Generation of Mouse for Conditional Expression of Forkhead Box A2

Abstract

Forkhead box A2 (FOXA2) is a pioneer transcription factor involved in organ development, function, and cancer. In the uterus, FOXA2 is essential for pregnancy and expressed specifically in the glands of the endometrium. Loss of FOXA2 function occurs during development of endometrial cancer in humans. The current study describes the development of a mouse model for conditional expression of mouse FOXA2. Using a system consisting of a minigene located at the Rosa26 locus, we generated a CAG-S-mFOXA2 allele in embryonic stem cells and subsequently in mice; before activation, the minigene is silent because of a floxed stop cassette inserted between the promoter and the transgene. To validate functionality, mice with the CAG-S-mFOXA2 allele were crossed with progesterone receptor (Pgr)–Cre mice and lactotransferrin (Ltf)-iCre mice that express Cre in the immature and adult uterus, respectively. In immature Pgr-Cre-CAG-S-mFoxa2 mice, FOXA2 protein was expressed in the luminal epithelium (LE), glandular epithelium (GE), stroma, and inner layer of the myometrium. Interestingly, FOXA2 protein was not observed in most of the LE of uteri from adult Pgr-Cre-CAG-S-mFoxa2 mice, although FOXA2 was maintained in the stroma, GE, and myometrium. The adult Pgr-Cre-CAG-S-mFoxa2 females were completely infertile. In contrast, Ltf-iCre-CAG-S-mFoxa2 mice were fertile with no detectable histological differences in the uterus. The adult uterus of Pgr-Cre-CAG-S-mFoxa2 mice was smaller, contained few endometrial glands, and displayed areas of partially stratified LE and GE. This transgenic mouse line is a valuable resource to elucidating and exploring FOXA2 function.

A mouse model for conditional expression of FOXA2 was developed and validated with Cre drivers that target the uterus.

Forkhead box (FOX) proteins are a family of transcription factors that play important roles in regulating the expression of genes involved in cell growth, proliferation, differentiation, and longevity (1, 2). FOX proteins have pioneering transcription activity by being able to bind and open condensed chromatin during cell differentiation processes. Forkhead box A2 (FOXA2) has important roles in the genesis, differentiation, and function of many endoderm-derived organs, such as the liver, lung, and gut (1–3), and differentiation and function of uterine glands (4–6). After birth, Foxa2 is expressed exclusively in glandular epithelium (GE) cells of the neonatal mouse uterus (4, 6). In the human uterus, FOXA2 protein is also expressed in the GE (7). Homozygous Foxa2 null embryos die shortly after gastrulation (8, 9) because Foxa2 is necessary for the development of several endoderm-derived organs (2, 10). To understand the role of FOXA2 in the uterus, Foxa2 was conditionally deleted with the progesterone receptor (Pgr)–Cre mouse model, in which Cre recombinase is expressed only after birth in most cells of the uterus (11). Conditional deletion of Foxa2 in the neonate inhibited uterine gland genesis, resulting in aglandular adult mice that were infertile because of defects in blastocyst implantation and stromal cell decidualization stemming from a lack of LIF and other GE secretions (4, 6). To determine the role of FOXA2 in the adult uterus, Foxa2 was conditionally deleted with the lactotransferrin (Ltf)-iCre mouse model in which Cre recombinase is expressed only after puberty in the luminal epithelium (LE) and GE cells of the uterus (12). The Ltf-iCre conditional deletion of Foxa2 in the adult uterus rendered the mice infertile due defects in blastocyst implantation stemming from a lack of LIF (13).

In addition to roles in organ development and function, FOXA2 also has a role in disease and cancer of many organs, including the liver and pancreas (14, 15). In women, FOXA2 is downregulated in ectopic as compared with eutopic endometrium in patients with endometriosis and also implicated in the molecular pathophysiology of endometriosis and endometrial adenocarcinoma (7, 16–18). FOXA2 is a proposed tumor suppressor gene in endometrioid endometrial carcinomas (19) and regulates tumor metastasis (20, 21). Furthermore, FOXA2 is somatically mutated in uterine carcinomas, particularly by frameshift and nonsense mutations (22). Thus, FOXA2 is a pathogenic driver gene in some of the most clinically aggressive forms of uterine cancer. The objective here was to develop a mouse model for conditional expression of FOXA2 in vivo by using an established approach (23) and validate the usefulness of the mouse model by determining the consequences of FOXA2 overexpression on development and function of the mouse uterus.

Materials and Methods

Generation of Foxa2 conditional expression mouse

An established approach for generating conditional expression mice was used as described by Wu et al. (23). The Shuttle Vector RfNLIII was generously provided by Ming-Jer Tsai (Baylor College of Medicine, Houston, TX). To generate an embryonic stem cell targeting construct (Supplemental Fig. 1), the mouse Foxa2 complementary DNA was amplified by polymerase chain reaction (PCR) and inserted into the targeting construct downstream of a Lox-Stop-Lox (LSL) cassette. The gene targeting in AB2.2 embryonic stem (ES) cells and production of chimeras from those ES cells was performed by the Genetically Engineered Mouse Core at Baylor College of Medicine. Chimeras were bred with C57BL/6 mice and founder mFoxa2^LSL mice [Gt(ROSA)26Sor^{tm1(CAG-Foxa2)TES}] screened by PCR genotyping. Primers for the gene replacement allele are 5′-GGA GCG GGA GAA ATG GAT ATG-3′ (forward) and 5′-GCT TTC TGG CGT GTG ACC-3′ (reverse), with a product size of 0.6 kb. PCR primers for the wild-type ROSA26 locus are 5′-GGA GCG GGA GAA ATG GAT ATG-3′ (forward) and 5′-AAA GTC GCT CTG AGT TGT TAT-3′ (reverse), with a product size of 0.6 kb. PCR genotyping was performed with tail DNA and 35 cycles of 95°C for 1 minute, 56°C for 1 minute, and 72°C for 1 minute.

Animals

All animal procedures were approved by the Institutional Animal Care and Use Committee of the University of Missouri and were conducted according to National Institutes of Health Guide for the Care and Use of Laboratory Animals. The mFoxa2^LSL mice were bred with Pgr^Cre (11) and Ltf^iCre (12) mice to generate Pgr^Cre/+mFoxa2^LSL/+ and Ltf^iCre/+mFoxa2^LSL/+ mice with conditional overexpression (cOE) of mouse Foxa2. The Pgr^Cre mice were generously provided by John Lydon (Baylor College of Medicine, Houston, TX). The Ltf^iCre mice (stock no. 022620) were obtained from the Jackson Laboratory (Bar Harbor, ME). Heterozygous mFoxa2^LSL/+ mice were used as controls.

For the fertility trial, female mice were housed individually and continuously with CD-1 male mice of proven fertility. Fertility was assessed by monitoring litter frequency and size for 6 months. Gestational time points were obtained by the mating of 8- to 10-week-old females to CD-1 male mice of known fertility, and the day of vaginal plug observation was designated as gestational day (GD) 0.5. Implantation sites on GD 5.5 were visualized by intravenous injection of 1% Evans blue dye (Sigma-Aldrich, St. Louis, MO) into the tail vein 5 minutes before euthanasia. Other studies involved subcutaneous injections of vehicle (sesame oil), 17β-estradiol (E2; 100 ng per mouse per day), or progesterone (P4; 1 mg per mouse per day). Sesame oil and hormones were sourced from Sigma-Aldrich.

RNA extraction and real-time PCR

Total RNA was isolated from uteri with a standard TRIzol-based protocol. To eliminate genomic DNA contamination, extracted RNA was treated with deoxyribonuclease I and purified with an RNeasy MinElute Cleanup Kit (Qiagen, Germantown, MD). Total RNA (1 μg) from each sample was reverse transcribed in a total reaction volume of 20 μL with iScript RT Supermix (Bio-Rad). Real-time PCR was performed with a CFX384 Touch Real-Time System with SsoAdvanced Universal SYBR Green Supermix (Bio-Rad, Hercules, CA) using Bio-Rad PrimePCR primers (Supplemental Table 1). Mus musculusGapdh and Rplp0 were used as reference genes.

Histology and immunohistochemistry

Paraformaldehyde-fixed, paraffin-embedded mouse tissues were sectioned (5 μm), mounted on slides, deparaffinized, and rehydrated in a graded alcohol series. For immunohistochemistry, sections were subjected to antigen retrieval in boiling 10 mM citrate buffer (pH 6.0) for 10 minutes, followed by an incubation with 1.0% (volume-to-volume ratio) hydrogen peroxide in methanol for 15 minutes. Sections were blocked with 10% (volume-to-volume ratio) normal goat serum (PCN500; Life Technologies, Carlsbad, CA) in phosphate-buffered saline (PBS) (pH 7.2) for 30 minutes and then incubated with the primary antibody in PBS with 1% bovine serum albumin overnight at 4°C. Antibody information is provided in Table 1. Sections were washed in PBS and incubated with 5 μg/mL biotinylated secondary antibody (Vector Laboratories, Inc., Burlingame, CA) in PBS for 1 hour at 37°C. Immunoreactive protein was visualized with a Vectastain ABC kit (Vector Laboratories, Inc.), with diaminobenzidine tetrahydrochloride as the chromagen. Sections were lightly counterstained with hematoxylin before dehydrating, and coverslips were affixed with Permount (Thermo Fisher Scientific).

Table 1.

Antibodies Used

Primary Antibody	Species	Manufacturer	Catalog No.	RRID	Dilution Ratio	Application
β-Actin	Rabbit	Cell Signaling Technology	4970	AB_2223172	1:3000	WB
FOXA2	Rabbit	LifeSpan Biosciences	LS-C 138006	AB_10946909	1:1000 (IHC)/1:2000 (WB)	IHC, WB
CXCL15	Rabbit	Abcam	ab197016		1:200	IHC
ESR1	Rabbit	Santa Cruz Biotechnology	sc-542	AB_631470	1:500	IHC
Ki-67	Rabbit	Abcam	ab15580	AB_443209	1:1000	IHC
P63	Rabbit	Cell Signaling Technology	4981s	AB_2286372	1:200	IHC
PGR	Rabbit	Thermo Fisher	MA5-14505	AB_10980030	1:1000	IHC

Abbreviations: IHC, immunohistochemistry; RRID, Research Resource Identifier; WB, Western blot.

In situ hybridization

In situ hybridization was performed with the RNAscope 2.5 HD Assay-Red kit from Advanced Cell Diagnostics (Newark, CA). Briefly, 4% paraformaldehyde-fixed, paraffin-embedded mouse tissues were sectioned (5 μm), mounted on slides, and deparaffinized in xylene and alcohol. The sections were hybridized at 40°C for 2 hours with a RNAscope Probe-Mm-Foxa2 that is specific for mouse Foxa2 messenger RNA (mRNA; Advanced Cell Diagnostics). The signal was visualized with chromogenic RNAscope 2.5 HD Detection Reagent (Advanced Cell Diagnostics). Sections were briefly counterstained with hematoxylin before dehydrating, and coverslips were affixed with Permount (Thermo Fisher Scientific).

Western blot

Total protein was isolated from frozen adult mouse uteri with Radioimmunoprecipitation assay buffer [50 mM tris(hydroxymethyl)aminomethane, 5 mM EDTA, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate, pH 7.5] containing Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific). Total uterine protein (30 mg) was separated on a denaturing 12% sodium dodecyl sulfate gel and transferred to a nitrocellulose membrane (Millipore, MilliporeSigma, Burlington, MA). The membrane was blocked and incubated overnight at 4°C with primary antibody (Table 1). After incubation with peroxidase-conjugated goat anti-rabbit secondary antibody (Thermo Fisher Scientific), immunocomplexes were visualized by enhanced chemiluminescence (Thermo Fisher Scientific) and a Bio-Rad Chemidoc Imaging System.

Statistical analysis

All quantitative data were subjected to least-squares analysis of variance with the general linear model procedures of the Statistical Analysis System (SAS Institute, Cary, NC) to determine the effects of genotype. In all analyses, error terms used in tests of significance were identified according to the expectation of the mean squares for error. Significance (P < 0.05) was determined by probability differences of least-squares means.

Results

Generation of the conditional expression allele for mouse Foxa2

As illustrated in Supplemental Fig. 1(a), a shuttle vector was made to deliver a single copy of minigene consisting of a ubiquitous CAGGS promoter, loxP-STOP-loxP cassette, mouse Foxa2 coding sequence (NCBI CCDS16836.1), and polyadenylation signal. The CAGGS promoter is a strong synthetic promoter widely used in mammalian cell lines and active in almost all tissues in vivo (24). The LSL cassette was placed between the CAGGS promoter and Foxa2 coding sequence to silence expression of the mouse Foxa2 transgene (25). In the presence of Cre recombinase, the LSL cassette is removed, which allows the CAGGS promoter to drive the expression of the mouse Foxa2 coding sequence. The minigene was inserted into the ROSA26 locus, which is widely used for constitutive, ubiquitous gene expression in mice (26). ES cell lines harboring one allele of mFoxa2^LSL at the ROSA26 locus of the mouse genome were generated by homologous recombination. Chimeras from the ES cells were bred to C57BL/6 mice to establish founder mice [Supplemental Fig. 1(b)]. Founder mice heterozygous (mFoxa2^LSL/+) and homozygous (mFoxa2^LSL/LSL) for the Foxa2 conditional allele were viable and fertile. To verify the function of mFoxa2^LSL locus, we generated Pgr^CreFoxa2cOE (Pgr^Cre/+mFoxa2^LSL/+) mice by crossing mFoxa2^LSL mice with Pgr^Cre mice (11). The Cre excision activity in the Pgr^Cre mouse model is restricted to cells that express PGR after birth, including the uterus, ovary, oviduct, pituitary gland, and mammary gland (11). Expression of Pgr is initiated in the reproductive tract only after birth, and PGR is present in the uterine LE by postnatal day (PD) 3 and in the stroma by PD 6 (27, 28). Immunohistochemical analysis of uteri from control (mFoxa2^LSL/+) PD 30 and adult GD 3.5 mice found that FOXA2 protein was only in the endometrial glands [Fig. 1(a)]. In contrast, FOXA2 protein was present in the LE, GE, stroma, and inner layer of the myometrium of immature PD 30 Pgr^CreFoxa2cOE mice. In adult Pgr^CreFoxa2cOE mice, FOXA2 protein was observed in the stroma, glands, and myometrium; however, many of the LE cells were not FOXA2 positive. In control (mFoxa2^LSL/+) mice, FOXA2 was observed only in the epithelia of oviduct and cervix and not in the ovary (Supplemental Fig. 2). In Pgr^CreFoxa2cOE mice, FOXA2 was observed in the epithelia and stroma of the oviduct and cervix but not in the ovary. In situ hybridization localized Foxa2 mRNA in the same cells as FOXA2 protein in both control and Pgr^CreFoxa2cOE adult mice [Fig. 1(c)]. Real-time quantitative PCR analysis found that Foxa2 mRNA levels were substantially higher (P < 0.01) in the uterus of Pgr^CreFoxa2cOE than in those of control mice on both PD 30 and GD 3.5 [Fig. 1(d)]. Western blot analysis found that the FOXA2 protein level was substantially higher in the uterus of adult Pgr^CreFoxa2cOE mice as compared with control uteri [Fig. 1(e)].

Figure 1.

FOXA2 expression in control and Foxa2 cOE females. (a) Immunoreactive FOXA2 in cross-sections of uteri from control and Pgr^CreFoxa2cOE female mice on PD 30, GDs 2.5, 3.5, and 4.5 (n = 4 mice per group). (b) Immunoreactive FOXA2 in cross-sections of uteri from control and Ltf^iCreFoxa2cOE female mice on PD 30 and GD 3.5 (n = 4 mice per group). (c) In situ localization of Foxa2 mRNA in cross-sections of uteri from control, Pgr^CreFoxa2cOE, and Ltf^iCreFoxa2cOE mice on GD 3.5. (d) Quantitative analysis of Foxa2 mRNA in uteri of control, Pgr^CreFoxa2cOE, and Ltf^iCreFoxa2cOE mice on PD 30 and GD 3.5 (n = 4 mice of each genotype per day; *P < 0.01). (e) Western blot analysis of FOXA2 in uteri from control, Pgr^CreFoxa2cOE, and Ltf^iCreFoxa2cOE mice on GD 3.5 (n = 2 mice per genotype). ge, glandular epithelium; le, luminal epithelium; m, myometrium; s, stroma.

Next, mFoxa2^LSL mice were crossed with Ltf^iCre mice, which express Cre recombinase only in the luminal and glandular epithelia of the uterus after puberty (12). Before puberty in immature PD 30 mice, FOXA2 was present only in the GE of the uterus of both control (mFoxa2^LSL/+) and Ltf^iCreFoxa2cOE (Ltf^iCre/+mFoxa2^LSL/+) mice [Fig. 1(b)]. In the uterus of adult control mice, FOXA2 was also solely expressed in the GE. In the Ltf^iCreFoxa2cOE mice, FOXA2 protein was in the GE as well as in a few cells in the LE [Fig. 1(b)]. As expected, Foxa2 mRNA levels were not different (P > 0.10) in the uterus of immature PD 30 control and Ltf^iCreFoxa2cOE mice and were greater (P < 0.01) in the adult uterus of Ltf^iCreFoxa2cOE mice [Fig. 1(d)]. Thus, conditional expression of FOXA2 is limited primarily to the glands of the uterus using the Ltf^iCre driver model.

In Ltf^iCre females, Cre expression is driven by the endogenous promoter of Ltf, which is an E2-responsive gene in the mouse uterus (29), and Cre recombinase activity in immature Ltf^iCre mice can be induced by estrogen receptor α (ESR1) agonists (30). Immature Ltf^iCreFoxa2cOE mice were injected subcutaneously with either vehicle (sesame oil) or E2 (100 ng per mouse per day) on PDs 20 and 21 and collected on PD 22. Both iCre and Foxa2 mRNA levels were clearly increased by E2 treatment [Fig. 2(a) and 2(b)]. FOXA2 protein was present in the GE of Ltf^iCreFoxa2cOE mice receiving vehicle as well as E2 treatment [Fig. 2(c)]. As in adult Ltf^iCreFoxa2cOE mice [Fig. 1(b)], a few cells in the LE were also FOXA2 positive [Fig. 2(c)], which could be immune cells rather than LE cells. Thus, the mFoxa2^LSL allele is effectively recombined and FOXA2 overexpressed in the GE of the uterus in the Ltf^iCre model, but expression is not observed in most LE cells.

Figure 2.

Effects of neonatal estrogen treatment on iCre and FOXA2 expression in Ltf^iCreFoxa2cOE female mice. Mice were injected subcutaneously with either vehicle (sesame oil) or E2 (100 ng per mouse per day) on PDs 20 and 21 (n = 3 mice per group). Uteri were collected on PD 22. (a) PCR analysis of iCre and Gapdh mRNA in the uterus. (b) Quantitative analysis of Foxa2 mRNA in uteri. Foxa2 mRNA levels were higher (*P < 0.01) in E2-treated mice. (c) Immunoreactive FOXA2 in cross-sections of uteri. Note the presence of immunoreactive FOXA2-positive cells in the LE (arrowheads) of E2-treated mice. ge, glandular epithelium; le, luminal epithelium.

Effects of FOXA2 conditional expression on female fertility

Both Pgr^CreFoxa2cOE and Ltf^iCreFoxa2cOE females displayed normal mating behavior, indicated by the presence of copulatory plugs in the vagina after mating. A 6-month fertility trial was conducted with control and Foxa2cOE mice (Table 2). As compared with control females, the Pgr^CreFoxa2cOE females were infertile, whereas the Ltf^iCreFoxa2cOE females displayed normal fertility. Next, mice were bred to intact fertile CD-1 males. Embryo implantation was assessed in nulliparous females on GD 5.5 by intravenous injection of a macromolecular blue dye that accumulates at sites of increased vascular permeability and can be used to visualize the location of embryo implantation (31). Implantation sites were easily discernible on GD 5.5 in the uterus of control and Ltf^iCreFoxa2cOE mice, but Pgr^CreFoxa2cOE mice had no visible implantation sites [Fig. 3(a)]. After the uterine lumen of mated control (n = 6) and Pgr^CreFoxa2cOE (n = 7) females was gently flushed with saline on GD 3.5, an average of six embryos were recovered from the uterine lumen of each control female, but only one embryo was recovered from the Pgr^CreFoxa2cOE females (data not shown).

Table 2.

Female Fertility Assessed in a 6-Month Breeding Trial

Genotype	n	Total Litters	Total Pups	Pups per Litter
Control mFoxa2LSL/+	6	48	432	9.0 ± 2.4
PgrCr/+eFoxa2LSL/+	6	0	0	0
LtfiCre/+Foxa2 LSL/+	6	46	404	8.8 ± 1.8

Figure 3.

Effects of FOXA2 overexpression on uterine function and histoarchitecture. (a) Embryo implantation sites (arrows) were observed in control and Ltf^iCreFoxa2cOE mice but not in Pgr^CreFoxa2cOE mice on GD 5.5 (n = 5 per genotype). (b) Uterine wet weight on GD 3.5 was lower (*P < 0.01) in Pgr^CreFoxa2cOE than control and Ltf^iCreFoxa2cOE mice (n = 4 mice per genotype). (c) Immunoreactive FOXA2 in the uterus of adult Pgr^CreFoxa2cOE mice during early pregnancy (n = 4 mice per genotype per GD). (d) Uterine gland number on GD 3.5 is substantially lower (*P < 0.01) in Pgr^CreFoxa2cOE than in control and Ltf^iCreFoxa2cOE mice (n = 6 mice per genotype). ge, glandular epithelium; le, luminal epithelium; s, stroma.

Conditional expression of FOXA2 in the uterus elicits epithelial stratification

Uterine wet weight was lower (P < 0.01) in Pgr^CreFoxa2cOE compared with control or Ltf^iCreFoxa2cOE mice on GD 3.5 [Fig. 3(b)]. Histological analysis revealed that the uterus of Pgr^CreFoxa2cOE mice appeared to have fewer uterine glands than control mice [Figure 3(c)], whereas the Ltf^iCreFoxa2cOE mice had normal uterine histoarchitecture. Indeed, the number of glands per transverse cross-section of the uterus was substantially reduced (P < 0.01) in the uterus of Pgr^CreFoxa2cOE as compared with control or Ltf^iCreFoxa2cOE mice [Fig. 3(d)]. In addition to reduced gland number, several areas of the endometrium from adult Pgr^CreFoxa2cOE mice displayed areas of uncharacteristic bilaminar or stratified epithelium [Fig. 3(c)]. Of note, the stratified regions of epithelium were not present throughout the entire uterus of nulliparous adult Pgr^CreFoxa2cOE mice. The upper layer of cells in the stratified areas of LE were FOXA2 positive in the Pgr^CreFoxa2cOE uterus, which is abnormal because FOXA2 is never present in the LE of control mice [Fig. 3(c)]. As shown in Supplemental Fig. 2, FOXA2 is normally expressed in the upper layer of the stratified squamous type epithelium of the cervix (13) but not vagina (32).

The abnormal stratified LE and GE in the uterus of Pgr^CreFoxa2cOE mice was confirmed by immunostaining for tumor protein 63 (TP63) and keratin 14 (KRT14), two markers of epithelial stratification and basal cells (33, 34). Immunostaining revealed that KRT14- and P63-positive basal cells could be observed in discrete areas of stratified epithelia in the immature PD 30 uterus from Pgr^CreFoxa2cOE mice [Fig. 4(a) and 4(b)]. The KRT14- and P63-positive basal cells persisted into adulthood in the uterus of Pgr^CreFoxa2cOE mice. Of note, embryo implantation can be compromised in mice whose uteri contain a stratified squamous LE or lack uterine glands (35).

Figure 4.

Basal cells are present in areas of stratified LE in uteri of Pgr^CreFoxa2cOE mice. (a) Immunoreactive KRT14 in the uterus of control and Pgr^CreFoxa2cOE mice on PD 30 and GDs 2.5, 3.5, and 4.5 (n = 4 mice per genotype per day). (b) Immunoreactive P63 in the uterus of control and Pgr^CreFoxa2cOE mice on PD 30 and GDs 2.5, 3.5, and 4.5 (n = 4 mice per genotype per day). Note the presence of KRT14- and P63-positive basal cells in the LE of the Pgr^CreFoxa2cOE uterus. ge, glandular epithelium; le, luminal epithelium; m, myometrium; s, stroma.

Temporal and spatial alterations in expression of the PGR, ESR1, and Ki67, a marker of cell proliferation (36), was conducted in the uterus of early pregnant mice (Fig. 5). Overall, the patterns of PGR and ESR1 expression were not different in the uterus of control and Pgr^CreFoxa2cOE mice. Cell proliferation appeared to be increased in the uterine LE and GE of GD 3.5 Pgr^CreFoxa2cOE mice, and Ki67-positive cells remained in the stratified areas of the LE on GD 4.5. Stromal cell proliferation was noticeably suppressed in the uterus of Pgr^CreFoxa2cOE mice on GDs 3.5 and 4.5. Expression of CXCL15, a gland-specific gene (37), declined in the GE of both control and Pgr^CreFoxa2cOE mice after GD 3.5 but was expressed in the areas of stratified LE in the Pgr^CreFoxa2cOE uterus (Fig. 5).

Figure 5.

Expression of steroid receptors, cell proliferation (Ki67), and CXCL15 in adult control and Pgr^CreFoxa2cOE uteri. Control and Pgr^CreFoxa2cOE mice were analyzed on GDs 2.5, 3.5 and 4.5 (n = 4 mice per genotype per day). ge, glandular epithelium; le, luminal epithelium; s, stroma.

Genes regulated by estrogen and progesterone and involved in uterine receptivity for embryo implantation were measured in the early pregnant uterus (Fig. 6). Expression of a number of E2-responsive genes (C3, Clca3, Cxcl15, Ltf, Muc1) was higher (P < 0.01) in the uterus of Pgr^CreFoxa2cOE as compared with control mice. With the exception of Areg, expression of progesterone-regulated genes (Gata2, Hoxa10, Ihh) was not different (P > 0.01) in the control and Pgr^CreFoxa2cOE uterus during early pregnancy. Consistent with the decreased numbers of uterine glands in Pgr^CreFoxa2cOE mice, expression of GE-specific genes (Lif, Prss29, Spink3, Ttr) was substantially decreased (P < 0.01) in the uterus of Pgr^CreFoxa2cOE during early pregnancy. Although CXCL15 is a GE-specific gene in the normal mouse uterus (37) (Fig. 6), levels of Cxcl15 mRNA were substantially higher in the Pgr^CreFoxa2cOE uterus throughout early pregnancy (Fig. 6), probably because of expression in the areas of stratified LE (Figs. 4 and 5).

Figure 6.

Expression of E2- and P4-responsive and gland-specific genes in the uterus during early pregnancy. Control and Pgr^CreFoxa2cOE mice were analyzed on GDs 2.5, 3.5, and 4.5 (n = 4 mice per genotype per day). *P < 0.01.

Effects of ovariectomy and steroid hormones in immature Pgr^CreFoxa2cOE mice

Expression of FOXA2 was clearly observed in the LE of uteri from immature PD 30 Pgr^CreFoxa2cOE mice, but the majority of LE cells in adult Pgr^CreFoxa2cOE mice do not express FOXA2 [Fig. 1 and Fig. 7(a)]. To determine the effect of ovarian hormones on FOXA2 overexpression in the LE, PD 30 Pgr^CreFoxa2cOE mice were ovariectomized and rested for 10 days. Beginning on PD 40, ovariectomized mice were treated daily with either vehicle, P4 (1 mg) or E2 (100 ng), and then necropsied on PD 50. Expression of FOXA2 was maintained in the uterus of ovariectomized Pgr^CreFoxa2cOE mice receiving either vehicle or P4 [Fig. 7(b)]. In contrast, most of the LE cells were FOXA2 negative in E2-treated mice; however, the stroma, GE, and myometrial cells remained FOXA2 positive. Note that expression of the PGR and Ki67 cell proliferation marker was increased in the LE by E2 treatment.

Figure 7.

Effects of puberty and steroid hormones on FOXA2 expression in Pgr^CreFoxa2cOE mice. (a) FOXA2 expression is lost from the LE of the uterus between PD 30 and 70. (b) Pgr^CreFoxa2cOE mice were ovariectomized on PD 30, rested for 10 days, and treated daily with subcutaneous injections of vehicle (sesame oil), P4 (1 mg), or E2 (100 ng) for 10 days (n = 4 mice per genotype per day). Immunoreactive FOXA2, PGR, Ki67, P63, and KRT14 were evaluated with immunohistochemistry.

Discussion

In the current study, conditional overexpression of FOXA2 was achieved in the neonatal uterus with the Pgr-Cre model and in the adult uterus with the Ltf-iCre model. In the uterus, the Pgr-Cre driver produced conditional FOXA2 expression in the LE, GE, stroma, and inner circular layer of myometrium, which phenocopies expression of the PGR (11). In wild-type mice, FOXA2 expression is specifically restricted to the differentiating and developing GE of the mouse uterus (38, 39). Curiously, expression of FOXA2 was not present in most of the LE cells of the adult uterus of Pgr^CreFoxa2cOE mice despite those cells uniformly expressing PGR. The loss of FOXA2 expression was observed by PD 70. The ovariectomy experiment suggests that estrogen is involved in the loss of LE cells that overexpress FOXA2. Although some LE cells remain FOXA2 positive in the adult uterus of adult Pgr^CreFoxa2cOE mice, those cells are clearly stratified and not normally present in the uterus. On PD 30, only a few cells in the LE of the Pgr^CreFoxa2cOE uterus were FOXA2-negative. Thus, one plausible explanation is that the subset of FOXA2-negative LE cells selectively proliferate in the prepubertal uterus under the influence of estrogen, which is supported by findings that FOXA2 is a tumor suppressor and controls the proliferation of endometrial epithelial cells (7). Indeed, FOXA2 is downregulated or inactivated by mutation during the development of endometrial epithelial hyperplasia and cancer (7, 22, 40). A second possibility is that an unknown mechanism silenced expression of PGR-A, which is used to drive the Cre transgene in Pgr-Cre mice (11). A third possibility is that the mFoxa2 transgene is progressively and selectively silenced in the LE cells of the Pgr^CreFoxa2cOE mice as the mice age and become adults. In mice, the Ltf gene is estrogen-responsive and specifically expressed in the luminal and GE of the adult uterus (30), and Cre expression in the Ltf-iCre driver mouse model recombines floxed alleles in both the luminal and GE (12). However, FOXA2 overexpression was observed only in the GE of adult Ltf^iCreFoxa2cOE mice. Indeed, expression of Foxa2 mRNA or FOXA2 protein was not observed in the LE cells of the uterus in adult Ltf^iCreFoxa2cOE mice. Those results support the idea that the FOXA2 transgene is silenced specifically in the LE by some unknown epigenetic mechanism. Indeed, the only current known mechanisms regulating FOXA2 gene expression involve microRNA targeting of the 3′ UTR and control of FOXA2 mRNA stability (41). Additional analysis of the conditional FOXA2 overexpression models developed here could provide insights into FOXA2 regulation in vivo.

The development of a stratified LE with basal cells in the uterus of Pgr^CreFoxa2cOE mice was an unexpected finding, particularly because the majority of LE cells in those adult mice were negative for FOXA2 expression. The presence of basal cells was confirmed by coexpression of TP63 and KRT14, which are basal cell markers and expressed in those cells in the cervix and vagina (42, 43). The expression of TP63 is essential for the stratified squamous differentiation because the epithelium of the Müllerian vagina in Tp63 null mice formed a uteruslike layer of columnar epithelium (42). In the current study, FOXA2 expression in the LE of adult Pgr^CreFoxa2cOE mice was confined to the nuclei of the upper, non–basal cell layer of the stratified epithelia. This same pattern of FOXA2 expression was observed in the cervix of control mice. The biological role, if any, of FOXA2 in the cervical epithelium is not known. Epithelial stratification is observed in the uterus of conditional deletion mutants of Wnt4 and Fgfr2 and conditional overexpression of Notch1 created with the Pgr^Cre model (35, 44–46). The development of stratified LE in the Pgr^CreFoxa2cOE uterus is probably caused by altered stromal-epithelial communication due to misexpression of FOXA2 in the stroma and myometrium (35). Stratified LE was not observed in the uterus of adult Ltf^iCreFoxa2cOE mice that were completely fertile.

In contrast to Ltf^iCreFoxa2cOE mice, adult Pgr^CreFoxa2cOE mice were infertile. The underlying cause of this infertility may be oviductal or cervical dysfunction, because few embryos were found in the uterine flush of GD 3.5 Pgr^CreFoxa2cOE mice or in their uteri on GD 4.5. The ciliary epithelium appeared less folded in Pgr^CreFoxa2cOE mice, but defects in cervix or uterine function may also be responsible for the infertility. In addition to areas of stratified LE, the uterus of Pgr^CreFoxa2cOE mice exhibited reduced numbers of uterine glands, based on histology, and reduced or absent expression of Lif, Prss29, Spink3, and Ttr, which are expressed only in the GE of GD 2.5 to 4.5 uteri (38, 47). The glands of the uterus begin to differentiate from the LE and develop after PD 5 in the uterus of neonatal mice (39, 48, 49). Conditional deletion of Foxa2 using Pgr-Cre driver mice prevents the genesis and differentiation of endometrial glands in the neonatal uterus, rendering a glandless uterus in the adult (4, 5). Of note, conditional expression of Notch1 in the uterus of Pgr-Cre driver mice aberrantly upregulates FOXA2 expression in the LE, and the uterus is glandless in adult PgrCre-NotchcOE mice (44). In addition to an absence of glands, E2- and P4-responsive genes were dysregulated in the Pgr^CreFoxa2cOE uterus. Thus, misexpression of FOXA2 in the LE or stroma of the developing neonatal mouse uterus alters or inhibits normal differentiation and genesis of endometrial glands and function of the adult uterus.

The current study used homologous recombination in ES cells to generate mice in which the expression of FOXA2 can be activated in a spatial- and temporal-specific fashion. As noted by Wu et al. (23), advantages of this system include: (1) fast generation of recombination construct; (2) no expression in the absence of Cre recombination; (3) a single mouse line to express a gene of interest in any given tissue or cell type of interest with available tissue-specific Cre recombinase mouse lines; (4) feasible temporal expression when inducible expression of Cre is used (e.g., tamoxifen- or tetracycline-inducible Cre); and (5) uniform expression levels wherever activated. Results of the current study and others (23, 50–52) confirm the utility of this overexpression strategy for FOXA2 and other transcription factors in studies of uterine development and function. Applications of this tool can be multiple, including mimicking human diseases, directing cell differentiation, reprogramming cell identity, and dissecting genetic pathways. For example, FOXA2 is essential for differentiation and development of many endoderm-derived structures in the embryo, such as the pancreas and lung (2), and glands in the uterus in the neonate after birth (4). Besides its importance in regulating organ differentiation, FOXA2 also plays a key role in regulating cell-specific gene expression in and function of adult organs, including the liver and uterus (2, 13). Furthermore, FOXA2 is a tumor suppressor that is involved in a number of different cancers including those of the bladder, colon, liver, lung, and uterus (19, 22, 53). Thus, the FOXA2 conditional expression allele could be useful to study FOXA2 involvement in organ-specific tumor progression and metastasis. Furthermore, in combination with genetic engineered alleles of other genes that pose as cancer risk factors, this model can be used to study effects of genetic interaction or dissect signal hierarchy between FOXA2 and other genes. Finally, the conditional overexpression allele approach could be used to create a humanized mouse model to analyze FOXA2 variants in development and tumorigenesis and to screen drugs that directly target FOXA2, accelerating the development of new therapeutic modalities.

Glossary

Abbreviations:

cOE

conditional overexpression

E2

17β-estradiol

ES

embryonic stem

ESR1

estrogen receptor α

FOX

forkhead box

FOXA2

forkhead box A2

GD

gestational day

GE

glandular epithelium

LE

luminal epithelium

KRT14

keratin 14

LSL

Lox-Stop-Lox

Ltf

lactotransferrin

mRNA

messenger RNA

P4

progesterone

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

PD

postnatal day

Pgr

progesterone receptor

TP63

tumor protein 63