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Molecular Endocrinology 20 (11): 2796-2805
Copyright © 2006 by The Endocrine Society

FOXL2 in the Pituitary: Molecular, Genetic, and Developmental Analysis

Buffy S. Ellsworth, Noboru Egashira, Jodi L. Haller, Darcy L. Butts, Julie Cocquet, Colin M. Clay, Robert Y. Osamura and Sally A. Camper

Department of Human Genetics (B.S.E., D.L.B., S.A.C.), The University of Michigan, Ann Arbor, Michigan 48109-0638; Department of Pathology (N.E., R.Y.O.), Tokai University, Isehara, Kanagawa 259-1193, Japan; Animal Reproduction and Biotechnology Laboratory (J.L.H., C.M.C.), Department of Biomedical Sciences, Colorado State University, Fort Collins, Colorado 80523; and Institut National de la Santé et de la Recherche Médicale Unité 709 (J.C.), Reproduction et Physiopathologie Obstetricale, Hopital Cochin, Paris, France 75014

Address all correspondence and requests for reprints to: Sally A. Camper, The University of Michigan Medical School, 4909 Buhl Building, Ann Arbor, Michigan 48109-0618. E-mail: scamper{at}umich.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FOXL2 is a forkhead transcription factor expressed in the eye, ovary, and pituitary gland. Loss of function mutations in humans and mice confirm a functional role for FOXL2 in the eye and ovary, but its role in the pituitary is not yet defined. We report that FOXL2 colocalizes with the glycoprotein hormone {alpha}-subunit ({alpha}GSU) in quiescent cells of the mouse pituitary from embryonic d 11.5 through adulthood. FOXL2 is expressed in essentially all gonadotropes and thyrotropes and a small fraction of prolactin-containing cells during pregnancy, but not somatotropes or corticotropes. The coincident expression patterns of FOXL2 and {alpha}GSU suggested that the {alpha}GSU gene (Cga) is a downstream target of FOXL2. We demonstrate that FOXL2 regulates mouse Cga transcription in gonadotrope-derived ({alpha}T3-1, LßT2), thyrotrope-derived ({alpha}TSH) and heterologous (CV-1) cells in a context-dependent manner. In addition, a FOXL2-VP16 fusion protein is sufficient to stimulate ectopic Cga expression in transgenic animals. Normal FOXL2 expression requires the transcription factors Lhx3 and Lhx4 but not of Prop1. Thus, FOXL2 expression is affected by mutations in early pituitary developmental regulatory genes, and its expression precedes that of genes necessary for gonadotrope-specific development such as Egr1 and Sf1 (Nr5a1). These data place FOXL2 in the hierarchy of pituitary developmental control and suggest a role in regulation of Cga gene expression.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FORKHEAD FACTORS ARE a class of transcription factors that are important for diverse developmental processes (1). Deficiencies of individual forkhead genes have revealed roles in speech and language disorders, diabetes, immunodeficiency, cleft palate, and eye development (1). One member of the forkhead family, Foxl2, is expressed in the eye, the ovary, and the pituitary (2, 3). It is one of the earliest markers of ovarian differentiation in mammals (4). Loss of function mutations in FOXL2 cause blepharophimosis, ptosis, and epicanthus inversus syndrome, a dominant disorder characterized by eyelid malformations and premature ovarian failure (POF) in some women (2, 5, 6). The POF is revealed as either primary or secondary amenorrhea (2, 5, 6). POF results from excessive attrition of ovarian follicles in fetal life such that follicles remaining at birth are insufficient to sustain a full reproductive lifespan (2, 4, 5, 6). Thus, Foxl2 appears to represent a new member of the expanding class of forkhead DNA-binding proteins (FOXO1, FOXO3, FOXO4) implicated in the regulation of follicular growth and development (7).

Foxl2, also known as P-frk, is expressed in the developing pituitary gland at embryonic d 10.5 (e10.5) and e12.5 (3, 8). However, key questions remain concerning the onset of expression, which pituitary cell types express Foxl2, and the nature of the upstream regulators and downstream targets of Foxl2. The only known pituitary target is Gnrhr (9), although several genes are implicated as potential targets in the ovary (10, 11). Individuals with blepharophimosis, ptosis, and epicanthus inversus syndrome have one normal allele of FOXL2 and do not have an obvious pituitary phenotype. Thus, FOXL2 may have a role in pituitary development that is not evident in heterozygotes if the pituitary is less sensitive than either the eye or the ovary to reduction in FOXL2 levels. This dosage effect is observed with other transcription factors such as PITX2 (12). Mouse knockouts of Foxl2 reveal that most of the homozygous mutant mice are not viable and have severe craniofacial abnormalities (13, 14). The surviving mice are smaller than their wild-type littermates and have reduced levels of serum IGF-I (13), which could be indicative of pituitary hormone insufficiency. Studies of the viable mutant mice have focused on the role of FOXL2 in the ovaries and, to date, no analysis of the pituitary phenotype has been reported.

We report a comprehensive analysis of the developmental regulation and cell-specific expression of FOXL2 protein in the pituitary gland. Its expression precedes that of most gonadotrope and thyrotrope markers, and it regulates glycoprotein hormone {alpha}-subunit ({alpha}GSU) expression in cell lines derived from gonadotropes and thyrotropes, and in transgenic mice. These results suggest a role for FOXL2 in early pituitary development and regulation of glycoprotein hormone production. In addition, we establish the position of FOXL2 in the genetic hierarchy of transcription factors that are essential for pituitary development and function.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
FOXL2 Protein Is Present in Quiescent Cells of the Anterior Lobe of the Pituitary from e11.5 through Adulthood
We performed immunohistochemical analysis of FOXL2 expression using the C-FOXL2 antibody (4) during embryonic mouse pituitary development. We did not detect FOXL2 protein at e10.5 (Fig. 1AGo). FOXL2 immunoreactivity was first detected at e11.5 in the developing anterior lobe of the pituitary and persisted in the anterior lobe throughout development (Fig. 1Go, C, E, and H) and into adulthood (Fig. 1NGo), which is consistent with a possible maintenance role in pituitary function.


Figure 1
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  		Fig. 1. FOXL2 Is Detected in Quiescent Anterior Pituitary Cells from e11.5 through Adulthood

Immunohistochemistry was performed with an antibody directed against the C terminus of FOXL2 (4 ) on midsagittal paraffin sections from C57 BL/6J mice. White brackets indicate the anterior lobe. A–H and N, FOXL2 and {alpha}GSU proteins are first detected at e11.5 and persist through adulthood. G and H, Mice were injected with BrdU 2 h before being killed to label cells in S-phase. BrdU incorporation is detected with immunohistochemistry and development with rhodamine (red). FOXL2 is labeled with FITC (green), and cell nuclei are labeled with 4',6-diamidino-2-phenylindole (DAPI) (blue). FOXL2 is confined to the nonproliferating cells of the anterior lobe at e12.5 (panel G) and is still not expressed in proliferating cells at e18.5 (panel H). I, This section is from an e18.5 embryo. J–M, These sections are from e17.5 embryos. I–M, FOXL2 immunoreactivity is shown in red and the following hormones are shown in green: {alpha}GSU (panel I), LHß (panel J), K) TSH (panel K), ACTH (panel L), and GH (panel M). N, This section is from a pregnant female 12.5 d after conception. PRL is labeled with alkaline phosphatase (dark purple), and FOXL2 is labeled with DAB (brown). FOXL2 is expressed in gonadotropes, thyrotropes, and a small fraction of PRL-producing cells in pregnant females. A–H, Scale bars, 50 µm. I–N, Scale bars, 1 µm. aGSU, {alpha}GSU; LHb, LHß; TSHb, TSHß.

 
To determine whether FOXL2 is expressed in undifferentiated, proliferating cells or differentiated, nonproliferating cells, we costained with antibodies to FOXL2 and bromodeoxyuridine (BrdU), a marker for actively dividing cells in S-phase. FOXL2 does not colocalize with BrdU labeled cells at e12.5 (Fig. 1GGo) or e18.5 (Fig. 1HGo). FOXL2 is found below a band of nonproliferating, undifferentiated cells and is colocalized with nonproliferating, differentiating cells, marked by {alpha}GSU (Fig. 1Go, E, F, and I). This is consistent with the idea that FOXL2 has a role(s) in promoting differentiation in the pituitary.

FOXL2 immunoreactivity spatially and temporally coincides with that of {alpha}GSU immunoreactive cells (Fig. 1Go, A–F and I). Consistent with this, FOXL2 colocalizes with LHß and TSHß, but not with ACTH or GH (Fig. 1Go, J–M). FOXL2 does not colocalize with prolactin (PRL) in males or nonpregnant females (data not shown). During pregnancy, however, FOXL2 is detectable in a small fraction of lactotropes (Fig. 1NGo). These cells may be in the process of converting to lactotropes to meet the requirements for increased PRL production during pregnancy (15).

FOXL2 Regulates {alpha}GSU Gene (Cga) Expression
Because FOXL2 is found in gonadotropes and thyrotropes, we tested for regulation of the hormone subunit genes expressed in these cells (Lhb, Fshb, Tshb, Cga). We detected no obvious regulation of Lhb, Fshb, or Tshb by FOXL2 in cell culture (data not shown), although the promoter fragments we tested are regulated by other transcription factors in cell culture (16, 17, 18). However, we found that the Cga promoter is regulated by FOXL2 in a context- and dose-dependent manner. High doses of FOXL2 repress, whereas low levels stimulate, Cga expression in the gonadotrope-derived {alpha}T3-1 (19) (Fig. 2AGo). In the thyrotrope-derived cell line, {alpha}TSH (20) low doses had no effect whereas high doses repressed promoter activity (Fig. 2BGo). In the gonadotrope-derived cell line, LßT2 (21, 22), which is thought to be more differentiated than {alpha}T3-1, and a heterologous cell line, CV-1 (23, 24), both low and high doses stimulate Cga expression (Fig. 2Go, C and D). The obligate activator, FOXL2-VP16, stimulated Cga in all cell lines (Fig. 2Go, A–D). The different effects of FOXL2 in these cell lines do not appear to be due to varying levels of endogenous FOXL2 expression because the levels are similar in {alpha}T3-1, {alpha}TSH, and LßT2 as measured by real time RT-PCR (data not shown).


Figure 2
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  		Fig. 2. FOXL2 Regulates Cga in Cell Culture

A vector containing 341 bp of the Cga promoter fused to luciferase was transiently transfected into {alpha}T3-1 (panel A), {alpha}TSH (panel B), LßT2 (panel C), or CV-1 cells (panel D) along with 2, 10, or 50 ng of a vector expressing FOXL2 or FOXL2 fused to the activation domain of the viral protein, VP16, or the empty expression vector (pcDNA3.1+). Asterisks (*) represent values that are significantly different from the pcDNA control (P < 0.05). Experiments were performed in triplicate and repeated three times. At least two different plasmid preparations were used.

 
To test these findings in vivo, we generated transgenic mice that overexpress FOXL2. Because the context and concentration of FOXL2 influences its function as an activator or repressor in cell culture, we fused the FOXL2 coding sequences with the activation domain from the viral protein VP16. Fertilized eggs were injected at the one-cell stage with a construct containing the Cga promoter and enhancer fused to the Foxl2-Vp16 coding sequence and an intron and polyadenylation sequences from mouse protamine 1 (mP1). These mice exhibit primarily appropriate expression of FOXL2 within the anterior lobe, with some ectopic expression in the prospective intermediate lobe and nearby tissue as indicated by mP1 in situ hybridization (Fig. 3Go, A, D, and G) and FOXL2 immunostaining (Fig. 3Go, B, E, and H). {alpha}GSU is expressed in all areas that express the FOXL2 transgene including ectopic regions (Fig. 3Go, C, F, and I). This demonstrates that FOXL2-VP16 is sufficient to stimulate expression of the Cga gene in vivo, even at ectopic sites.


Figure 3
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  		Fig. 3. FOXL2 Stimulates Expression of {alpha}GSU in Transgenic Mice

Transgenic mice were generated that overexpress FOXL2 fused to the activation domain of VP16 under the control of the Cga promoter and enhancer sequences. A mouse protamine 1 intron and polyadenylation sequences follow the Foxl2-Vp16 coding sequence. In situ hybridization for mP1 was used to assess expression of the transgene in midsagittal paraffin sections from e14.5 transgenic mice (panels A, D, and G). A–C, Nonexpressor. D–I, Expressors. Immunohistochemistry was performed to assess FOXL2 (red) (B, E, and H) and {alpha}GSU (green) (C, F, and I) expression. Ectopic expression of the Foxl2 transgene and {alpha}GSU is denoted by arrows. Ectopic FOXL2 is accompanied by ectopic {alpha}GSU, suggesting that activated FOXL2 is sufficient to stimulate ectopic expression of the Cga gene. Expression of {alpha}GSU is enriched in the anterior lobe of the Foxl2-Vp16 transgenic mice as compared with wild-type samples.

 
FOXL2 Is Downstream of Lhx3 and Lhx4 in the Genetic Hierarchy of Pituitary Developmental Control
We and others have established a genetic hierarchy of the transcription factors that orchestrate pituitary development (25, 26, 27). To determine how Foxl2 fits into this hierarchy, we analyzed FOXL2 expression in mice with mutations in several critical genes that are already placed into the genetic hierarchy. Lhx3 and Lhx4 are LIM homeodomain factors that are expressed at e9.5 in the oral ectoderm that will form Rathke’s pouch, the primordial for the anterior and intermediate lobes. By e12.5 Lhx4 expression is restricted to the developing anterior lobe, and it diminishes progressively until e15.5. In contrast, Lhx3 expression is maintained throughout Rathke’s pouch and the prospective anterior and intermediate lobes throughout pituitary development (28). Lhx3 and Lhx4 have overlapping functions, and Lhx4 is required for timely activation of Lhx3 and for cell survival (28, 29). We analyzed FOXL2 expression in the pituitaries of Lhx3 and Lhx4 knockout mice and found that FOXL2 is not expressed in Lhx3 knockout mice at e12.5 (Fig. 4Go, A and B) or just before birth at e18.5 (Fig. 4Go, C and D). In contrast, Lhx4 mutant mice exhibit reduced FOXL2 expression at e12.5 (Fig. 4Go, E and F), but FOXL2 expression recovers by e16.5 (Fig. 4Go, G and H), when Lhx3 expression is strong. Thus, Foxl2 expression is enhanced by LHX4 and apparently requires LHX3 directly or indirectly.


Figure 4
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  		Fig. 4. Foxl2 Is Downstream of Lhx3 and Lhx4 and Independent of Prop1 in the Genetic Hierarchy of Pituitary Developmental Control

FOXL2 expression was analyzed by immunohistochemistry in mice homozygous for mutations (n = 3) in Lhx3 (panels A–D), Lhx4 (n = 1) (panels E–H), or Prop1 (n = 3) (panels I–L). FOXL2 immunoreactivity is shown in green. The developing anterior lobe is indicated by brackets. Dotted lines outline the pituitary gland (B, D, and F). White scale bars, 50 µm. FOXL2 expression is lost in the absence of Lhx3 and delayed in the absence of Lhx4, but it is not affected in Prop-deficient mice. M, This model illustrates the place of Foxl2 place in the genetic hierarchy of pituitary developmental control. These genetic relationships do not imply direct transcriptional regulation. Lhx3 expression is lost in double knockouts of the genes encoding homeodomain factors PITX1 and PITX2, indicating that Lhx3 is genetically downstream of these PITX genes (56 ). Mutations in Lhx3 eliminate FOXL2 expression, whereas mutations in Lhx4 delay expression of FOXL2. Prop1 is necessary for differentiation of the Pit1 lineage (somatotropes, lactotrope, and thyrotrope cells) but is not necessary for appropriate expression of FOXL2. FOXL2 stimulates expression of the Cga gene ({alpha}GSU) in cell culture and transgenic mice. Cells expressing Cga will differentiate into thyrotropes and gonadotropes. Cartoon representations illustrate the pituitary expression patterns of different transcription factors and gene products at e12.5 and e14.5.

 
Prop1 is a paired-like homeobox transcription factor gene expressed only in the mouse pituitary anlage from e10–e15. It is important for expansion of Rathke’s pouch and the Pit1 lineage, but it is not essential for gonadotrope commitment (26). The expression patterns of Prop1 and FOXL2 are mutually exclusive at e12.5 (30, 31) exhibiting dorsal and ventral expression, respectively. Lhx3 and Isl1 recognize a similar dorsal ventral boundary that requires Lhx4 (29). In Lhx4-deficient mice, Lhx3 is expressed only in the most dorsal tip of Rathke’s pouch, whereas expression of Isl1, a transcription factor normally expressed only in the developing anterior lobe, spreads dorsally. To test whether the same is true for Foxl2 and Prop1, we looked for changes in FOXL2 expression in Ames dwarf mice (Prop1df) during pituitary development. These mutants have a missense mutation in the homeodomain of Prop1 resulting in essentially no activity in vivo (30, 31). There are no obvious alterations in the level or spatially restricted pattern of FOXL2 expression at e12.5 or e15.5 in mutants relative to wild-type mice (Fig. 4Go, I–L), demonstrating that FOXL2 expression is independent of Prop1 (Fig. 4MGo).

We extended this line of investigation by assessing FOXL2 expression in mice that overexpress Prop1 (32), knockout mice for the transcription factors Gata2 (33) and Tcf7l2 (Tcf4) (34), and the signaling factors Wnt4 (35) and Wnt5a (36). FOXL2 expression was not altered in the pituitaries of any of these mice, suggesting that Foxl2 expression is independent of each of these genes in the genetic hierarchy of pituitary development (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
To understand the role of FOXL2 in pituitary gland development and function, we have characterized its spatial and temporal expression patterns, cell specificity of expression, and genetic requirements for normal expression. We report that FOXL2 is expressed early in pituitary development, before the gonadotrope transcription factors Sf1 (Nr5a1) and Egr1, and the thyrotrope factor Pit1. Its expression is spatially and temporally consistent with expression of Gata2 and Isl1, factors involved in gonadotrope and thyrotrope function (8, 33, 37). Thus, Foxl2 is one of the earliest markers of differentiating pituitary cells (Fig. 4MGo). Foxl2 is expressed ventrally in the pituitary with quiescent differentiating cells, suggesting a possible role in suppressing cell proliferation and/or promoting cell differentiation. FOXL2 was also seen in nonproliferating cells in the ovary where it is essential for differentiation of granulosa cells (13, 14).

Many forkhead proteins regulate proliferation and act as tumor suppressors. For example, FOXO factors function as tumor suppressors by blocking proliferation and promoting apoptosis (38, 39), and FOXL1 inhibits proliferation in the gut through activation of the Wnt pathway (40). Forkhead factors also promote differentiation, including FOXD3 (41), FOXA1 and 2 (42), and FOXO1 (43). Like these forkheads, FOXL2 may play a role in cell cycle regulation, tumor suppression, and/or cell differentiation.

Endogenous Cga expression is concomitant with that of FOXL2 (Fig. 1Go), consistent with the idea that Cga is a FOXL2 target gene. We demonstrate that FOXL2 regulates Cga gene expression in a context-dependent manner. FOXL2 represses Cga expression at high concentrations and stimulates expression at low concentrations in pregonadotrope-like cells. This duality of function is similar to the related factor FOXG1, which inhibits expression of the neuronal differentiation marker, N-tubulin, when present at high levels, but increases expression at lower concentrations (44). In more differentiated gonadotrope-like cells and in heterologous CV-1 cells, FOXL2 stimulates Cga at all concentrations tested. In fact, in CV-1 cells the level of FOXL2 stimulation is equal to or greater than that of a FOXL2-VP16 fusion protein. The difference in concentration-dependent function among cell types is likely due to the complement of corepressors and/or coactivators present in each cell type. FOXL2 acts as a direct repressor of the gene encoding steroidogenic acute regulatory protein (10) and a direct activator of Gnrhr (9). The concomitant expression patterns of Foxl2 and Cga suggest that FOXL2 is a positive regulator of Cga expression during pituitary development, although the ability of it to repress Cga expression, when present at high concentrations in less differentiated pituitary cell lines, suggests it could be a context-dependent modulator of Cga expression.

We prove that FOXL2-VP16 is sufficient for expression of Cga in transgenic mice. Surviving Foxl2 knockout males are fertile, however, suggesting that Foxl2 is not required for adequate {alpha}GSU synthesis (13). However, pituitary gonadotropin production has not been quantified in surviving male or female knockout mice, because the examination of female infertility focused on ovarian failure (13, 14). Moreover, the fatality of most Foxl2 mutant mice suggests the possibility that modifier genes, whose expression overlaps with FOXL2, may be compensating for Foxl2 deficiency in the surviving mice, masking some important roles of Foxl2 in the pituitary and other organs. Clearly, further analysis of Foxl2 mutants will be necessary to resolve these issues.

Our studies show that Foxl2 expression is influenced by both Lhx3 and Lhx4, suggesting that Foxl2 is downstream of these genes in the genetic hierarchy of pituitary developmental control. Lhx4 deficiency causes a delay in FOXL2 expression, whereas Lhx3 deficiency results in failure to activate FOXL2 expression. This is consistent with the timing of expression; Lhx4 expression is down-regulated from e12.5 until e15.5, whereas Lhx3 is expressed at high levels in the pituitary throughout development (28). We have shown previously that timely activation of Lhx3 transcription requires Lhx4, although Lhx3 can be activated eventually in the absence of Lhx4 (29). This explains the basis for the observation that Lhx3 mutants have a more severe phenotype than Lhx4 mutants (28). These findings make sense in the context of our data, namely that Lhx3 is absolutely required for Foxl2 expression and Lhx4 deficiency simply results in a developmental delay, consistent with the time it takes for Lhx3 to be activated in the absence of Lhx4. Thus, Lhx3 and Lhx4 have overlapping functions that are necessary for normal activation of Foxl2 expression. The lack of FOXL2 protein in Lhx3 and Lhx4 mutants may result from the pituitary hypoplasia (absence of cells) or because the LIM transcription factors are direct activators of Foxl2. Several lines of evidence suggest that Foxl2 regulatory sequences are a considerable distance from the gene (>100 kb) (45, 46). Identification of these distant cis-acting elements may be necessary to assess whether LHX3 or LHX4 act as direct transcriptional activators of FOXL2.

The reciprocal expression patterns of Foxl2 and Prop1 expression suggest that FOXL2 may play a role in cell differentiation, whereas PROP1 is important for transitioning cells from active cycling to migration and differentiation. In fact, Prop1 is required for differentiation of cells in the Pit1 lineage (somatotropes, lactotropes, and thyrotropes), but is not necessary for gonadotrope specification in mice (26, 47). The opposing expression patterns of Foxl2 and Prop1 suggest the possibility that they inhibit each other’s expression, but the polarity of FOXL2 expression is unaltered in Prop1df mice, suggesting that the mechanism involved in limiting FOXL2 expression to the ventral aspect of the pituitary is independent of Prop1. This contrasts with the ectopic expression of Tle3 and Hesx1 in Prop1-deficient mice (31, 48).

Numerous transcription factors have been shown to play important roles in pituitary development by analysis of loss of function mouse models and humans with MPHD (25, 49). One of the challenges is to understand the complexity of this interacting network, to define the hierarchy of regulation, and to identify downstream targets. Here we show that Foxl2 is downstream of the LIM family and independent of Prop1. Few direct downstream targets of FOXL2 are known (9, 10); however, we have identified the gene encoding {alpha}GSU as a downstream target of FOXL2 in the pituitary. {alpha}GSU is essential for gonadotrope function and reproduction as well as thyrotrope and thyroid function. Thus, FOXL2 may contribute to modulating expression of an essential hormone gene.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Animals
Embryos were obtained from matings of wild-type mice or an intercross of heterozygous mice with mutations in Prop1df, Pitx2, Lhx3, or Lhx4; (12, 29, 50, 51). Prop1df, Pitx2, and Lhx3 mutant mice were on a C57 BL/6J background. Lhx4 mutant mice were on a mixed CF1, 129, C57 BL/6J background. The day the copulatory plug was detected was taken as e0.5. Embryos or adult pituitaries were fixed for 20 min to 24 h (depending on stage of development) in 4% paraformaldehyde in PBS (pH 7.2). All samples were washed in PBS, dehydrated in a graded series of ethanol, and embedded in paraffin. Sections (6 µm) were prepared and processed as described below. Murine ovarian tissue was immersion fixed in 4.0% paraformaldehyde. The fixed tissue was washed, dehydrated, and embedded in paraffin. Slides were prepared of 6 µm thick tissue sections to be processed for immunohistochemistry or in situ hybridization.

To detect cell proliferation, pregnant mice were injected ip with BrdU at 100 µg/g body weight, 2 h before embryo collection (52). The BrdU epitope was detected with an anti-BrdU antibody (1:100 dilution; Immunologicals Direct, Oxford, UK) and a tetramethylrhodamine isothiocyanate-conjugated antirat secondary antibody (1:200 dilution).

The University of Michigan Committee on Use and Care of Animals approved all procedures using mice. All experiments were conducted in accord with the principles and procedures outlined in the NIH Guidelines for the Care and Use of Experimental Animals.

Immunohistochemistry and in Situ Hybridization
To visualize FOXL2 in the pituitary, immunohistochemistry was performed as follows. Slides were deparaffinized in xylene, and 1.5% peroxide in methanol was used to remove endogenous peroxidases. After epitopes were unmasked by boiling in 10 mM citric acid for 10 min, slides were incubated at room temperature with an antibody directed against the C terminus of FOXL2 (4) (1:2500 dilution). A biotinylated antirabbit secondary (1:200 dilution; Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) was used in conjunction with streptavidin-conjugated horseradish peroxidase and the fluorescein isothiocyanate (FITC) fluorophor (trichostatin A kit; PerkinElmer, Norwalk, CT).

Colocalization of FOXL2 with pituitary hormones was performed as described above except that epitopes were unmasked by boiling in 10 mM citric acid for 5 min and immediately rinsing in PBS. Slides were incubated simultaneously with the FOXL2 antibody and antibodies against either GH [1:1000; National Hormone and Peptide Program (NHPP)] or LHß (1:500; NHPP) 1 h at room temperature and then the appropriate secondary antibodies: antihuman-FITC (1:50; Sigma Chemical Co., St. Louis, MO) or antiguinea pig-FITC (1:100; Sigma). To costain with FOXL2 and ACTH, TSHß or {alpha}GSU slides were incubated with the FOXL2 antibody, biotinylated antirabbit secondary antibody, horseradish peroxidase and FITC as described above then blocked with antirabbit Fab' (1:100, Jackson ImmunoResearch Laboratories). Slides were then incubated with anti-ACTH (1:2000; DAKO Corp., Carpinteria, CA), anti-TSHß (1:500; NHPP), or anti-{alpha}GSU (1:150; NHPP) for 1 h at room temperature and with FITC-conjugated antirabbit antibodies (1:100; Jackson ImmunoResearch Laboratories) for 30 min at room temperature.

For costaining with FOXL2 and PRL, slides were deparaffinized and rehydrated, endogenous peroxides were removed, and epitopes were retrieved as described above. Slides were then incubated with the C-FOXL2 antibody [1:2000 (4)] overnight at 4 C and incubated with a biotinylated antirabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories) for 30 min at room temperature. The addition of avidin and biotinylated peroxidase (Vector Laboratories, Inc., Burlingame, CA) and then diaminobenzidine (Sigma) resulted in a brown precipitate. Slides were then blocked with antirabbit Fab' (1:100; Jackson ImmunoResearch Laboratories) and biotin (1:12; Vector Laboratories) for 1 h at room temperature. An antibody that recognizes PRL (1:2500; NHPP) was added and incubated with slides for 1 h at room temperature before the addition of a biotinylated antirabbit secondary antibody (1:200; Jackson ImmunoResearch Laboratories). Addition of alkaline phosphatase (1:100; Vector Laboratories) and nitrobluetetrazolium bromochloroiodyl phosphate (1:50; Roche Applied Science, Indianapolis, IN) resulted in a purple color.

In situ hybridization was performed using a riboprobe labeled with digoxigenin (Roche) as described elsewhere (53). An mP1 antisense riboprobe was generated by using T3 polymerase and a linearized plasmid containing an intron and poly A tail from the mouse protamine 1 gene. The riboprobe was hybridized to sections overnight at 55 C.

Vector Construction
A plasmid containing 341 bp of proximal promoter from the Cga gene fused to luciferase was generated as described (54). Cmv-Vp16-Foxl2 uses the cytomegalovirus (CMV) promoter to drive expression of a fusion protein containing FOXL2 and the activation domain from the viral protein VP16 (9). To generate the {alpha}GSU-Foxl2 transgene, Cmv-Vp16-Foxl2 was digested with BglII and HincII to release a 1.5-kb fragment containing the VP16 activation domain and the entire Foxl2 coding sequence. The ends were filled in with Klenow and ligated into a SmaI site in pBluescript SK+ (Stratagene, La Jolla, CA). A 4.5-kb fragment of the Cga promoter was digested with Asp718 and HindIII and ligated into the KpnI and HindIII sites of pBluescript SK+, upstream of the Foxl2-Vp16 coding sequence. Finally, a plasmid containing an intron and polyadenylation sequences from mouse protamine 1 (mP1) was digested with BamHI and BglII and ligated into the BamHI site of pBluescript SK+ downstream of the Foxl2-Vp16 coding sequence.

Generation and Genotyping of Transgenic Mice
A 7.2-kb {alpha}GSU-Foxl2-mP1 fragment was generated by digestion of the {alpha}GSU-Foxl2-mP1 plasmid with BciVI and KpnI. The insert was isolated and microinjected into F2 zygotes from (C57BL/6J x SJL/J)F1 parents as described previously (32, 55). At e14.5, embryos were collected from recipient females. Genomic DNA was prepared from tail biopsies of all embryos and screened for the {alpha}GSU-Foxl2-mP1 transgene. Transgenic mice were identified by PCR using oligonucleotides (5'-CATTGCTCATACTGGGACCACG-3' and 5'-TCACGCAGGAGTTTTGATGGAC-3') that amplify a 526-bp product. Reactions were heated to 94 C for 3 min and then 40 cycles of denaturation at 94 C for 30 sec, annealing at 60 C for 30 sec, and extension at 72 C for 30 sec, with a final extension of 72 C for 10 min. Reactions were carried out using standard conditions as described previously (32).

Cell Culture
All cell cultures were maintained in a humidified atmosphere of 5% CO2 at 37 C. Cells (19) were cultured in high-glucose DMEM containing 2 mM glutamine, 10% fetal bovine serum. Cells were transfected using FuGENE (Roche Applied Science). Briefly, 1 d before transfection, 6 x 104 cells ({alpha}T3-1), 1 x 105 cells ({alpha}TSH, LßT2), or 1 x 104 cells (CV-1) were plated in a 24-well tissue culture plate. Transfections included 0.6 µl of transfection reagent, 50 ng of the reporter vector, 2–50 ng of the expression vector, and 5 ng of pRL-TK (Promega Corp., Madison, WI) that was used as the internal control for transfection efficiency. This mixture, together with 20 µl of serum-free media, was incubated at room temperature for 15 min and added directly to cell cultures containing 0.5 ml of complete media. After 48 h of incubation, the transfected cells were harvested using the Dual-Luciferase Reporter Assay System (Promega). Lysates were immediately assayed for luciferase activity using the Lmax microplate luminometer (Molecular Devices, Sunnyvale, CA). The transfection data were analyzed using a paired t test to test for significance (P < 0.05) between FOXL2- or FOXL2-VP16-stimulated and unstimulated values.


   ACKNOWLEDGMENTS
 
We thank Dr. Pamela Mellon for providing the {alpha}T3-1 and {alpha}TSH cell lines; Dr. Steve Potter (University of Cincinnati), and Dr. Heiner Westphal [National Institutes of Health (NIH)] for the Lhx3 and Lhx4 mutant mouse stocks; Michelle Brinkmeier, Dr. Kelly Cha, Mike Charles, Mary Anne Potok, and Dr. Lori Raetzman for providing samples of various mouse mutants; and Dr. Audrey Seasholtz for her comments on the paper. We thank the National Hormone and Pituitary Program, the National Institute of Diabetes and Digestive and Kidney Diseases, and the National Institute of Child Health and Human Development for providing antibodies for LHß, TSHß, and GH. We also thank Dr. Margaret Van Keuren, Dr. Wanda Filipiak, Galina Gavrilina, Dr. Thom Saunders, and the University of Michigan Transgenic Animal Model Core for generating the transgenic mice.


   FOOTNOTES
 
This work was supported by: National Institutes of Health (NIH) Grant RO1-HD34283 (to S.A.C.), NIH Grants F32-HD046300-01 and T32-HD7048-28 (to B.S.E.), the University of Michigan Undergraduate Research Opportunity Program and Summer Research Opportunity Program (to D.A.B.), the Foundation of Tokai University School of Medicine, and the Research Abroad Fellowship 2003 from the Japan Brain Foundation (to N.L.) and NIH Grant RO1-HD32416 (to C.M.C.). The University of Michigan Transgenic Animal Model Core received support from NIH Grants CA46592, AR20557, and DK07367, The University of Michigan Center for Organogenesis, the Michigan Economic Development Corporation, and the Michigan Technology Tri-Corridor (Michigan Animal Models Consortium Grant 085P1000815).

None of the authors have anything to disclose.

First Published Online July 13, 2006

Abbreviations: BrdU, Bromodeoxyuridine; CMV, cytomegalovirus; e10.5, embryonic d 10.5; FITC, fluorescein isothiocyanate; {alpha}GSU, glycoprotein hormone {alpha}-subunit; POF, premature ovarian failure; PRL, prolactin.

Received for publication July 22, 2005. Accepted for publication June 26, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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