TCF and Groucho-Related Genes Influence Pituitary Growth and Development
Michelle L. Brinkmeier,
Mary Anne Potok,
Kelly B. Cha,
Thomas Gridley,
Stefano Stifani,
Jan Meeldijk,
Hans Clevers and
Sally A. Camper
Department of Human Genetics (M.L.B., M.A.P., K.B.C., S.A.C.), University of Michigan Medical School, 4301 MSRBIII, Ann Arbor, Michigan 48109-0638; The Jackson Laboratory (T.G.), Bar Harbor, Maine 04609; Center for Neuronal Survival (S.S.), Montreal Neurological Institute, McGill University, Montreal, Quebec, Canada H3A 2B4; and Department of Immunology (J.M., H.C.), University Medical Center Utrecht, Heidelberglaan 100, 3584 CX Utrecht, The Netherlands
Address all correspondence and requests for reprints to: S. A. Camper, Department of Human Genetics, University of Michigan Medical School, 4301 MSRBIII, 1500 West Medical Center Drive, Ann Arbor, Michigan 48109-0638. E-mail: scamper{at}umich.edu.
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ABSTRACT
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Mutations in the prophet of PIT1 gene (PROP1) are the most common cause of multiple pituitary hormone deficiency in humans; however, the mechanism of PROP1 action is not well understood. We report that Prop1 is essential for dorsally restricted expression of a Groucho-related gene, transducin-like enhancer of split 3 (Tle3), which encodes a transcriptional corepressor. Deficiency of a related gene, amino terminal enhancer of split (Aes), causes pituitary anomalies and growth insufficiency. TLE3 and AES have been shown to interact with TCF/LEF (transcripiton factors of the T cell-specific and lymphoid enhancer specific group) family members in cell culture systems. In the absence of TCF4 (Tcf7L2), Prop1 levels are elevated, pituitary hyperplasia ensues and palate closure is abnormal. Thus, we demonstrate that Tcf4 and Aes influence pituitary growth and development, and place Tcf4 and Tle3 in the genetic hierarchy with Prop1.
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INTRODUCTION
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PITUITARY ORGANOGENESIS IS dependent on the expression of spatially and temporally regulated factors from the oral ectoderm, the ventral diencephalon, and the pituitary primordium. One of the earliest markers of pituitary development is sonic hedgehog (Shh), which is expressed throughout the oral ectoderm except in the region that becomes the pituitary placode (1). Bone morphogenetic protein 4, fibroblast growth factor (FGF) 8, and FGF10, produced by the infundibulum, constitute dorsal signals that contribute to establishing region-specific expression of the critical pituitary transcription factors Isl1 and Lhx3, and promote pituitary cell survival (2, 3, 4). The dorsal FGF signals must be opposed by ventral bone morphogenetic protein 2 signaling for initial dorsal-ventral patterning of the pituitary and for specification of the first two hormone-producing cell types, corticotropes, and rostral-tip thyrotropes, that appear at embryonic day (e) 10.512.5 (3). Fully differentiated somatotropes and gonadotropes appear several days later, in distinct dorsal and ventral regions, at e14.5 and e16.517.5, respectively (5, 6). Little is known about the influence of signaling molecules on these later differentiation steps.
During ontogeny, hormone-producing cells are located ventrally and rostrally to the proliferating cells that line the lumen of Rathkes pouch (7, 8). After birth, proliferating cells are dispersed throughout the anterior lobe of the pituitary gland, but only a subset of the proliferating cells express pituitary hormones (9, 10, 11, 12). The molecules that constitute hypothalamic input and feedback from end organs are well documented in their ability to act as mitogens for expansion of individual pituitary cell lineages, but these processes only come into play after birth (13, 14, 15). For example, GHRH receptor (Ghrhr) and Ghrh are not necessary for somatotrope commitment or expansion of the lineage before birth. However, after birth, both genes are necessary for expansion of the somatotrope population, and without them, mice exhibit pituitary hypoplasia and growth insufficiency (16, 17).
Prop1 encodes a paired homeodomain transcription factor that is the first pituitary-specific player in the hierarchical control of pituitary organogenesis (18). Mutations in PROP1 are the most common cause of multiple pituitary hormone deficiency in humans (19). Although the features of PROP1 deficiency vary, the clinical hallmarks are lack of gonadotropins and the pituitary hormones whose production depends on the transcription factor PIT1, namely GH, TSH, and PRL (20). Ames dwarf mice have a missense mutation, Ser83Pro, in the homeodomain of Prop1 that causes reduced DNA binding and transactivation, and deficiencies of the same hormones as humans with PROP1 mutations (21, 22, 23, 24). The pituitary glands of adult Prop1df/df mice are extremely hypocellular due to the near absence of somatotropes and lactotropes, which normally represent about 75% of the organ bulk (23). These mutant mice do not exhibit any defects in the initial dorsal-ventral patterning steps, and the first two cell types expected, corticotropes and thyrotropes, appear to be allocated normally at e12.5 (8). After this time, the effects of Prop1 deficiency are apparent in mutant embryos. The dorsal aspect of Rathkes pouch begins to appear thickened and branched and becomes profoundly dysmorphic, whereas the ventral aspect, or prospective anterior lobe is hypoplastic (8, 25). These abnormalities are not readily explained by changes in cell proliferation or cell death rates. Prop1 appears to control the decision of cells to leave the proliferative zone that surrounds the lumen of Rathkes pouch and move ventrally into the prospective anterior lobe where hormone-producing cells differentiate (7, 8).
Prop1 is required for transcriptional suppression of Hesx1, or Rpx, a paired-like transcription factor (25), that when mutated causes pituitary hormone deficiency and septo-optic dysplasia in mouse and man (26, 27). HESX1 interacts with the corepressor TLE1 [transducin-like enhancer of split 1 or Groucho-related gene (Grg) 1] to antagonize the actions of Prop1 (28). Intriguingly, abnormal branching of Rathkes pouch is also evident in Hesx1-deficient animals, although the dysmorphology is detectable earlier in Hesx1 mutants than Prop1 mutants, at e10.5 instead of e14.5 (25, 26). Little is known about the direct transcriptional targets or upstream regulators of either of these genes.
We characterized the expression profile of the developing pituitary gland during the peak action of Prop1, e12.514.5, and identified transcripts for TCF transcription factors and Aes (amino terminal enhancer of split, also known as Grg5) (29, 30). Aes and Tle1 are both members of the Grg family, which encodes corepressors that interact with DNA binding proteins that contain an engrailed repressor homology domain (eh1) (31). There are five members of the mouse Tle gene family, Tle14 and Aes. Tle14 each contain a WD-40 domain, 40 amino acid tandem repeats including conserved tryptophan (W) and aspartate (D) residues, that is responsible for interaction of Tle with eh1 containing transcription factors (32). Aes lacks the WD-40 domain, but it contains the amino terminal glutamine-rich domain that is necessary for interaction between Tle family members. In the neural tube, ectopic expression of Aes results in the dorsal expansion of ventral markers by blocking the corepressor functions of Tle14 (33). Because one function of AES is to repress the actions of the other Tle family members, it can be effectively an activator of TCF (34). In the developing eye, the amino terminal glutamine-rich domain of AES interacts with the eh1-like motif in the Six domain of SIX3 to repress
-crystallin transcription, resulting in the failure of lens formation (35). These data suggest that in addition to its derepression functions, AES can also act as a corepressor.
Members of the Tle family and Nkx genes are expressed in a spatially restricted pattern along the dorsal-ventral axis of the neural tube, and are essential for cell fate determination there (33). We hypothesized that spatially restricted expression of these same genes may be involved in cell fate determination in the pituitary gland. This idea is supported by the fact that several genes containing eh1 domains are expressed in the pituitary gland; the TCF/LEF family, Nkx3.1 and the pituitary transcription factors Hesx1, Six3, and Six6 (transcription factors of the T cell-specific and lymphoid enhancer-specific group) (2, 28, 34, 35, 36, 37). Moreover, two of these eh1-containing genes have proven roles in pituitary cell fate determination: Hesx1 and Six6 (28, 38).
We found that TLE3 and Aes are localized to the dorsal portion of Rathkes pouch and TLE4 is localized in the infundibulum, in contrast to the broad expression of Tle1 in the pouch. Prop1 is necessary for spatial restriction of TLE3 expression, but not Aes. Aes deficiency results in Rathkes pouch abnormalities that bear some similarities to those observed in Prop1df/df and Hesx1-/- mice. In addition, Aes is a modest repressor of pituitary growth. We have found that TCF4 (officially known as Tcf7L2) has a major role in controlling pituitary growth by limiting the expansion of the anterior lobe. Furthermore, TCF4 is essential for down-regulation of Prop1 transcription. These data prove that TCF4 and AES have roles in pituitary development and begin to place these genes in the genetic hierarchy that includes Prop1 and Tle3.
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RESULTS
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Tle1 (Grg1) is expressed throughout Rathkes pouch, coincident with the expression pattern of the critical eh1 transcription factor Hesx1 (28). TLE1 is thought to act as a corepressor with HESX1 to antagonize the action of PROP1. The spatial and temporal pattern of expression of other Tle genes has not been reported in the pituitary gland. We found that the expression of Aes, Tle3, and Tle4 is dynamic and regionally restricted, in contrast to the broad expression of Tle1 in the pouch. Immunoreactive TLE3 (GRG3) is first detectable at e10.5-e12.5 in the ventral diencephalon and presumptive hypothalamus, but by e14.5 TLE3 is specifically located dorsally and caudally relative to the lumen of Rathkes pouch (Fig. 1A
). This restricted zone of TLE3 expression differentiates into the intermediate lobe at e14.5, expressing pro-opiomelanocortin, which is cleaved to produce MSH and endorphins in this region of the organ.

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Fig. 1. Ectopic Expression of TLE3 in Prop1 Mutants
A, Spatial and temporal expression of corepressors. In situ hybridization with an Aes probe and immunohistochemistry with TLE3 and TLE4 antibodies were used to determine the spatial and temporal expression in sagittal sections of the developing pituitary gland. Embryonic time points used to determine the developmental expression of each corepressor were analyzed in the same experiment for optimal comparison of expression levels. Brackets denote the region of strongest expression at each developmental stage, e12.516.5. B, Prop1 is necessary for proper Tle3 expression. Prop1df/df mice exhibit strong ventral expression of TLE3 immunoreactivity at e14.5 relative to wild-type littermates (n = 3 for each genotype). No differences were observed in Aes expression between Prop1+/+ and Prop1df/df mice at e14.5 (n = 4 for each genotype).
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Aes expression is barely detectable in Rathkes pouch at e12.5; however, by e14.5 it is clearly detectable in the dorsal region of Rathkes pouch, and expression is diminished by e16.5 (Fig. 1A
). The domain of Aes expression partly overlaps the area that expresses TLE3. This dynamic expression pattern is similar to that of Prop1, which peaks at e12.5, is absent from cells in the rostral tip of the anterior lobe, and diminishes rapidly to undetectable levels by birth (21).
Tle4 expression begins around e12.5 in the developing infundibulum, or posterior lobe, of the pituitary gland (Fig. 1A
). By e14.5, TLE4 protein is concentrated in the developing posterior lobe. TLE4 expression is diminished by e16.5 and is absent at birth (Fig. 1
, data not shown). TLE4 is not detectable in the prospective anterior or intermediate lobes from e12.5-e16.5. This expression pattern suggests that TLE4 is likely to play a role in the development of the posterior lobe of the pituitary gland, and any influence it may have on the anterior lobe would be indirect.
Prop1 has essential roles in regulation of cell specification and growth of the pituitary gland along the dorsal-ventral axis, although its mechanism of action is not understood (8). The dorsally restricted patterns of Tle3 and Aes expression suggested that they could be involved in suppressing the growth of the dorsally located prospective intermediate lobe, a process that goes awry in Prop1 mutants. We analyzed the expression of Tle3, Tle4, and Aes in Prop1df/df mice and found that mutant embryos express TLE3 throughout the entire Rathkes pouch instead of restricting it to the dorsal aspect of the pouch (Fig. 1B
). Ventral expansion of the TLE3 expression domain is not attributable to developmental delay in Prop1 mutant mice, as normal embryos do not express TLE3 in the ventral pouch at any time (Fig. 1A
). Thus, Prop1 is necessary for dorsal restriction of TLE3. In addition, even though the spatial and temporal expression of TLE3 overlaps that of Hesx1, TLE3 is not altered in Hesx1-/- mice (data not shown). Aes and Tle4 expression is similar in Prop1df/df and normal embryos, indicating that spatially restricted expression of these genes is independent of Prop1 (Fig. 1B
).
The importance of Aes in modeling the shape of Rathkes pouch and the growth of the anterior lobe is evident by analysis of Aes-/- embryos. Bifurcation of the dorsal aspect of the pouch is detectable at e12.5 in Aes-/- mice (data not shown). All mutants exhibit a varying degree of dorsal dysmorphology at e14.5, and it persists through e16.5 and postnatal d 1 (P1) (Fig. 2
). Using three-dimensional reconstruction, we determined that the volume of the pituitary gland is larger in the Aes-/- mice [11.8E6 arbitrary units (A.U.)3 ± 4.2E6 A.U.3, n = 4] than their wild-type littermates (5.4E6 A.U.3 ± 0.9E6 A.U.3, n = 3) at P1. Thus, Aes normally restricts pituitary growth, in addition to its role in regulating organ shape.

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Fig. 2. Aes Deficiency Causes Persistent Pituitary Dysmorphology
Pituitaries of Aes-/- embryos and their wild-type littermates were stained with hematoxylin and eosin at e14.5, e16.5, and P1. Dorsal dysmorphology in the pituitaries of Aes-deficient mice cause bifurcations and abnormalities in the shape of the lumen of Rathkes pouch. Sagittal sections are depicted at e14.5 and 16.5 and coronal sections are illustrated for P1. Three-dimensional reconstruction of P1 pituitaries also demonstrates the severe degrees of dysmorphology. Yellow (anterior and intermediate lobes), blue (posterior lobe), red (lumen), green (cells within the anterior lobe of indeterminate cell fate).
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All five pituitary hormone producing cell types are present in Aes-/- mice, and they appear in the expected domains (Fig. 3
). For example, the gonadotropes are found near the midline in the most ventral aspect of the anterior lobe in both mutant and wild-type mice. Therefore, Aes is not required for either cell specification or the patterning steps that cause the differentiated cells to appear in predictable domains.

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Fig. 3. Aes Deficiency Does Not Affect Cell Specification
The pituitary glands of Aes+/+ and Aes-/- neonates were examined by immunohistochemistry of coronal sections at postnatal d 1. All five hormone producing cell types were detected in the pituitary glands of mutant mice and their littermates. A region of the anterior lobe in Aes-/- mice fails to stain with any of the hormone antibodies except POMC, which does not distinguish between anterior and intermediate lobe cell fates (asterisk).
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Aes mutant mice have a reasonably well-differentiated intermediate lobe, located appropriately at the most dorsal aspect of the gland, but their pituitaries contain a substantial patch of cells of indeterminate fate (Fig. 3
). Cells located in the midsection of the pituitary gland dorsal to the lumen are comprised of primarily POMC-positive cells with only scattered cells positive for the hormones of the anterior lobe. It is not clear whether the POMC cells in this region are fated to become corticotropes of the anterior lobe or melanotropes of the intermediate lobe. The POMC protein is processed differently in these two cell types, yielding ACTH and MSH respectively, and the POMC antibodies do not distinguish the different peptides. Using three-dimensional reconstruction, we determined that this region is relatively small, comprising approximately 6% of the total mutant pituitary volume. The fraction of the pituitary that is clearly anterior lobe is similar between Aes-/- (67%) and Aes+/+ (72%) mice at P1.
It has been reported that Aes-/- mice exhibit a mild, transient, growth insufficiency (39). We have investigated GH production in Aes-/- mice. The number of cells producing GH in individual pituitary sections appears reduced in mutant relative to wild-type mice by immunohistochemistry (Fig. 3
). The overall size of the mutant pituitary is larger, however, and Western blot analysis confirms comparable total pituitary GH content at P1 (data not shown). This suggests that the growth deficiency in Aes mutants is not attributable to failure in somatotrope commitment.
To address the mechanism whereby Aes deficiency affects pituitary gland growth and shape, we examined expression of several genes proven, or suspected, to be involved in pituitary development. Prop1 and Aes deficiencies both cause pituitary dysmorphology, but Aes deficiency has no apparent influence on initiation of Prop1 expression. Prop1 transcripts are appropriately excluded from cells in the ventral region that have left the proliferative zone around the lumen (Fig. 4
). Thus, Prop1 does not appear to be involved in the Aes mutant phenotype. In addition, the expression of Tle4 and Hesx1 is not altered in Aes-/- mice (Fig. 4
, data not shown).

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Fig. 4. Aes Deficiency Does Not Affect Prop1 Expression
Sagittal sections were prepared from Aes+/+ and Aes-/- mice collected at e12.5 and e14.5. Levels of Prop1 transcripts appeared similar by in situ hybridization at e12.5 (n = 2 for each genotype) and no differences in TLE3 immunoreactivity were noted at e14.5 (n = 3 for each genotype). The brackets denote a portion of the anterior lobe in both Aes+/+ and Aes-/- mice that are devoid of Prop1 transcripts.
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TLE and AES proteins interact with members of the HMG-box family of repressors (34). We discovered a variety of TCF4 (Tcf7L2) isoforms in the developing pituitary gland, including ones that can act as endogenous inhibitors of Wnt signaling because they lack a DNA binding domain (30). To assess the contribution of TCF4 to pituitary development, we analyzed pituitary gland morphology, growth, and cell specification of Tcf4-/- mice. Tcf4-/- mice were generated through a targeted disruption of the DNA binding domain, eliminating all isoforms that affect downstream gene expression in response to Wnt signaling (40). Isoforms lacking the DNA binding domain may be expressed. The anterior pituitary lobes of Tcf4 mutants are enlarged to nearly twice the normal height by e14.5 (Fig. 5
). The prospective intermediate lobe and infundibulum are both displaced dorsally, protruding into the space normally occupied by the hypothalamus. Three-dimensional reconstruction of P1 pituitary glands documented a 3-fold increase in the combined volume of the anterior and intermediate lobes in Tcf4-/- mice (23.3E6 A.U.3 ± 4.8E6 A.U.3, n = 4) compared with Tcf4+/+ mice (9.0E6 A.U.3, 7.9E6 A.U.3, n = 2). The overgrowth of the prospective anterior lobe in most TCF4-deficient mice apparently interferes with palate development, as the pituitary is expanded ventrally between the edges of the cartilage plate in the midline (Fig. 5
, arrow).

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Fig. 5. TCF4 Deficiency Causes Pituitary Hyperplasia
Mid-sagittal and coronal sections of Tcf4+/+ and Tcf4-/- mice were analyzed at e14.5 and e18.5, respectively, by hematoxylin and eosin staining. The mutant, hyperplastic pituitary presses ventrally into the cartilage plate in Tcf4-/- mice at e18.5 (arrow). Three-dimensional reconstruction was used to visualize the hyperplasia of the anterior lobe in P1 mutant and wild-type mice. Yellow, Anterior and intermediate lobes; blue, posterior lobe; red, lumen.
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The five anterior pituitary hormone producing cell types are present in apparently normal proportions and normal spatial distribution in the anterior lobes of TCF4-deficient mice (Fig. 6
). In addition, TCF4 is not required for the spatial expression of TLE3, TLE4 or Aes (Fig. 7
, data not shown). These genes are expressed at similar levels and in the same spatial pattern in Tcf4-/- mice compared with wild-type littermates. Absence of TCF4, however, causes a dramatic increase in Prop1 transcripts at e14.5 relative to normal mice. Prop1 expression normally peaks at e12.5 and diminishes from e14.5 onwards. Thus, TCF4-deficient mice express Prop1 longer than normal. Despite the prolonged expression of Prop1, appropriate spatial expression is maintained, as indicated by the exclusion of transcripts from the ventral aspect of the developing anterior lobe. This suggests that extinction of Prop1 expression is accomplished by active repression, rather than loss of upstream activators, and that TCF4 is necessary for down-regulation of Prop1 expression.

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Fig. 6. Cell Specification in TCF4-Deficient Mice
Pituitaries of Tcf4-/- mice and their wild-type littermates were analyzed at e18.5 by immunohistochemistry with pituitary hormone antibodies. All five pituitary hormone-producing cell types were identified.
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Fig. 7. Prop1 Expression Is Increased in TCF4-Deficient Mice
Pituitaries of Tcf4-/- mice and their wild-type littermates were examined at e14.5 in sagittal sections. No differences were detected in the spatial or temporal expression of TLE3, using immunohistochemistry (n = 4 for each genotype). Prop1 transcripts are barely detectable by in situ hybridization in the pituitaries of normal mice at e14.5, but TCF4-deficient mice exhibit readily detectable Prop1 transcripts relative to wild type (n = 3 for each genotype). The most ventral-rostral portion of the anterior lobe that is circled represents a region where Prop1 is not expressed in either Tcf4+/+ or Tcf4-/- embryos.
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DISCUSSION
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Several genes are known to be important for pituitary gland development, and we demonstrate here that Aes and TCF4 (Tcf7L2) both have roles in pituitary organogenesis. Aes-/- mice display pituitary dysmorphology that bears some similarities to Prop1df/df and Hesx1-/- mice. The overall size of the pituitary gland in Aes-/- mice is larger than wild-type littermates. TCF4 deficiency either directly or indirectly causes elevated and prolonged expression of Prop1, and profound pituitary hyperplasia. Together with our previous demonstration that constitutive Prop1 expression increased the frequency of pituitary tumors; this suggests that this pathway may have an important role in formation of pituitary adenomas, which are the most common intracranial tumor in humans (41, 42). Thus, our findings are of potential significance for human clinical medicine in addition to advancing our understanding of pituitary organogenesis.
Growth insufficiency is evident in Aes mutants within 2 wk after birth (39). The most severely affected mutants weigh only 40% of their wild type littermates and die within 5 wk after birth, and the mildly affected mice weigh about 70% that of their wild-type littermates at weaning and 80% that of their wild-type littermates in adulthood. The bones of Aes mutant mice have smaller zones of proliferative and hypertrophic chondrocytes in the growth plates (43). GH is known to increase longitudinal bone growth by stimulating the expansion of prechondrocytes in the proliferative zone of the growth plate both directly and indirectly through induction of IGF-I secretion, suggesting that the growth defect in Aes mutants could be a result of pituitary GH deficiency (44). Consistent with this hypothesis, Aes is expressed at readily detectable levels at e14.5, a critical time in pituitary development. We see somatotrope commitment in Aes mutants and normal pituitary GH content at birth, which argues against a pituitary based growth insufficiency. We cannot rule out the possibility that Aes-deficient mice have reduced GH secretion, but the variability in growth insufficiency among affected mice and the lethality of unknown etiology makes this technically difficult to investigate. Because Aes is broadly expressed in the developing embryo, the growth plate abnormalities in Aes mutants may be attributable to affects of Aes on liver IGF production or intrinsic bone defects (45, 46).
Prop1 is required for undifferentiated pituitary cells to leave the peri-luminal proliferative zone and initiate development of glandular tissue in the prospective anterior lobe (25). Lack of PROP1 causes dorsal overgrowth of cells that are of undetermined cell fate, deficiency of the PIT1 lineage, and reduced gonadotrope function (8, 22). The mechanism of Prop1 action has been elusive, although a few target genes of Prop1 have been identified. Prop1 is known to act as a repressor of Hesx1, steroidogenic factor 1 (SF1 or Nr5a1) and Brn4, and it is an activator of Pit1 (8, 21, 25). None of these target genes were revealing in terms of the disruption in the growth of the anterior lobe at e14.5, which is the earliest discernable phenotype in Prop1 mutant mice. To better understand this aspect of Prop1 action, we previously carried out a differential expression analysis of pituitary development and found members of the Wnt signaling pathway, including the corepressors of the Grg family as candidate genes (29, 30). Here we show that Prop1 is required to restrict expression of the corepressor Tle3 to the dorsal aspect of the gland. Thus, Tle3 is genetically downstream of Prop1. The abnormal presence of TLE3 in the ventral pituitary could contribute to the failure of the ventral differentiation program to be activated in Prop1 mutants.
In normal mice, TLE3 is likely to act as a corepressor for eh1-containing transcription factors that are located with it in the dorsal aspect of the pituitary primordium. HESX1 contains an eh1 domain that can interact with corepressors like TLE3, and excess or persistent Hesx1 expression is not significant unless a corepressor is present (28). Prop1 deficiency has already been shown to cause persistent expression of Hesx1, although the significance of this is unclear (25). In conjunction with persistent Hesx1 expression, the misregulation of Tle3 in Prop1-deficient mice may contribute to the anterior lobe hypocellularity and failed cell specification that is characteristic of Prop1df/df mice.
There are several intriguing candidates for interaction with the corepressor TLE3 in addition to HESX1 and TCF4. Nkx family members contain eh1 domains and have been shown to act as corepressors with TLE14 to regulate cell fate in the neural tube (33), and the pattern of NKX3.1 expression overlaps perfectly with TLE3 (Fig. 8A
) (2). SIX3 is also a candidate for interaction with TLE3, based on the presence of an eh1 domain and its pituitary expression pattern (2). Intriguingly, SIX3 functions as a corepressor with AES in eye development, and the patterns of Aes and Six3 expression overlap in the pituitary. This provides support for the idea that AES and SIX3 could interact during pituitary organogenesis (35, 36). Finally, TLE3 and AES could also interact with SIX6 in its role of regulating exit from the cell cycle (38). Because there are multiple candidates for interaction with TLE3, and because AES can function either as a corepressor or as an antagonist of repressor function, additional studies will be necessary to unravel the next steps in this pathway.

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Fig. 8. Aes, Prop1, and Tcf4 (Tcf7L2) Regulate Pituitary Gland Size and Shape
A, The spatial expression of Aes, Prop1, TLE3, and the overlapping corepressors Six3 and Nkx3.1 are indicated at e12.5 and e14.5 by the length and thickness of the corresponding arrows. The region of highest expression is noted at the start of the arrow and the region of lowest expression is noted at the end of the arrowhead. The thickness of the line corresponds the relative level of expression throughout the pituitary gland at the indicated time point. The red circles are analogous to the region of proliferating cells at e12.5 and e14.5 in Rathkes pouch. Areas in blue represent the developing anterior lobe. Representative drawings of mutant pituitary glands are shown at the bottom. The arrows next to the pituitary glands correspond to a ventral expansion of TLE3 in Prop1df/df embryos and an increase of Prop1 in Tcf4-/- embryos. B, A basic model of Wnt signaling in pituitary organogenesis is based on published data (48 ) and incorporates Tcf4, Prop1, and Tle3 from data in this paper.
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Canonical Wnt signaling is the cause of increased nuclear ß-catenin, which activates TCF/LEF-mediated gene transcription (47). Two lines of evidence suggest that ß-catenin has an important role in growth of the pituitary gland. Recently ß-catenin, LEF1, and LiCl have been shown to regulate expression of Pitx2, a paired-like homeobox gene that controls pituitary cell proliferation in a dosage sensitive manner (48) (Fig. 8B
). In addition, ß-catenin is associated with pituitary adenomas in humans (49, 50, 51). Our data show that TCF4 down-regulates the expression of Prop1, and failure to down-regulate Prop1 disrupts differentiated cell function and predisposes the mouse to pituitary tumors (42). These findings demonstrate that TCF4 controls growth of the pituitary gland during development and suggest that misregulation of this process can contribute to pituitary tumorigenesis.
Our data suggest that Tcf4 is involved in repression of Prop1 and that Prop1 is necessary for regulating spatial expression of the TCF4 corepressor, TLE3. We also show that AES, which can antagonize TLE3, is important for normal pituitary organ size and shape. The correlation between the mouse mutant phenotypes and human disease features is exceptional (20, 41), suggesting that AES and TCF4 are worth investigating as causative genes in adult and pediatric human disease.
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MATERIALS AND METHODS
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Generation of Mice and Genotyping
Aes- and TCF4 (Tcf7L2)-deficient mice were obtained by gene targeting (27, 39, 40). Aes-deficient mice are maintained on a C57BL6/J genetic background at the Jackson Laboratory and at the University of Michigan. TCF4-deficient mice were maintained at the University Medical Center Utrecht on a mixed genetic background (40). Prop1 df/df mice were obtained from A. Bartke (Southern Illinois University, Carbondale, IL) and maintained as a stock, DF/B, at the University of Michigan. Mice were housed on a 12-h light, 12-h dark cycle, with unlimited access to tap water and Purnia 5008 chow. All procedures using mice were approved by the University of Michigan Committee on Use and Care of Animals, and all experiments were conducted in accord with the principles and procedures outlined in the NIH Guidelines for the Care Use of Experimental Animals.
Aes -/- and Prop1 df/df embryos were obtained through heterozygote matings. Timed pregnant females were used to generate embryos with noon on the day the copulatory plug was detected considered e0.5. The day of birth was considered postnatal d 1. DNA was prepared from limbs as previously described (52). Mice were genotyped by PCR as previously described (39, 42).
Embryo Preparation
Embryos were removed and fixed in 4% buffered paraformaldehyde: e12.5 and e14.5 for 1.5 h, e16.5 and e18.5 heads for 2 h, and P1 heads for 1624 h. After fixation, the samples were rinsed in PBS, dehydrated, and embedded in paraffin (Citadel 1000, Shandon, Cheshire, UK). Six-micrometer sections were used for immunohistochemistry, in situ hybridization and staining with hematoxylin and eosin.
In Situ Hybridization
In situ hybridization was performed on paraffin sections using digoxygenin labeling (Roche, Indianapolis, IN) as described (30). The 388-bp Aes clone was obtained through cDNA subtraction of e14.5 Prop1+/+ vs. Prop1df/df Rathkes pouch (accession no. BE692765) and contains from 864-1106 bp of the full-length cDNA (30). The antisense Aes probe was linearized with SpeI and labeled with T7 polymerase. A 1:50 dilution of the probe was hybridized to 6-µm paraffin sections overnight at 50 C. An 846-bp Prop1 clone containing the entire coding sequence was linearized with SpeI and the antisense probe was generated with T7 polymerase. A 1:100 dilution of the probe was hybridized to 6-µm paraffin sections overnight at 55 C. A 400-bp Hesx1 clone was linearized with HindIII and the antisense probe was generated with T7 polymerase. A 1:50 dilution of the probe was hybridized to 6-µm paraffin sections overnight at 55 C. Probes generated from the sense strand were used as negative controls.
Histology and Immunohistochemistry
Embryo sections were deparaffinized, rehydrated and stained for 40 sec in hematoxylin (Fisher Scientific, Pittsburgh, PA) and 20 sec in eosin (Sigma, St. Louis, MO). The pituitary hormone antibodies were received from the National Hormone Pituitary Program. Immunohistochemistry for the pituitary hormone antibodies was performed on paraffin sections as previously described (53). The TSA (tyramide signal amplification)-fluorescein isothiocyanate (FITC) system (Perkin-Elmer Life Sciences, Boston, MA) was used for immunohistochemistry with the rabbit anti-Grg3 (TLE3) affinity purified polyclonal antibody (Chemicon International, Inc., Temecula, CA) and the antichicken TCF4 antibody (provided by Dr. Greg Dressler, Department of Pathology, University of Michigan). Antibodies to TLE1 and TLE4 were visualized by fluorescent microscopy using secondary antibodies conjugated to FITC (54, 55). Embryo sections were deparaffinized, rehydrated and rinsed in 1x PBS/0.5% Tween 20 (PBST). Endogenous peroxidases were quenched by a 20 min incubation in a 1:1 solution of methanol:H2O2 and rinsed in PBST. Sections were then incubated in a boiling 0.1 M citric acid bath for 10 min followed by three 10-min washes in PBST. The blocking reagent included in the TSA-FITC System kit, consisting of 0.1 M Tris-HCl (pH 7.5)/0.15 M NaCl/0.5% Tween-20 (TLE3, TCF4) or 3% BSA in PBS/0.5% Tween-20 (TLE1, TLE4), was applied to sections for 30 min. The primary antibodies were diluted in their respective blocking solutions: TLE3 at 1:300, TCF4 at 1:50, TLE1 at 1:1000, and TLE4 at 1:500. One hundred microliters of the primary antibody was applied to sections under a parafilm coverslip in a humidity chamber overnight at 4 C. After reaction of the primary antibody, sections were washed three times in PBST, 10 min each followed by a 40-min incubation a 1:200 dilution of the secondary antibody in blocking reagent. Biotin goat antirabbit IgG Fab' fragment was used as the secondary antibody for TLE3, biotin-SP conjugated donkey antichicken Ig Y F(ab')2 fragments (Jackson ImmunoResearch Laboratories, West Grove, PA) was used as the secondary antibody for TCF4 and an antirabbit-FITC (Jackson ImmunoResearch Laboratories) was used as the secondary antibody for TLE1 and TLE4. Sections hybridized with either TLE1 or TLE4 primary antibody were then washed three times in PBST, mounted under a glass coverslip using a Gelvatol mounting media (56) and viewed using fluorescent microscopy. For sections hybridized with either TLE3 or TCF4 primary antibodies, a 30-min incubation in a 1:100 dilution of Streptavidin-horseradish peroxidase reagent from the TSA-FITC System kit in blocking solution was preceded and followed by three 10-min washes in PBST. Sections were then incubated with a 1:50 dilution of TSA-FITC in the dilution buffer included with the kit, washed three times for 10 min each in PBST, then mounted under a glass coverslip using a Gelvatol mounting media (56) and viewed using fluorescent microscopy.
Digital images of pituitary sections were captured with a Leitz DMRB microscope (W. Nuhsbaum, Inc., McHenry, IL) and an Optronics camera (Goleta, CA) and processed with Surfdriver software (Kailua, HI) for three-dimensional reconstructions and volume estimates.
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ACKNOWLEDGMENTS
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We would like to thank Dr. Greg Dressler for the TCF4 antibody, Petra Moerer for her help in collecting Tcf4-/- embryos, Dr. Kathy Mahon for the Hesx1 probe, and Dr. Juan Pedro Martinez for the Hesx1 mutant mice. We would also like to thank Dr. Kristin Douglas for her contributions to this work, Dr. Ken Cadigan and Dr. Gary Hammer for helpful comments, and the National Hormone Pituitary Program and Dr. Parlow for the pituitary hormone-specific antibodies.
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FOOTNOTES
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This work was supported by the NIH (HD30428 and HD34283, to S.A.C.), Physiology Training Grant (K.B.C.) 2-T32-GM08322-13 and the University of Michigan Summer Research Opportunity Program (M.A.P.).
Abbreviations: Aes, Amino terminal enhancer of split; e, embryonic day; FGF, fibroblast growth factor; FITC, fluorescein isothiocyanate; Grg, Groucho-related gene; LEF, transcription factor, lymphoid enhancer specific; P1, postnatal day 1; Prop1, prophet of PIT1; TCF, transcription factor, T cell specific; Tle3, transducin-like enhancer of split 3.
Received for publication June 11, 2003.
Accepted for publication July 30, 2003.
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