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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The epidermal growth factor receptor (EGFR) and its ligands EGF and transforming growth factor-{alpha} (TGF{alpha}) are expressed in the anterior pituitary, and overexpression of TGF{alpha} 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: 600–613, 2001)


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 Rathke’s 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-{alpha} (TGF{alpha}), are expressed in multiple cell types in the pituitary (21, 22, 23, 24), and TGF{alpha}, 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{alpha} can modulate hormone production; EGF can stimulate the secretion of ACTH, GH, LH, and TSH (31, 32, 33, 34, 35), and TGF{alpha} can stimulate LHRH (36). Conversely, hormones can modulate the EGFR system; GH can enhance EGFR concentration in mice (37, 38), and TGF{alpha} 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{alpha} antibodies (39), and the pubertal development of the mammary ductal system requires EGFR signaling (40). In the pituitary, the up-regulation of TGF{alpha} expression precedes the lactotrope hyperplasia that is induced by estrogen in rats (38). Furthermore, overexpression of TGF{alpha} in the lactotropes of transgenic mice results in lactotrope hyperplasia and lactotrope adenomas, implying that the signaling system downstream of TGF{alpha} 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{alpha} 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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Pituitary-Specific Transgenic Models
Four lineages of transgenic mice were generated for this study and the schematic diagrams are shown in Fig. 1Go, A–C. 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).

 
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. 2Go, A–D). 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 [8–1, 3–9 lines for Prl-EGFR-tr (A), 6–5 for Prl-tTA (B), 3–6 and 4–3 for GH-rtTA (C), and 2–9 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 2–4 and 3–5 in panel A are nontransgenic mouse lines.

 
EGFR-tr Expression in Pituitary Directed by the PRL and GH Promoters
For lactotrope-specific expression, either the constitutive 3–9 or 8–1 PRL-EGFR-tr lines, or the 6–5 PRL-tTA and 2–9 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. 3CGo) 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. 3AGo). 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. 3DGo, lane 1). When doxycycline was given for 7 days to turn off the transgene expression, the expression of EGFR-tr became undetectable (Fig. 3DGo, 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. 3DGo, lanes 3–5). 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. 3BGo, lane 4) and male (data not shown), but not in wild-type or single positive transgenic mice (Fig. 3BGo, lanes 1–3, 5, and 6). At the exposure used, the endogenous EGFR mRNA, with a predicted size of 4.7–4.9 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. 3EGo, 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 2–6, female mice from the 6–5 transgenic mouse line. Lanes 2–4, 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 1–4, female; lanes 5 and 6, male mice from the 3–6 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, 3–9 line; lane 3, 8–1 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.

 
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. 4AGo). 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. 4BGo. 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. 4BGo). 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.

 
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. 5AGo), 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. 5BGo). 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 2–3% 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, {blacksquare}) and compared with the eight offspring of a lactating nontransgenic littermate (Con, {diamondsuit}). 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.

 
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 2–3 days. By 6 weeks of age, the bitransgenic mice had an obvious dwarf phenotype (Fig. 6AGo). The rate of weight gain (Fig. 6BGo) 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, {diamondsuit}) was compared with its bitransgenic littermate (TG, {blacktriangleup}), and both mice received Dox.

 
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. 7AGo, 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. 7AGo, 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. 7BGo). ACTH- and TSH-containing cells showed normal patterns of staining in these mice (Fig. 7BGo). 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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The fully developed anterior pituitary gland expresses components of the EGFR signaling system including the EGFR itself, its ligands, TGF{alpha}, 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{alpha} (22, 23). An adaptive role of this system has been suggested by the finding that TGF{alpha} is up-regulated in the pituitary by estrogen before the development of lactotrope hyperplasia (38). Furthermore, TGF{alpha} 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{alpha} (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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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 manufacturer’s 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 2–3 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, 4–5 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 Gill’s 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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 ABSTRACT
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 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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