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Endocrinology Vol. 149, No. 7 3446-3451
Copyright © 2008 by The Endocrine Society

Loss of Glucocorticoid Receptor Function in the Pituitary Results in Early Postnatal Lethality

Gitta Erdmann, Günther Schütz and Stefan Berger

German Cancer Research Center, Division Molecular Biology of the Cell I, 69120 Heidelberg, Germany

Address all correspondence and requests for reprints to: Professor Dr. Günther Schütz, German Cancer Research Center, Division Molecular Biology of the Cell I, Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. E-mail: g.schuetz{at}dkfz.de.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Glucocorticoid action in the brain is mediated by the glucocorticoid receptor (GR) and the mineralocorticoid receptor, thereby affecting physiological processes such as neurogenesis, synaptic plasticity, and neuroendocrine control. To examine GR function in the regulation of the hypothalamic-pituitary-adrenal axis, we generated GR mutant mice that are homozygous for a conditional GR allele and heterozygous for a transgene that expresses the Cre recombinase under control of the regulatory elements of the mouse calcium/calmodulin-dependent protein kinase II{alpha} gene, resulting in Cre-mediated recombination in the brain and pituitary. The GR mutants die about 1 wk after birth and display a fulminant increase in plasma corticosterone as well as a severe histopathological phenotype. To assess in which time frame targeting of the pituitary occurs during embryonic development, we used a transgenic line expressing an inducible CreERT2 fusion protein under the control of the regulatory elements of the calcium/calmodulin-dependent protein kinase II{alpha} gene. Cre reporter data show that pituitary targeting occurred during embryonic development at the time when glucocorticoid synthesis starts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
GLUCOCORTICOIDS (corticosterone in rodents, cortisol in men) influence many physiological functions in the brain including neurogenesis, synaptic plasticity, behavior, and neuroendocrine control (1, 2, 3). Glucocorticoid action is mediated by two homologous ligand-activated transcription factors, the glucocorticoid receptor (GR) and the mineralocorticoid receptor. Adrenal synthesis and release of corticosterone is controlled by the hypothalamic-pituitary-adrenal (HPA) axis that shows circadian activity and a strong increase in activity in response to stress. The activation of the HPA axis is initiated with the release of CRH and arginine-vasopressin from the paraventricular nucleus (PVN). These hormones induce the secretion of ACTH, a cleavage product of proopiomelanocortin (POMC), from the corticotroph cells of the adenohypophysis. ACTH stimulates adrenal synthesis and secretion of glucocorticoids. GR activation leads to repression of hypothalamic CRH and pituitary POMC expression, which results in the return to homeostasis (4). The tone of the HPA axis is controlled by limbic structures that are also involved in feedback inhibition. Lesioning and electrical stimulation studies suggest an overall inhibitory influence of the hippocampus on the activity of the HPA axis (5), whereas the amygdala appears to have an excitatory influence (4).

Investigation of GR function in the brain in vivo using targeted gene inactivation in the mouse was limited by the fact that GR knockout mice die perinatally (6). The Cre/loxP-recombination system, that allows tissue-specific inactivation of genes, made it possible to investigate GR function in the brain of adult mice (7). To achieve tissue-specific gene inactivation, an essential part of a gene is flanked by loxP sequences, the recognition site for the bacteriophage P1 Cre recombinase, without affecting the expression of the gene. The cell type/tissue-specific expression of Cre recombinase under control of specific promoter/enhancer elements results in deletion of the part that is flanked by loxP sequences and consequently in gene inactivation (8, 9).

Mice lacking GR in neurons and glial cells showed a strongly activated HPA axis causing Cushing-like symptoms, e.g. altered fat distribution and reduced bone density. In addition, the mutant mice displayed reduced anxiety (7). Postnatal acquired forebrain deficiency of GR produces depression-like changes in behavior and a mild HPA axis dysregulation and modulates anxiety-associated locomotor activity and adrenal responsiveness (10, 11). However, the particular role of GR in feedback regulation during development of the HPA axis has not been analyzed in detail. To investigate the contribution of GR expressed in the PVN, hippocampus, and pituitary to HPA axis regulation, we made use of a transgene expressing Cre recombinase under control of the regulatory elements of the calcium/calmodulin-dependent protein kinase II{alpha} (CaMKII{alpha}) gene (CaMKCre transgene) (12). Reporter data showed that the transgene is active in the brain already during embryonic development and mediates in addition recombination in the pituitary. Mutant mice (GRCaMKCre) lacking GR in neurons and pituitary die about 1 wk after birth and display a dramatic increase in plasma corticosterone as well as a severe histopathological phenotype. Investigation of the onset of pituitary recombination using the regulatory elements of the CaMKII{alpha} gene to drive an inducible Cre recombinase revealed prenatal activity of the CaMKII{alpha} locus in the pituitary around the time when glucocorticoid synthesis starts.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animal treatment
All animal experiments were performed at the German Cancer Research Center according to institutional and international standards and were approved by the local authorities (Regierungspräsidium) of the state Baden-Württemberg. Tamoxifen (Sigma, Munich, Germany) was dissolved in sunflower seed oil/ethanol (10:1) mixture at a final concentration of 10 mg/ml. Eight-week-old mice were injected ip with 1 mg of tamoxifen twice per day for 5 consecutive days. Animals for Cre reporter analysis were killed 10 d after the last injection. To induce CreERT2 activity during embryonic development, pregnant females were injected ip with 50 mg tamoxifen/kg body weight from embryonic day (E) 12.5 to E14.5. Pups were delivered by cesarean section and raised by foster mothers until d 12 after birth.

Hemalaun/eosin staining, immunohistochemistry, and β-galactosidase analysis
For hemalaun/eosin staining, organs were isolated and postfixed with 4% paraformaldehyde at 4 C overnight before embedding in paraffin. Paraffin sections were performed at a thickness of 6 µm using a microtome (Leica, Wetzlar, Germany). Sections were hydrated through an ethanol series followed by 5 min incubation in Mayers hemalaun (AppliChem, Darmstadt, Germany), 1 min 0.5% HCl in 70% EtOH, 5 min treatment with running tap water, and 1 min incubation in eosin (Sigma). Sections were dehydrated and mounted with Eukitt (O. Kindler, Freiburg, Germany).

For immunohistochemical analysis, animals were killed and dissected brains were postfixed with 4% paraformaldehyde at 4 C for 72 h. Coronal sections at a thickness of 50 µm were performed using a vibratome (Microm, Walldorf, Germany). The free-floating sections were processed for immunohistochemistry using the VECTASTAIN ABC system (Vector Laboratories, Peterborough, UK) and diaminobenzidine (Sigma) incubation. The following primary antibody was used: polyclonal CRF antibody (dilution 1:1000 Abcam, Cambridge, UK), polyclonal GR antibody (dilution 1:1000; Santa Cruz Biotechnology, Heidelberg, Germany).

For β-galactosidase whole-mount stainings, freshly prepared pituitaries were washed three times with washing buffer (1x PBS, 5 mM EGTA, 2 mM MgCl2, 0.01% sodium-deoxycholate, and 0.02% Nonidet P-40) and subsequently stained with X-gal staining solution (5 mM EGTA, 2 mM MgCl2, 0.01% sodium-deoxycholate, 0.02% Nonidet P-40, 10 mM K3[Fe(CN)6], 10 mM K4[Fe(CN)6], and 0.5 mg/ml X-gal in 1x PBS) at 37 C overnight. After staining, the pituitaries were postfixed with 4% paraformaldehyde at 4 C overnight and kept in 1x PBS at 4 C for analysis.

Corticosterone and ACTH measurement
To determine plasma corticosterone and ACTH at d 6 after birth, blood sampling was performed in the morning after decapitation. Serum from individual mutants was used, whereas serum from two control mice was pooled. Plasma corticosterone as well as ACTH was measured using commercially available RIA kits (MP Biomedicals, Heidelberg, Germany). Results are presented as mean ± SEM. Measurements were analyzed using unpaired, two-tailed Student’s t test.

POMC in situ hybridization
In situ hybridization was performed on paraffin sections as described previously (13), with the exception that the antidigoxigenin antibody dilution has been reduced to 1:1000. The probe was encompassing the open reading frame of the POMC gene (708 bp) in an antisense direction, whereas the sense transcript served as negative control.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The use of the regulatory elements of the CaMKII{alpha} gene for conditional targeting results in recombination besides the brain within the pituitary
The Cre/loxP-recombination system allows to restrict gene inactivation to certain cell populations by using cell type-specific promotors to drive Cre recombinase expression. So far, plasmid-derived transgenes that have been generated using up to 15-kb fragments of the CaMKII{alpha} promotor to drive Cre expression showed postnatal forebrain-specific Cre expression (14, 15). In contrast, a transgene harboring a 43-kb upstream and 100-kb downstream sequence of the CaMKII{alpha} locus to drive Cre recombinase expression (CaMKCre transgene) (12) was shown to be already active at E12.5 and gives rather widespread recombination in the entire brain including the hypothalamus (16, 17). To analyze whether CaMKCre-mediated recombination also occurs outside the brain, we used ROSA26 Cre reporter (R26R) mice (18). Surprisingly, whole-mount β-galactosidase staining of the pituitary isolated from R26R/CaMKCre mice at d 12 after birth demonstrated widespread Cre-mediated recombination (Fig. 1AGo). However, other peripheral organs like skin, spleen, and thymus did not reveal any recombination (data not shown). Subsequent immunohistochemical staining of paraffin sections prepared of pituitary whole-mount samples showed that Cre-mediated recombination is detectable among others in corticotroph cells expressing POMC (Fig. 1AGo, inset).


Figure 1
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  		FIG. 1. Conditional targeting using the CaMKII{alpha} gene for Cre expression results in recombination within the pituitary during prenatal development. Whole-mount β-galactosidase staining using ROSA26 Cre reporter mice demonstrated Cre-mediated recombination in the adenohypophysis of CaMKCre transgenic mice at P12 (A) but not in NesCre transgenic (P12) (B) and adult tamoxifen-treated CaMKCreERT2 (D) transgenic mice. Induction of Cre activity by tamoxifen treatment of R26R/CaMKCreERT2 transgenic mice during embryogenesis results in recombination within the pituitary (C). Subsequent immunohistochemistry of paraffin sections of the whole-mount samples shows POMC expression in β-galactosidase-positive cells of CaMKCre transgenic animals (A, inset) and CaMKCreERT2 transgenic animals that were treated with tamoxifen during embryogenesis (C, inset). In NesCre transgenic mice and CaMKCreERT2 transgenic animals that were treated with tamoxifen at the adult stage, only POMC expression is detectable (B and D, insets).

 
Prenatal onset of GR ablation at all feedback sites of the HPA axis impairs survival
The PVN and the pituitary as well as limbic structures are responsible for feedback inhibition of the HPA axis (1). Increased glucocorticoid levels result in shutdown of the stress response by binding to the GR expressed in the respective structures (3, 4). To investigate the role of GR-mediated feedback inhibition of the HPA axis in vivo during development, we made use of the CaMKCre transgene that allows prenatal gene inactivation in all regulatory regions of the HPA axis. By crossing mice harboring a conditional GR allele (GRflox) (7) with mice expressing the CaMKCre transgene (12), we obtained GRflox/flox/CaMKCre (GRCaMKCre) mice. Within litters containing mutant and control pups, we observed animals dying between d 6 and 10 after birth that were genotyped as GRCaMKCre mice exclusively (Fig. 2AGo). Genotyping of the litters at postnatal day (P) 6 revealed a normal Mendelian distribution, indicating that only postnatal mortality occurs (data not shown). Therefore, phenotypic analysis was performed with 6-d-old GRCaMKCre mice and their control littermates. At P6 the mutant animals showed a strong visible phenotype with 50% reduced weight gain and almost no pigmentation (Fig. 2AGo), whereas perinatally they were indistinguishable from their control littermates. At P6 GR protein expression in control mice was detectable only in neurons of the hippocampal CA1 region, the central amygdala, and the PVN. Within these regions GRCaMKCre mice express Cre protein and display consequently loss of GR protein (data not shown).


Figure 2
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  		FIG. 2. Inactivation of the GR gene in cells expressing CaMKII{alpha} causes early postnatal lethality and a severe dysregulation of the HPA axis. A, Mice homozygous for the GRflox allele and heterozygous for the CaMKCre transgene (GRCaMKCre) die between d 6 and 10 after birth (n = 17). At P6, mutant animals display a weight reduction of about 50% and show strongly reduced pigmentation, compared with littermate controls. B, At this time point, GRCaMKCre mice show a 750-fold elevation of plasma corticosterone and a 16-fold increase in plasma ACTH, compared with controls. *, P < 0.005. C, In situ hybridization of sections of the pituitaries of GRCaMKCre and control mice shows an increase of POMC transcripts in the adenohypophysis of mutant animals (arrows: POMC-positive cells). D, Immunohistochemical staining of vibratome-sections demonstrates an elevation of CRH protein expression in the PVN of GRCaMKCre mice, compared with controls. The region of the PVN was defined by staining of consecutive sections with either GR (control) or Cre (mutant) antibody (data not shown).

 
To assess the consequences of GR ablation in GRCaMKCre mice for HPA axis activity, plasma corticosterone and ACTH levels as well as the expression of CRH protein in the PVN and POMC mRNA within the pituitary were determined. At P6, GRCaMKCre mice show a 750-fold elevation of plasma corticosterone and a 16-fold increase of plasma ACTH (Fig. 2BGo), compared with control littermates. In situ hybridization revealed a strong increase of POMC transcripts in the pituitary of GRCaMKCre mice (Fig. 2CGo), and immunohistochemical staining of vibratome sections showed a strong elevation of CRH protein in the PVN of mutant animals (Fig. 2DGo).

To evaluate the importance of GR in the pituitary for the regulation of the HPA axis activity during the early postnatal period, we compared GRCaMKCre mice with mice harboring an ablation of GR in precursors of neurons and glia cells during early embryogenesis using the NesCre transgene (GRNesCre mice). These mice did not show impaired survival and no change of GR protein expression in corticotroph cells of the pituitary (7). Additional analysis using Cre reporter mice confirmed that the NesCre transgene is not active in the pituitary (Fig. 1BGo). At P6, GRNesCre mice display only a 45-fold elevation of corticosterone, whereas GRCaMKCre mice show a 750-fold increase in plasma corticosterone (Table 1Go), even though both mutant lines show inactivation of the GR gene within the neuronal populations important for HPA axis regulation and a strong increase in CRH immunoreactivity (GRCaMKCre: Fig. 2DGo, GRNesCre: data not shown).


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  		TABLE 1. Morning levels of plasma corticosterone (nanograms per milliliter) in different brain-specific GR knockout mice at P6

 
In contrast to GRCaMKCre mice, GRNesCre mice are visually indistinguishable from their littermates at P6. Accordingly, hemalaun-eosin stained paraffin sections of several organs revealed a severe histopathological phenotype in GRCaMKCre mice only (Fig. 3Go, A–F). For example, the skin, compared with controls, showed a dramatic decrease of hair follicles and a strong reduction of the sc fat depot, a thinned epidermis, and an increase in the thickness of the stratum corneum (Fig. 3Go, A and D). The mutants also displayed a strongly reduced size of the thymus and the spleen that is hardly differentiated into red and white pulpa (Fig. 3Go, B, C, E, and F). Surprisingly, GRNesCre mice, despite increased corticosterone levels, show no histopathological changes in any of these organs (Fig. 3Go, G–I).


Figure 3
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  		FIG. 3. In contrast to GRNesCre mutants, GRCaMKCre mice display a severe histopathological phenotype. Comparison of hemalaun-eosin-stained paraffin sections from control (A–C), GRCaMKCre (D–F), and GRNesCre (G–I) mice at P6 reveals a severe histopathological phenotype in GRCaMKCre animals only. The skin displays a severe reduction of sc fat depot (asterisk), a thinner epidermal layer (rhomb), and a strong decrease in the number of hair follicles (A and D, arrows). The thymus (B and E) and spleen (C and F) of GRCaMKCre mutants are strongly decreased in size, and the spleen is hardly differentiated into red and white pulpa. In GRNesCre mice, no morphological changes in any of these organs are detectable (G–I).

 
Prenatal recombination in pituitary results from activity of the CaMKII{alpha} locus
To demonstrate that the Cre-mediated recombination within the pituitary using the CaMKCre transgene results from a transient developmental activity of the CaMKII{alpha} locus, we made use of an additional transgenic mouse line expressing an inducible Cre fusion protein (CreERT2) under control of the regulatory elements of the mouse CaMKII{alpha} gene (CaMKCreERT2 transgene) (17). The activity of the CaMKCreERT2 transgene is tightly controlled by the synthetic steroid tamoxifen. At the adult stage, strong Cre-mediated recombination is observed in the cortex, hippocampus, and PVN on tamoxifen induction (17) but not in the pituitary (Fig. 1DGo).

It has been shown that feedback regulation of the HPA axis is fully established at E16.5, even though glucocorticoid synthesis is already initiated around E14 (19). Analysis of recombination mediated by the constitutive CaMKCre transgene revealed that it is detectable in the brain as early as E12.5 (16). To determine whether the regulatory elements of the CaMKII{alpha} gene allow to target the pituitary during development in the time period when glucocorticoid synthesis starts, we treated pregnant females harboring R26R/CaMKCreERT2 embryos ip with 50 mg tamoxifen/kg body weight · d from E12.5 to E14.5. Postnatal analysis at P12 revealed that R26R/CaMKCreERT2 mice treated with tamoxifen during embryogenesis displayed indeed recombination in the pituitary (Fig. 1CGo). Moreover, immunohistochemical staining of paraffin sections derived from β-galactosidase-positive whole-mount samples revealed that the CaMKCreERT2 transgene targets POMC-expressing cells (Fig. 1CGo, inset).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Inactivation of the GR gene using the CaMKCre transgene causes postnatal lethality with severe dysregulation of the HPA axis
To investigate GR function in feedback regulation of the HPA axis, we used a conditional GR allele (7) and a transgene expressing the Cre recombinase under control of the regulatory elements of the CaMKII{alpha} gene. In contrast to the first analysis of this transgene showing that the loss of CREB protein starts within the first week after birth (12), it has been shown recently that this transgene causes gene inactivation already during embryonic development (20). Using Cre reporter mice we could show that the CaMKCre transgene mediates besides the brain also recombination in the pituitary.

The mutant mice (GRCaMKCre) with GR ablation in brain and pituitary die between d 6 and 10 after birth. They display a reduced weight gain and a fulminant increase in HPA axis activity with a 750-fold elevation of plasma corticosterone. The mutant mice are visually indistinguishable from their control littermates after birth and develop the apparent phenotype within the first postnatal week. Because we excluded recombination of the conditional GR allele in peripheral organs of GRCaMKCre mice, we assume that the observed histopathological changes in skin, thymus, and spleen result from the permanent stimulation of glucocorticoid signaling in GR-expressing cells of these tissues. However, we observed Cre activity in the anterior lobe of the pituitary. Besides loss of GR function in corticotrophs, inactivation of the GR in somatotroph cells might contribute to the growth retardation observed in GRCaMKCre mice.

In contrast to GR ablation in the brain and within the pituitary, the loss of GR in precursors of neurons and glia cells during early development in GRNesCre mice did not impair survival (7), and no histopathological changes in peripheral organs of these mutant mice could be observed, even though they show considerable elevation of plasma corticosterone. The absolute plasma corticosterone level in GRNesCre mice is in the range of basal peak levels in adults (20) and should therefore already permanently increase corticosterone signaling in the cells of peripheral organs that still express GR. However, it seems that higher plasma corticosterone levels, like the ones detected in GRCaMKCre mice, which are at least 5-fold higher than the levels measured in adults after restraint stress (20), are necessary to cause histopathological changes and lethality.

Inducible gene targeting reveals prenatal onset of recombination in the pituitary
It has been shown that the synthesis of glucocorticoids starts around E14 and that POMC expression in the pituitary is under negative feedback control as early as E16.5 (19). We have demonstrated that the CaMKCre-transgene mediates recombination in the pituitary and addressed the question whether recombination occurs before E16.5 using a transgene that, under control of the regulatory elements of the mouse CaMKII{alpha} gene, expresses a tamoxifen-inducible CreERT2 fusion protein allowing temporal control of Cre activity (17). Therefore, recombination can occur only if the CreERT2 fusion protein is expressed during the period of tamoxifen treatment. To elucidate whether Cre-mediated recombination and thereby loss of GR protein in the pituitary can be expected before glucocorticoid synthesis starts, we treated pregnant females harboring transgene- and reporter-positive embryos with tamoxifen between E12.5 and E14.5. Postnatal analysis indeed revealed Cre-mediated recombination in the pituitary of R26R/CaMKCreERT2 offspring, when the mother has been treated with tamoxifen during this time frame. It may appear surprising that the CaMKII{alpha} locus shows expression in the adenohypophysis because the primordial cells of the epithelial pituitary gland have been believed to be of extraneural origin (21). However, according to more recent reports, the adenohypophysis in vertebrates from amphibians to mammals is actually of neuroectodermal origin (22, 23). In addition, it was shown in vitro that the CaMKII protein is involved in CRH signaling and thus POMC expression in corticotroph cells of the anterior lobe of the pituitary (24).

In summary, we inactivated the GR gene in neuronal populations important for the HPA axis regulation and within the pituitary and found that the mutant mice show growth impairment and severe histopathological changes in peripheral tissues and actually die in the early postnatal period. Using a Cre-transgenic line expressing an inducible Cre fusion protein under the control of the regulatory elements of the CaMKII{alpha} gene in combination with a Cre reporter, we could show that pituitary targeting occurred already during embryonic development in the time frame when glucocorticoid synthesis starts. Our data demonstrate that GR function in pituitary is indispensible for the control of the HPA axis activity in the early postnatal period that is normally characterized by a hyporesponsiveness of the HPA axis to most stimuli that elicit HPA axis activation in adults (25) and very low corticosterone levels.


   Acknowledgments
 
We thank Dagmar Bock and Heike Alter for their excellent technical support.


   Footnotes
 
This work was supported by the Deutsche Forschungsgemeinschaft through Sonderforschungsbereiche 488 and 636, GRK 791/1.02, and Sachbeihilfe Schu 51/7-2; the Fonds der Chemischen Industrie; the European Union through Grant LSHM-CT-2005-018652 (CRESCENDO); the Bundesministerium für Bildung und Forschung through NGFN Grants FZK 01GS01117, 01GS0477, and KGCV1/01GS0416 and Project 0313074C (Systems Biology).

Disclosure Statement: The authors have no conflict of interest.

First Published Online March 27, 2008

Abbreviations: CaMKII{alpha}, Calcium/calmodulin-dependent protein kinase II{alpha}; E, embryonic day; GR, glucocorticoid receptor; HPA, hypothalamic-pituitary-adrenal; P, postnatal day; POMC, proopiomelanocortin; PVN, paraventricular nucleus.

Received December 21, 2007.

Accepted for publication March 17, 2008.


   References
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
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