chose to use the GH promoter to drive EGFR-tr
expression. By driving EGFR-tr expression to this earlier precursor, we
could test whether EGFR signaling plays a role earlier in somatotrope
and lactotrope cell development. To ensure that the EGFR-tr transgene
was expressed at the earliest possible time, doxycycline was
administered to the bitransgenic mice through maternal feeding from the
time of conception.
At the time of birth, there were no gross morphological differences
between GH-rtTA/TetRE-EGFR-tr transgenic mice and control wild-type or
single positive littermates. Nor were there any distinguishing features
evident during the first 10 days of postnatal life. However, by 14
days, the bitransgenic mice were obviously smaller in size than their
control wild-type or single positive littermates, and eye opening of
the induced bitransgenic mice was delayed by 23 days. By 6 weeks of
age, the bitransgenic mice had an obvious dwarf phenotype (Fig. 6A
). The rate of weight gain (Fig. 6B
) of
the mouse shown in this photograph indicated that this bitransgenic
mouse had a weight comparable to the control littermate at birth, but
its weight gain lagged significantly behind the littermate with a
cessation of growth at approximately 6 weeks of age. The observed
growth pattern is consistent with the role of GH during early postnatal
growth but not in utero. This mouse attained a weight of
approximately one-half the weight of its littermate. Other bitransgenic
mice had weight gains ranging from one-half to two-thirds of their
control littermates. In addition, while female dwarf bitransgenic mice
were capable of developing and carrying a pregnancy to completion, all
of the offspring died consistently shortly after birth, probably as a
result of impaired lactation. To determine whether EGFR signaling is
also required for postnatal pituitary development or maintenance,
doxycycline induction was initiated at the time of birth by transfer
via the milk (45). The bitransgenic mice with the later onset of
EGFR blockade exhibited a growth rate indistinguishable from that of
the control littermates. However, when doxycycline was given from
embryonic day 14, the same dwarf phenotype was generated. Taken
together, these results suggest that EGFR signaling is required for
pituitary somatotrope development between embryonic day 14 and birth
but that signaling through this receptor is not required for postnatal
pituitary development, maintenance, or the adaptation to pregnancy.

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Figure 6. Gross Phenotype of GH-rtTA/TetRE-EGFR-tr
Bitransgenic Mice
A, Six-week-old bitransgenic mouse (smaller one), and its single
transgene positive (GH-rtTA) littermate. B, The growth of the mice was
followed by serial weights. The GH-rtTA monotransgenic mouse (Con, )
was compared with its bitransgenic littermate (TG, ), and both mice
received Dox.
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Analysis of the Pituitaries from the GH-rtTA/TetRE-EGFR-tr
Transgenic Mice
The anterior lobes of pituitary from bitransgenic mice with
EGFR-tr expression directed to somatotropes either from conception or
from embryonic day 14 were markedly smaller than the pituitaries from
control littermates at 6 weeks, whereas the sizes of posterior and
intermediate lobes were normal (Fig. 7A
, upper panel). The reticulin architecture both in single
positive control and bitransgenic mice was normal as shown by the
Golden-Sweet Silver (G.S.) staining method (Fig. 7A
, lower
panel). Immunohistochemical staining indicated a near complete
absence of GH-staining cells, and the number of PRL-staining cells was
markedly reduced in GH-rtTA/TetRE-EGFR-tr transgenic animals (Fig. 7B
).
ACTH- and TSH-containing cells showed normal patterns of staining in
these mice (Fig. 7B
). In these 6-week-old animals, gonadotropin
staining was present, although reduced relative to normal adult animals
and somewhat reduced relative to normal animals of the same age (data
not shown). The fact that female dwarf bitransgenic mice were capable
of developing and carrying a pregnancy to completion supports the
notion that gonadotrope development is ultimately sufficient for normal
reproductive function and fertility. Furthermore, any effect on
gonadotrope function does not appear to result from a direct effect of
the transgene on gonadotropes in that the transgene is expressed only
in the GH lineage. Rather, there is evidence that gonadotrope function
is impaired in GH-deficient mice such as the Ames dwarf (53, 54) and
other models (55, 56), suggesting an indirect mechanism of GH
deficiency on the gonadotrope. Taken together, these results imply that
EGFR signaling is directly required early in the development of
somatotropes and lactotropes but not after these cells have emerged and
differentiated in the pituitary.

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Figure 7. Histological Analysis of Pituitary Glands and
Immunostaining for GH, PRL, ß-TSH, and ACTH
A, Hematoxylin-eosin (H&E), and Golden Sweet silver (G.S.)-stained
section of a pituitary gland from a 6-week-old GH-rtTA/TetRE-EGFR-tr
transgenic mouse receiving Dox as compared with a single transgene
control mouse. Notice the size difference between the bitransgenic and
the single positive control mouse. G.S. silver staining was done to
show the reticulum fiber network. B, Immunostaining for GH, PRL,
ß-TSH, and ACTH in the pituitaries from a 6-week-old bitransgenic and
a control littermate, respectively. GH-staining cells were nearly absent, and the
number of PRL-staining cells was significantly reduced in the
bitransgenic mouse pituitary. However, ß-TSH and ACTH staining showed
normal staining patterns compared with those of a single positive
control littermate. A, upper panel, 44x; lower
panel, 70x; B, 90x.
|
|
 |
DISCUSSION
|
---|
The fully developed anterior pituitary gland expresses components
of the EGFR signaling system including the EGFR itself, its ligands,
TGF
, and EGF. However, the role of this system in pituitary
development, maintenance, and adaptation has not been explored. The
EGFR is known to be important for normal embryo development; targeted
disruption of the EGFR in mice revealed receptor involvement in a broad
range of developmental processes especially in epithelial tissues
(56). Ultimately, these EGFR-deleted mice displayed epithelial
phenotypes ranging from peri- implantation death to short lived progeny
suffering from abnormalities in multiple organs depending on the
genetic background (58, 59). The lethal and multisystem phenotypes in
these mice precluded studies on the role of the EGFR in specific
tissues such as the pituitary. To obviate this problem, we directed the
expression of a truncated form of the EGFR, lacking most of its
cytoplasmic domain, to cells of interest using an inducible system that
also allowed temporal control of expression. This EGFR-tr has proved to
act as a dominant negative mutant because of its ability to form
nonfunctional heterodimers with endogenous full-length EGFR both in
cultured cells and in a transgenic mouse model (40). This strategy for
EGFR blockade was useful in the study of the role of EGFR in the
mammary gland (40) and skin (42). When we used this dominant negative
strategy to express the EGFR-tr in the mammary glands of transgenic
mice, these animals showed a marked inhibition of mammary gland ductal
development (40).
We chose to focus this dominant negative strategy on the somatotropes
and lactotropes of the pituitary. In bovine and rat pituitary, these
cell lineages have been shown to express the EGFR ligand, TGF
(22, 23). An adaptive role of this system has been suggested by the finding
that TGF
is up-regulated in the pituitary by estrogen before the
development of lactotrope hyperplasia (38). Furthermore, TGF
overexpression in the lactotropes of transgenic mice results in
lactotrope hyperplasia and pituitary adenoma formation (60), suggesting
that the lactotropes are capable of a proliferative response to the
overexpression of an EGFR ligand. Together, these studies suggest that
EGFR activation may occur under certain physiological conditions, and
this activation can result in the selective expansion of the lactotrope
cell population. In this study, we found that blockade of the EGFR
early in somatomammotrope development results in a near complete
failure of this pituitary subpopulation to expand. Thus, the mice
expressing the EGFR-tr under GH promoter control at this early time
point develop as dwarfs incapable of lactation during subsequent
pregnancy. These observations suggest that EGFR signaling is necessary
for this early step in pituitary organogenesis when proliferation of
the newly differentiated cells is required (41). However, we also find
that if the EGFR blockade is imposed later in development, either when
the somatotropes have emerged or later still, the somatotropes and
lactotropes continue to function normally. Indeed, the lactotrope
expansion that occurs during pregnancy appeared normal despite EGFR
blockade.
The dwarf phenotype of the GH-EGFR-tr mice appears
superficially similar to a number of other dwarf syndromes. Those mouse
syndromes, such as the Snell and Jackson dwarfs, that result from
mutations in Pit-1 (61, 62, 63), a transcription factor involved in
pituitary cell differentiation, also display an absence of those
pituitary cell lineages that require Pit-1 for differentiation. The
dwarf syndrome caused by the expression of the GH-EGFR-tr transgene
requires activation of the GH promoter for expression of the dominant
negative receptor. For this promoter to be expressed, primary
differentiation of the somatomammotrope lineage must have taken place.
Thus, the failure of development of the somatomammotropes in the
GH-EGFR-tr mice did not result from a primary failure of
differentiation but must have resulted from a failure of expansion of
the somatomammotrope population, a process requiring EGFR activation.
The geno-type of another dwarf syndrome, the little
mouse, involves a single point mutation in the GH-releasing hormone
(GHRH) receptor gene, and this mutation also results in a reduced
population of somatotropes and a dwarf phenotype (64). It is not
established whether this reduced population of somatotropes is a result
of impaired primary differentiation or population expansion. However,
there is evidence that GHRH receptor activation, which is coupled to
adenylate cyclase, results in somatotrope growth. Indeed, adenylate
cyclase activation resulting from mutations in G protein, Gs
(65, 66, 67), or in transgenic animals expressing the activator of
adenylate cyclase, cholera toxin, in somatotropes (68) does result in
somatotrope hyperplasia and adenomas. Thus, the little
mutation probably results in a failure of somatotrope lineage expansion
similar to the GH-EGFR-TR mice. Whether the GHRH-adenylate cyclase and
EGFR systems interact to control this population expansion during and
post-developmentally remains to be seen.
 |
MATERIALS AND METHODS
|
---|
Generation of Transgenic Mice
Four separate lineages of transgenic mice were generated for
this study: in one lineage, PRL-EGFR-tr, the transgene directly drives
expression of cytoplasmic tyrosine kinase- deleted mutant form of
EGFR (EGFR-tr) in PRL-producing pituitary cells. In two lineages,
PRL-tTA and GH-rtTA, the transgene drives expression of
tetracycline-controlled transactivator (tTA) or reverse
tetracycline-controlled transactivator (rtTA) in PRL- or GH-producing
cells in the pituitary, respectively. The fourth lineage,
TetRE-EGFR-tr, expressed EGFR-tr under the control of the tetracycline
responsive element (TetRE). The PRL-tTA or GH-rtTA mouse lines were
cross-bred with TetRE-EGFR-tr mouse line to establish
PRL-tTA/TetRE-EGFR-tr or GH-rtTA/TetRE-EGFR-tr bitransgenic mouse
lines. To generate PRL-EGFR-tr transgene, the 3.0-kb rat PRL promoter
(a gift from Dr. H. Elsholtz) was placed in front of the 2.23-kb mouse
cDNA encoding the truncated EGFR that we have previously described to
behave as a dominant negative regulator of EGFR function (40). The
0.8-kb SV40 polyA sequence and an intron were placed downstream of the
EGFR-tr. For PRL-tTA or GH-rtTA transgenes, 1.1-kb tTA or rtTA
(CLONTECH Laboratories, Inc., Palo Alto, CA) was placed
behind the 3.0-kb rat PRL- or 1.7-kb rat GH promoter (rat GH promoter
was a gift from Dr. Arthur Gutierrez-Hartmann), followed by the SV40
polyA sequence and intron. TetRE-EGFR-tr transgene was generated by
placing 2.23-kb mouse EGFR cDNA (40) downstream of the 0. 44-kb
tetracycline-responsive element (TetRE), followed by the 1.6-kb SV40
intron/polyA sequence. The following transgenes were excised from the
various constructs: The 6.1-kb BamHI fragment containing
PRL-EGFR-tr, 4.9-kb HindIII-EcoRI fragment
containing PRL-tTA, 4.3-kb ClaI-NotI fragment
containing GH-rtTA, and 4.2-kb BamHI fragment containing
TetRE-EGFR-tr. The transgene DNA was purified using the Qiaquick
purification kit (QIAGEN). Microinjection into one-cell
B6xSJL mouse zygotes at a concentration of 2 ng/ml was carried out at
the University of Alabama at Birmingham transgenic animal facility.
Identification of Transgenic Mice
Genomic DNA from tail biopsies was isolated as described
previously (40). Transgene-positive founder mice were identified by PCR
using the following sets of transgene-specific oligonucleotides as
primers; forward primer from rat PRL cDNA (5'-AGTTGAAGTCAACATACC) and
from rat GH cDNA (5'-AATCATGGGGAAAATACC), and a reverse primer from tTA
(5'-TCTTTAGCGACTTGATGC) for PRL-tTA and GH-rtTA, and forward primer
from TRE (5'-AGCAGAGCTCGTTTAGTG) and a reverse primer from N-terminal
mouse cDNA of EGFR (5'-GGGGCACAGATGATTT) for TetRE -EGFR-tr
identification. Slot blot and Southern blot analysis, using a
transgene-specific probe derived from the SV40, tTA, or EGFR sequences,
confirmed the presence of the transgene in PCR-positive mice and the
integration of transgene into chromosomal DNA. Briefly, 10 µg of
mouse liver genomic DNA was digested overnight with transgene-unique
enzymes (EcoRI for PRL-EGFR-tr, SalI and
SacII for PRL-tTA, SalI for GH-rtTA and TetRE
-EGFR-tr, respectively) and subjected to Southern blotting on Hybond-N
+nylon membrane (Amersham Pharmacia Biotech, Arlington Heights, IL) as described in the
manufacturers protocol using the following 32P-
labeled probes: 1.1-kb BamHI-EcoRI tTA
fragment, 2.23-kb N-terminal EcoRI EGFR cDNA, and 1.6-kb
SalI-EcoRI fragment of the SV 40 sequences.
Doxycycline Administration
Doxycycline (Sigma, St. Louis, MO) was diluted in
5% sucrose in water to a final concentration of 2 mg/ml, supplied to
the mice in black-painted bottles as drinking water, and changed
every 23 days.
Northern Blot Analysis
Pituitary glands were processed for total RNA isolation using
the RNeasy total RNA isolation kit (QIAGEN, Chatsworth,
CA). The RNA from all the other tissues was prepared using the
guanidine-thiosulfate method (69). RNA was separated on 1%
formaldehyde-agarose gel, transferred onto the Hybond-N
+nylon membrane (Amersham Pharmacia Biotech), and hybridized with 32P-labeled
cDNA as indicated in the figures.
In Situ Hybridization
Paraffin sections(10 µm) were used. The probes were the 700-bp
GH mouse cDNA, or 2.23-kb EGFR mouse cDNA. The
35S-UTP labeling of the antisense cRNA riboprobe
and hybridization was carried out as described previously (45). After
hybridization, emulsion dip, exposure, and developing, the sections
were counterstained with hematoxylin and eosin and subjected to
microscopic examination under bright- and dark-field illumination.
Histological Analysis, BrdU Labeling, and
Immunohistochemistry
Transgenic and age- and sex-matched control mice were killed by
decapitation with appropriate observation of the institutional animal
care protocols. At autopsy, the pituitaries were removed, fixed in 10%
phosphate-buffered formaldehyde for 4 h, and embedded in paraffin.
Sections, 45 µm thick, were stained with hematoxylin and eosin and
with the Gordon-Sweet silver method to demonstrate the reticulin fiber
network. In vivo BrdU labeling was performed by ip injection
of BrdU as described (40). Formaldehyde fixed and paraffin embedded
sections were prepared and immunostained with a rat monoclonal
anti-BrdU antibody MSA250P (Accurate Chemical & Scientific Corp., Westbury, NY) at 1:100 dilution and
Vectastain Elite ABC Kit (Vector Laboratories, Inc., Burlingame, CA). 3'-Diaminobenzidine tetrahydrochloride
(DAB) was used as the chromogen, and sections were counterstained with
Gills Hematoxylin (Vector Laboratories, Inc.). To
facilitate BrdU detection, sections were pretreated with 2
M HCl for 20 min at 37 C and exposed to 0.01%
trypsin at 37 C for 3 min. Immunohistochemical stains to localize
adenohypophysial hormones were performed using the
avidin-biotin-peroxidase complex technique. The duration of exposure to
primary antiserum was 24 h at 4 C. The primary polyclonal antisera
were directed against the following antigens and were used at the
indicated dilutions: ACTH (DAKO Corp., Carpinteria, CA,
prediluted 1:15), rat GH, PRL, ßTSH, ßFSH, and LH [all donated by
the National Hormone and Pituitary Program (NHPP), NIDDK, NICHHD,
Bethesda, MD], 1:2500, 1:2500, 1:3000, 1:1600, and 1:2500,
respectively.
 |
ACKNOWLEDGMENTS
|
---|
The authors wish to thank Dr. Arthur Gutierrez-Hartmann for
providing the rat GH promoter, Dr. Harry Elsholtz for providing the rat
PRL promoter, and Kelvin So for technical assistance.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jeffrey Kudlow, Department of Medicine/Endocrinology and Cell Biology, University of Alabama at Birmingham, 1807 7th Avenue South, Room 756, Birmingham, Alabama 35294.
These studies were supported by NIDDK Grant DK-43652.
Received for publication July 19, 2000.
Revision received December 8, 2000.
Accepted for publication January 5, 2001.
 |
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