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Molecular Endocrinology, doi:10.1210/me.2005-0052
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Molecular Endocrinology 19 (7): 1893-1903
Copyright © 2005 by The Endocrine Society

PITX Genes Are Required for Cell Survival and Lhx3 Activation

Michael A. Charles1, Hoonkyo Suh1, Tord A. Hjalt, Jacques Drouin, Sally A. Camper and Philip J. Gage

Department of Human Genetics (M.A.C., S.A.C.), Neuroscience Program (H.S., S.A.C.), University of Michigan, Ann Arbor, MI 48109-0638; Department of Cell and Molecular Biology (T.A.H.), Lund University, SE-22184 Lund, Sweden; and Laboratoire de Genetique Moleculaire (J.D.), Institut de Recherches Cliniques de Montreal, Montreal, Quebec, Canada H2W 1R7

Address all correspondence and requests for reprints to: Sally A. Camper, 4301 MSRB III, 1500 West Medical Center Drive, University of Michigan Medical School, Ann Arbor, Michigan 48109-0638. E-mail: scamper{at}umich.edu.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The PITX family of transcription factors regulate the development of many organs. Pitx1 mutants have a mild pituitary phenotype, but Pitx2 is necessary for the development of Rathke’s pouch, expression of essential transcription factors in gonadotropes, and expansion of the Pit1 lineage. We report that lack of Pitx2 causes the pouch to undergo excessive cell death, resulting in severe pituitary hypoplasia. Transgenic overexpression of PITX2 in the pituitary can increase the gonadotrope population, suggesting that the absolute concentration of PITX2 is important for normal pituitary cell lineage expansion. We show that PITX1 and PITX2 proteins are present in similar expression patterns throughout pituitary development and in the mature pituitary. Both transcription factors are preferentially expressed in adult gonadotropes and thyrotropes, suggesting the possibility of overlap in maintenance of adult pituitary functions within these cell types. Double knockouts of Pitx1 and Pitx2 exhibit severe pituitary hypoplasia and fail to express the transcription factor LHX3. This indicates that these PITX genes are upstream of Lhx3 and have compensatory roles during development. Thus, the combined dosage of these PITX family members is vital for pituitary development, and their persistent coexpression in the adult pituitary suggests a continued role in maintenance of pituitary function.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
TWO MEMBERS OF THE PITX family of homeobox-containing transcription factors, Pitx1 and Pitx2, have been implicated in several aspects of pituitary development and transcriptional regulation (1, 2). Pitx1 and Pitx2 have nearly identical homeodomains and conserved C termini (3). Both transcription factors can bind the same bicoid sites in vitro (4, 5) and have similar transactivational activity in cell culture (3). Hormone genes and lineage-specific transcription factors are among the downstream targets of the PITX genes in the pituitary. Both PITX proteins can bind and transactivate the promoters of most pituitary hormones including glycoprotein hormone {alpha}-subunit ({alpha}GSU), LHß, FSHß, GH, prolactin (PRL), TSHß, and proopiomelanocortin (POMC) as well as the GnRH receptor (5, 6, 7). PITX1 can synergize with both early growth response 1 (EGR1) and steroidogenic factor 1 (SF1) (officially known as NR5A1) to activate transcription of the LHß and Müllerian-inhibiting substance genes (8, 9). Antisense knockdown of PITX1 in cell culture leads to the extinction of Lhx3 expression (5). Cell culture studies also show that Pitx2 controls genes that regulate the cell cycle such as cyclin D1 (Ccnd1) and cyclin D2 (Ccnd2) as well as Wnt-dependent mRNA turnover (10). PITX2 is important for the expression of several transcription factors such as Hesx1, Prop1 (11), and Gata2 in intact animals (12). Little is known about the sensitivity of these target genes to PITX dosage or the functional overlap the two PITX genes may have in their activation.

Both Pitx1 and Pitx2 have been knocked out in mice, and both affect the development of many organs. Mice completely lacking Pitx1 have severe hindlimb defects, cleft palate, and die after birth (13, 14). The pituitary appears to be mildly affected, exhibiting slightly altered proportions of differentiated cell populations. Pitx1 is not associated with any known human disease; however, humans with a mutation in a single allele of PITX2 have Rieger syndrome. Rieger syndrome is characterized by defects in the eyes, teeth, umbilicus, and heart as well as rare cases of isolated GH deficiency and is thought to be primarily a disease of haploinsufficiency (15, 16). Mice heterozygous for a Pitx2 null allele recapitulate some Rieger syndrome features at low penetrance. Loss of both Pitx2 alleles is lethal to the early mouse embryo due to profound heart defects (11, 17, 18, 19). The pituitaries of Pitx2–/– mice develop a definitive Rathke’s pouch, but the pouch fails to expand and develop an anterior lobe. Mechanistically, this has been attributed to reduced proliferation and failure to maintain Prop1 and Hesx1 expression (11, 17). It is not clear, however, that the Pitx2 null phenotype is explained by these downstream effectors.

The creation of a hypomorphic, or reduced function, allele of Pitx2 (Pitx2neo) made it possible to study the role of Pitx2 in pituitary cell specification (17). Homozygotes for the Pitx2neo allele survive until birth. Their pituitaries lack gonadotropins and have a decrease in both somatotropes and thyrotropes, whereas corticotropes are apparently unaffected (12). The lineage-specific transcription factors GATA2, EGR1, and SF1 are not expressed in the pituitaries of these hypomorphs. This reveals the sensitivity of gonadotrope differentiation to PITX2 dosage and highlights the reliance of other cell types on this transcription factor for establishing the appropriate populations of differentiated cells at birth.

Many haploinsufficiency disorders are semidominant, resulting in more severe effects in homozygotes (20). Surprisingly, the effect of overexpressing a normal allele can sometimes be as deleterious as reduced expression. For example, with a complete loss of the transcription factor paired box homeotic gene 6 (PAX6), eyes fail to develop; however, both haploinsufficiency and excess expression of the transcription factor result in small eyes (21). Recent evidence suggests that eye development may respond in a similar way to PITX2 dosage. Although most Rieger mutations appear to cause loss of function, symptoms of Rieger syndrome have been noted in a human patient whose mutation in PITX2 endows increased activational ability in cell culture assays (22). In addition, transgenic mice overexpressing PITX2 in the cornea exhibit corneal hypertrophy similar to that seen in Pitx2–/– mice, and features that are similar to those of Rieger syndrome patients including irido-corneal adhesions and severe retinal degeneration (23). Taken together, this suggests that excess PITX2 dosage is deleterious to eye development. The consequences of excess PITX2 expression may not be the same in all PITX2-dependant organs because the organs vary considerably in their response to Pitx2 haploinsufficiency.

An allelic series containing different combinations of Pitx2 wild-type, hypomorphic, and null alleles showed a proportional dependence of both pituitary size and cell differentiation on PITX2 dosage (12). Although loss of PITX1 alone has no effect on pituitary size, lack of PITX1 combined with reduced levels of PITX2 results in an extremely severe and early arrest in pituitary expansion (12). Pituitary growth and differentiation seems to be dependent on the combined dosage of these two PITX proteins, with PITX2 having a more prominent role than PITX1.

Interestingly, another family of molecules, the LIM (Lin-1, Isl-1, Mec-3) homeodomain transcription factors, exhibit dosage effects on pituitary growth that are similar to those observed for the PITX family (24). Single gene knockouts showed that both Lhx3 and Lhx4 are important for pituitary gland growth and cell specification (24, 25). Classical genetic analysis revealed that Lhx3 and Lhx4 have overlapping roles in early development and possess dosage sensitive properties. The similar expression pattern of PITX and LHX genes, similar hypocellularity phenotype of the mutants (25, 26), and failure to express LHX3 in cell culture with knockdown of PITX1 (5) suggest that the PITX and LHX genes may function in the same pathway.

Here we report that lack of PITX2 causes excessive cell death in the pituitary, whereas an excess of PITX2 causes an increase in the gonadotrope population, implying that the dosage of PITX2 is critical for normal pituitary cell lineage expansion. We also show that PITX1 and PITX2 proteins have remarkably similar expression patterns during pituitary development and are expressed in identical cell types in the adult pituitary. Animals bearing disruptions of both PITX genes have severe pituitary hypoplasia and loss of LHX3 expression. This suggests that Pitx1 and Pitx2 are upstream of Lhx3 and that their combined dosage is necessary for LIM gene expression and pituitary development.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Cell Death Is Increased in Pitx2 Null Pituitaries
Cell death plays an important role in the development of many parts of the embryo such as the limb and developing nervous system (27). No longitudinal studies of cell death have been reported for the developing pituitary gland. To address the role of cell death in normal pituitary organogenesis, we carried out terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining at multiple, early developmental time points. At embryonic day (E) 10.5, during the first stages of invagination of the oral placode, cell death is limited to the border between the invaginating pouch and surrounding oral ectoderm near the putative pinch off point (Fig. 1CGo, arrowhead). No dying cells are detected in Rathke’s pouch at this time. As development continues and the pouch begins to detach from the oral ectoderm (E11.5), cell death remains relegated to the pouch’s pinch off point (Fig. 1EGo, arrowhead). Later, at E12.5, cell death is rarely found in the pouch or the expanding anterior lobe (Fig. 1GGo). This pattern of TUNEL staining suggests that cell death could have an early and transitory role that is important for separation of the pouch from the rest of the oral ectoderm. To explore the mechanism underlying the hypocellular anterior pituitary of Pitx2 null homozygotes (12, 17), we compared cell proliferation and cell death in Pitx2–/– animals with their wild-type littermates.



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  		Fig. 1. Increased Cell Death Is Observed in the Pituitaries of Pitx2 Null Animals

Bromodeoxyuridine incorporation after 2-h pulse (red) and histone H3 label (green) show proliferation in E10.5 wild-type (WT) and Pitx2 null animals (A and B). TUNEL assays performed at E10.5, E11.5, and E12.5 show increased and inappropriate cell death in Pitx2 null animals as compared with wild-type littermates (normal cell death pattern indicated by arrowheads). During the early invagination of Rathke’s pouch (C and D), increased and dorsally mislocalized cell death is observed. This altered cell death pattern continues through E11.5 (E and F) leaving a stunted and hypocellular pouch by E12.5 (G and H).

 
At E10.5, proliferating cells are abundant in both the normal and mutant pouch (Fig. 1Go, A and B). In contrast, much more cell death is detected In Pitx2–/– mice than in normal mice. In mutants, apoptotic cells extend from the oral ectoderm much farther dorsally into the invaginating pouch at E10.5 (Fig. 1DGo). This dorsally expanded and mislocalized pattern of cell death continues until E11.5 (Fig. 1FGo) and extends almost as far as the dorsal tip of the pouch. The pouch pinches off later in Pitx2 null animals, occurring between E11.5 and E12.5 instead of E10.5 to E11.5 (Fig. 1Go, F and H). At E12.5, the mutant pouch is markedly hypocellular (Fig. 1HGo). This series of observations suggests the pituitary primordium in Pitx2–/– animals is derived from only the dorsal tip of the pouch, and that cell death is an important aspect of the mechanism of pituitary hypoplasia in Pitx2 null mice.

Overexpression of PITX2 Increases the Number of Gonadotropes
To distinguish between a threshold model and a model requiring a narrow window of PITX2 dosage, we overexpressed the cDNA of Pitx2b in the anterior pituitary. This was accomplished by using the well-characterized {alpha}GSU promoter, which drives high-level expression early in the pituitary primordium and later in thyrotropes and gonadotropes, with lower level expression at other sites including skeletal and cardiac muscle (28, 29, 30). The {alpha}GSU promoter was placed upstream of the Pitx2b cDNA with a FLAG epitope fused to its N terminus followed by the polyA signal and intron from the rabbit ß-globin gene. The FLAG epitope was added to differentiate between endogenous and transgene-derived PITX2. Litters of transgenic mice were collected at E18.5 to avoid loss of severely affected fetuses if excess PITX2 caused perinatal lethality. Histology revealed no significant change in overall morphology between transgenic and nontransgenic mice (Fig. 2Go, A and B). Twenty-six mice were determined to be transgenic by genotyping tail DNA and were assayed for transgene expression. One embryo expressed PITX2 robustly, as determined by immunohistochemistry for both the FLAG epitope and PITX2 (Fig. 2Go, C–F). The unexpected low penetrance of high level expression is probably due to early embryonic lethality caused by PITX2 transgene expression in extrapituitary sites (30). Overexpression of PITX2B results in many more cells staining for both LHß and FSHß, suggesting that additional PITX2 increased the size of the gonadotrope population (Fig. 2Go, G–J). The gonadotropes expanded not only in number but also in their domain, which extended dorsally. We examined the expression of the gonadotrope-specific transcription factor SF1 to confirm the gonadotrope expansion implied by the LHß and FSHß stainings. Indeed, we saw an expansion of SF1-positive cells (Fig. 2Go, K and L), both in number and domain, mirroring the results of the hormone immunohistochemistry. We examined markers for other cell types including somatotropes, thyrotropes, and corticotropes (data not shown); however, no significant change was found between transgenic and wild-type littermates for these cell populations. We saw no significant loss of PIT1-positive cells, and no colocalization of SF1 and PIT1, indicating no changes in cell fate or failure of specification (Fig. 2Go, M and N). Therefore, the expansion of the gonadotropes in response to excess PITX2B does not arise at the expense of other cell populations.



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  		Fig. 2. Overexpression of PITX2 Increases the Number of Gonadotropes

A transgenic animal driving high expression of PITX2 in the gonadotropes and thyrotropes was analyzed by immunohistochemistry to determine the relative size of the different pituitary cell populations. The morphology of the pituitary in transgenic animals was similar to wild-type (WT) as seen with hematoxylin and eosin (H&E) stain (A and B). Overexpression of PITX2 was verified with immunohistochemistry by staining with antibodies to the FLAG epitope attached to the N terminus of the protein (C and D) and to PITX2 itself (E and F). Gonadotropes, marked with either LHß (G and H) or FSHß (I and J) were clearly increased in number and were expanded dorsally. The number of cells producing the gonadotrope-specific transcription factor SF1 were also increased in the transgenic animal as compared with the wild-type littermate (K and L). The domain of the dorsal transcription factor PIT1 was not decreased (M and N). i, Intermediate lobe; a, anterior lobe; p, posterior lobe.

 
PITX Proteins Have Overlapping Expression Patterns during Development
The expression pattern of PITX mRNA has been investigated for some developmental time points, but there is limited protein expression data (10, 12, 31, 32, 33, 34, 35, 36). To conduct a complete survey of PITX1 and PITX2 expression, we collected wild-type CD1 mouse embryos at five different time points spanning the entirety of pituitary development from initial Rathke’s pouch formation at E10.5 until the day before birth (E18.5) and traced the pituitary expression patterns using immunohistochemistry. At E10.5, (Fig. 3Go, A and G) both PITX1 and PITX2 are strongly expressed in the invaginating Rathke’s pouch and are excluded from the developing neurohypophysis and surrounding mesenchyme. This pattern is coincident with {alpha}GSU expression reported in developing rat pituitary (37). At E11.5 (Fig. 3Go, B and H) both PITX proteins are found throughout Rathke’s pouch as well as the newly expanding anterior lobe. This pattern continues through E12.5 (Fig. 3Go, C and I). By E14.5, PITX1 is detected throughout the entirety of Rathke’s pouch and anterior lobe, including the presumptive intermediate lobe on the dorsal side of the pouch. PITX2, however, is not highly expressed in the presumptive intermediate lobe (Fig. 3Go, D and J). PITX1is maintained in the intermediate and anterior lobes through E18.5 and into adulthood, whereas PITX2 immunoreactivity remains strong only in the anterior lobe (Fig. 3Go, E, F, K, and L). This trend is also observed in the adult pituitary (Fig. 3Go, F and L). PITX1 immunoreactivity becomes patchy after E14.5, indicating that it is no longer highly expressed in all cells. Some cells have very high levels of PITX1, whereas others have low levels and still others are below the limit of detection. This phenomenon is also observed with PITX2. The apparent lower expression level of PITX2 at E14.5 and E18.5 is most likely an artifact due to the suboptimal function of the PITX2 antibody in paraffin because strong staining has been previously reported in E15.5 pituitaries (38).



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  		Fig. 3. PITX1 and PITX2 Have Overlapping Expression Patterns during Pituitary Development

Adult pituitaries and embryos were collected at E10.5, E11.5, E14.5, and E18.5. The embryos and pituitaries were embedded in paraffin, sagitally sectioned and stained by immunohistochemistry (red) for PITX1 (A–F) or PITX2 (G–L). The sections were then counterstained with 4',6-diamidino-2-phenylindole (blue) to reveal morphology. A–F, PITX1 is expressed throughout the entire pituitary by E10.5 and maintains its expression for the entirety of pituitary development and into adulthood. PITX1 is found early in the invaginating Rathke’s pouch, later in the forming anterior lobe (a) and, notably, in the forming intermediate lobe (i), but not in the posterior lobe (p). The expression in the presumptive intermediate lobe is stable during development and in the adult pituitary (G–L). PITX2 has a similar expression pattern to PITX1 but is excluded from the intermediate lobe. Both PITX1 and PITX2 are uniformly expressed throughout the anterior lobe and Rathke’s pouch; however, they are not expressed in each cell at the same level. As development progresses, the expression patterns for both PITX1 and PITX2 become patchy. Adult pituitaries are stained (green) for hormone markers prolactin (F) and LH (L) to differentiate between anterior and intermediate lobes.

 
PITX Protein Is Primarily Expressed in Gonadotropes and Thyrotropes in the Adult Pituitary
The cell-specific expression data for PITX mRNA or protein in the adult pituitary are limited and contradictory (5, 13, 17, 34, 35, 39, 40). In the case of Pitx2, much of the data are centered on RNA expression in pituitary cell lines that are immortalized and do not necessarily express the appropriate complete hormone. They may resemble adenomas or prepituicytes rather than fully differentiated adult pituicytes.

To determine which of the anterior pituitary gland cell types express the PITX proteins, we carried out colocalization studies in wild-type adult pituitaries with immunohistochemistry for PITX1 or PITX2 and a specific pituitary hormone. The pituitary hormones are each synthesized by a specific cell type in the pituitary and, thus, can be used as markers for these cell types. ACTH marks corticotropes, LH marks gonadotropes, TSH marks thyrotropes, PRL marks lactotropes, and GH marks somatotropes. We found that most somatotropes do not express appreciable quantities of PITX1 or PITX2 (Fig. 4Go, B and G, arrowheads). A small fraction of corticotropes and lactotropes express detectable levels of these transcription factor proteins (Fig. 4Go, A, D, F, and I). In contrast, PITX1 and PITX2 were readily detected by immunohistochemistry at high penetrance in both thyrotropes and gonadotropes (Fig. 4Go, C, E, H, and J, arrows). We found that 83% of thyrotropes, 87% of gonadotropes, 33% of lactotropes, 12% of corticotropes and only 1% of somatotropes express PITX2, and 80% of thyrotropes, 90% of gonadotropes, 35% of lactotropes, 16% of corticotropes and only 4% of somatotropes contain readily detectable PITX1. By increasing the sensitivity of detection, we observed some PITX immunostaining in almost all cells in the adult anterior pituitary; however, there is a substantial difference in intensity between high expressing cells and minimally expressing cells. Interestingly, PITX1 and PITX2 appear to have the same protein expression patterns in the five major cell types of the anterior pituitary, although PITX1 predominates in the intermediate lobe of the adult pituitary. This is consistent with PITX1 immunohistochemistry reported for adult mice (41).



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  		Fig. 4. PITX1 and PITX2 Have Similar and Nonuniform Expression Patterns in Adult Pituitaries

Adult pituitaries were removed from wild-type 5-wk-old animals, embedded in paraffin, stained by immunohistochemistry (red) for either PITX1 (A–E) or PITX2 (F–J) and double-labeled (green) for pituitary hormones: ACTH (A and F), GH (B and G), LH (C,H), prolactin (Prl) (D and I), TSH (E and J). We found that colocalization patterns were comparable for both PITX1 and PITX2 in the adult anterior pituitary. Colocalization was highest (arrows) for gonadotropes and thyrotropes (C, E, H, and J), intermediate for lactotropes (D and I), and nearly absent (arrowheads) for corticotropes and somatotropes (A, B, F, and G).

 
Loss of PITX1 and PITX2 Leads to Extinction of LHX3 Expression
The LIM homeodomain transcription factor LHX3 is initially expressed in the developing pituitary during invagination of Rathke’s pouch at E10.5, coincident with PITX gene expression. There is evidence that antisense RNA inhibition of Pitx1 expression in cell culture causes reduced LHX3 expression (5). These experiments suggest that Lhx3 is downstream of Pitx1; however, Pitx1 knockout mice (Pitx1–/–) exhibit nearly normal pituitary development (14), and Pitx2 knockout mice (Pitx2–/–) express LHX3 normally in the pituitary rudiment (12). To clarify whether Lhx3 expression requires PITX genes, we examined developing mouse pituitaries deficient in both PITX genes for the presence of LHX3.

Pitx1+/– mice were bred to Pitx2+/– and Pitx2+/neo mice; however, very few double heterozygotes were obtained. From 130 live progeny of a cross between Pitx1+/– and Pitx2+/– mice, 32 double heterozygous pups would be expected, but only four viable pups were obtained (P value = 1.86 x 10–7). The Pitx1+/–, Pitx2+/neo double heterozygotes were also significantly underrepresented (12). The doubly heterozygous mice have been shown to have a hypoplastic pituitary with a decrease in all pituitary cell types (42). The cause of death is not known, and we observed no obvious defects in newborn double heterozygotes that explained the lethality. Despite the paucity of surviving double heterozygotes in either cross and the low frequency of double mutants expected from the intercross (1/16), we were successful in obtaining one mouse for each of the genotypes Pitx1–/–, Pitx2neo/– and Pitx1–/–, Pitx2–/– for analysis.

LHX3 is easily detectable in mutant mice lacking PITX1 (Fig. 5BGo). LHX3 is also easily detectable throughout the entire developing pituitary in mice with reduced PITX2 and mice with a complete loss of PITX2 (Fig. 5Go, C and D). In contrast, no LHX3 immunoreactivity was observed in doubly mutant mice with a loss of PITX1 and either a partial or complete loss of PITX2 (Fig. 5Go, E and F). Therefore, PITX1 and PITX2 have a functional overlap and are required for LHX3 expression in the pituitary.



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  		Fig. 5. PITX1 and PITX2 Are Required for LHX3 Expression

Embryos were collected at E10.5, embedded in paraffin, and sectioned in the sagittal plane. We selected embryos with genotypes of wild-type (A), Pitx1 null (B), Pitx2 null (C), Pitx2 hypomorph (D), Pitx1 null; Pitx2 hypomorph (E), or double null of Pitx1 and Pitx2 (F). The sections were stained (red) by immunohistochemistry for LHX3. LHX3 was found in wild-type sections (A) as well as those with less PITX2 (D) and a loss of either PITX1 or PITX2 alone (B and C); however, in animals with a loss of both PITX1 and PITX2 (F), or loss of PITX1 and low amounts of PITX2 (E), the expression of LHX3 was extinguished.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Loss of PITX2 Leads to Increased Cell Death
Decreased cell proliferation has been proposed as the mechanism of pituitary hypoplasia in Pitx2–/– mice (42). However, we did not detect a large difference in proliferation between mutant and wild-type mice at E10.5. The link between the PITX and LIM family suggested the possibility that cell death might contribute to the hypocellularity in Rathke’s pouch as observed in Lhx4 mutants (43). This cell death is transient and easily overlooked. We found that cell death is confined to the base of Rathke’s pouch in normal mice and hypothesize that this apoptosis contributes to normal separation of the pouch from the remaining oral ectoderm. In Pitx2 mutants, cell death is excessive during early development, and the location of the apoptotic cells in the pituitary is shifted dorsally. This abnormality in border formation between the pouch and the oral ectoderm apparently results in pinching off of only the most dorsal tip of the pouch. As a result, the primordium is composed of fewer cells, which undoubtedly accounts for the extreme hypocellularity of the mutant pituitary gland. It is interesting that the pattern and timing of apoptosis in PITX2-deficient mice differs from the abnormal apoptosis observed in Lhx4 and Prop1-deficient mice. This suggests that there are multiple inputs for cell survival.

PITX2 and the dependent LIM genes may function as survival signals for the early pituitary cells, or their loss may disrupt responsiveness to other developmental cues resulting in cellular suicide and stunted organ growth. This is consistent with instances of apoptosis in cells that do not undergo proper differentiation (44) or do not receive appropriate contextual signals (45).

Excess PITX2 Increases Number of Gonadotropes
Pitx2 null mice (Pitx2–/–) make up the most severely affected group of an allelic series and show no anterior lobe expansion. Pituitaries of mice with very little PITX2 (Pitx2neo/–) have only a small anterior lobe, whereas those with less than half dose (Pitx2neo/neo) have an anterior lobe that is nearly normal in size but fails to develop gonadotropes and has a reduced number of somatotropes and thyrotropes. Mice with full or half PITX2 dose (Pitx2+/+ or Pitx2+/–) undergo normal pituitary development (12). Recently, a Rieger syndrome patient was shown to carry a V45L mutation in the homeodomain of PITX2 that increased transactivational activity in cell culture (22). Although this was done with an artificial reporter, it suggests that excess PITX2 may be as detrimental to organ development as a deficit of PITX2. We speculated that increased dosage of PITX2 would cause a phenotype that mirrors the haploinsufficiency disease state as seen with increased dosage of PAX6 (21). In this paradigm, a small window of proper gene dosage is required for appropriate cellular specification and proliferation. This model is in contrast to a mechanism where a threshold of concentration must be reached and excess protein concentration has no effect. This mechanism is evident in contiguous gene syndromes where numerous genes are duplicated, but only one causes a disorder (46). In fact, only a small fraction of genes are sensitive to dosage effects, whereas most reach a threshold level and cause no pathology at elevated levels.

To test our hypothesis, we overexpressed PITX2 in the gonadotrope and thyrotrope cells of developing mice. We observed a marked increase in only one cell population: the gonadotropes. The number and location of the thyrotropes was unchanged, and there was no evidence for a change or loss of cell fate from other pituitary cell populations. The isolated expansion of gonadotropes in response to excess PITX2 corresponds to the high sensitivity of gonadotropes to the loss of PITX2.

Our findings are consistent with overexpression studies in which PITX2 expression in somatotropes precursors caused an increase in the number of somatotropes (42). These pituitary misexpression experiments and the phenotype of the PITX2 hypomorph emphasize the role of Pitx2 dosage in lineage expansion. However, the pituitary results contrast with findings in the eye where increased PITX2A causes many of the same phenotypes observed in Pitx2–/– animals (23). This disparity between transcription factor dosage effects in different organs highlights the complex nature of their interactions and the importance of the chromatin context in which they act. Many of the downstream targets of Pitx2 are likely to be different in the developing eye vs. the pituitary gland; thus, organ-specific differences in dosage sensitivity are not unexpected.

PITX Expression in the Embryonic and Adult Pituitary
PITX1 and PITX2 protein are found in an identical pattern in Rathke’s pouch and the forming anterior lobe at E12.5 (12). We investigated the coexpression of PITX1 and PITX2 in the pituitary throughout development and in adult mice. Because Pitx2 may be posttranscriptionally regulated (10) and because the mRNA data are not from one source, it is important to fully characterize the protein expression patterns of both PITX1 and PITX2 in a single study (10, 31, 32, 33, 34, 35, 36). We found both PITX1 and PITX2 protein expression in pituitary glands from E10.5 to E18.5. Both transcription factors are found in the thickened, invaginating placode of the oral ectoderm and in Rathke’s pouch. They are also found in cells of the expanding anterior lobe. PITX1 maintains its expression in the dorsal half of Rathke’s pouch and in the intermediate lobe in the adult mouse, which is derived from it. PITX2 expression diminishes to little or none in this part of the pouch after E12.5 and throughout adulthood. PITX1 activates POMC gene transcription in conjunction with TPIT in both the corticotropes in the anterior lobe and in melanotrophs in the intermediate lobe (47). Thus, it is logical that PITX1 expression is sustained in the intermediate lobe in differentiated melanotrophs to maintain POMC transcription.

After the anterior pituitary has begun its expansion the two PITX proteins are detected in certain cells at significantly higher levels than others. Both proteins are expressed throughout the anterior lobe but are no longer readily detected in all cells. In adults, we found that those cells with intense staining for PITX1 or PITX2 were always thyrotropes or gonadotropes. Only rarely was the strong nuclear PITX1 or PITX2 signal found in corticotropes, somatotropes, or lactotropes. This is consistent with the Drouin group’s findings for PITX1 (41). The paucity of PITX2-positive somatotropes was surprising given the report of IGHD in some Rieger patients (48). Perhaps IGHD occurs in some Rieger patients because PITX2 haploinsufficiency results in failure to establish the appropriate population size of the Pit1 lineage, as we reported in Pitx2neo/neo mice (12).

The coexpression of PITX1 and PITX2 in the embryonic and adult pituitary supports the hypothesis that they have a functional overlap (12). Furthermore, the dynamic aspect of their expression pattern in the course of development and their prolonged period of expression during development supports the hypothesis that there are multiple roles for the PITX genes during pituitary organogenesis (12). Our detection of pan-pituitary PITX expression is similar to the findings that PITX genes are expressed in all pituitary-derived cell lines. These cell lines may represent the earliest stages of differentiation and are more analogous to a state found developmentally than to fully differentiated, adult cells. We propose that PITX genes play a role in the maintenance of function or survival in gonadotropes and thyrotropes but may have less of a role for maintenance of somatotropes, corticotropes, and lactotrope function.

The null alleles for Pitx1 and Pitx2 and the hypomorphic allele of Pitx2 allowed the roles of these genes in early development to be tested. The "floxed" allele of Pitx2 in combination with inducible, tissue and cell type-specific Cre transgenes will be valuable for testing the role of PITX2 in gonadotrope, thyrotrope, and somatotrope function.

Loss of PITX Genes Leads to Extinction of LHX3 Expression
With the overlapping expression patterns of PITX1 and PITX2 defined, we searched for molecular changes in the developing mouse pituitary when both PITX genes were simultaneously inactivated. In the hindlimb, where PITX1 and PITX2 are also coexpressed, the two genes act cooperatively and can compensate for the other’s loss (49). We previously noted a similar phenomenon in the pituitary. Mice with two hypomorphic alleles of Pitx2 and no functional Pitx1 had smaller pituitaries than mice with only one of these defects (12). Although this morphological finding is consistent with compensation between the two transcription factors, there are no known molecular defects to explain the hypocellularity of the pituitary. This inability to find molecular defects is in part due to the low level of PITX2 expression allowed by its hypomorphic alleles. To find an early molecular defect in mice with PITX mutations, we generated mice with a complete loss of both Pitx1 and Pitx2. This proved challenging because of the significantly lowered viability of the compound heterozygotes. Because the LIM homeobox gene, Lhx3, is coexpressed with Pitx1 and Pitx2 in the forming pituitary and mice deficient in Lhx3 (26) resemble those with a complete loss of Pitx1 and partial loss of Pitx2, it is a tempting potential target of the PITX genes. Mice lacking Pitx2 or Pitx1 alone express LHX3 throughout the invaginating Rathke’s pouch at E10.5; however, in the absence of Pitx1 (Pitx1–/–) concurrently with a severe or total loss of Pitx2 (Pitx2neo/– or Pitx2–/–), no LHX3 expression is detectable. This suggests that LHX3 expression is dependent on a combinatorial threshold level of both PITX proteins in the pituitary, and that either transcription factor alone is sufficient to express LHX3 during early pouch invagination. Furthermore, this indicates that the PITX genes are upstream of Lhx3. Thus, the PITX family and the LIM family are intimately tied in pituitary development.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Wild-Type and Mutant Mice
Mice carrying the Pitx2neo, Pitx2–, and Pitx1– alleles were previously generated by gene targeting (13, 17) and bred at the University of Michigan. Mice were housed in ventilated cages under 14-h light and 10-h dark cycles. They were fed PMI 5020 mouse chow ad libitum. Pitx2 alleles were maintained after repeated backcrosses to C57BL/6J mice (n = 4–6). C57BL/6J mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and CD-1 mice were obtained from Charles River (Wilmington, MA). Timed pregnancies were generated using mice naturally cycling in estrus, and the morning after mating was designated as E0.5. 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 National Institutes of Health Guidelines for the Care and Use of Experimental Animals.

Transgene Construction
Pitx2b cDNA was subcloned from a Pitx2b expression construct (12). PCR was performed with a forward primer containing complementary sequence to the 5' end of the cDNA as well as an ATG start site preceding a FLAG-tag sequence (50, 51) (GAC TAC AAG GAC GAC GAT GAC AAG) and a reverse primer with complementary sequence to the 3' end of the cDNA and an EcoRI site (CTT GTC ATC GTC GTC CTT GTA GTC CAT TCA AGC TTG AAT TCT AGA AGG). This PCR fragment was cut and subcloned into the pBlueScript II SK+ vector (Stratagene, La Jolla, CA) containing the rabbit ß-globin polyA tail and intron from pCGN2 provided by Dr. David Gordon (Denver, CO). The {alpha}GSU promoter (officially known as chorionic gonadotropins {alpha} or Cga) was obtained from the {alpha}GSU containing pGEM7Zf+ vector (28). A total of 4.6 kb of the {alpha}GSU promoter/enhancer was excised with KpnI and ClaI and subcloned upstream of the Flag tagged Pitx2b cDNA in the pBlueScript II SK+ vector (Stratagene). The transgene was linearized with KpnI and NotI, and the resulting 6.75-kb fragment was isolated.

Generation and Genotyping of Transgenic Mice
The linearized Cga-Pitx2b construct was microinjected into F2 zygotes from (C57BL/6J X SJL/J) F1 parents at a concentration of approximately 2–3 ng/µl (52). Embryos at the two-cell stage were transferred to 0.5 post coitum pseudopregnant CD-1 females. Embryos were removed at E18.5. Genomic DNA was prepared from tail biopsies of the embryos by a simple salting out procedure (53), and PCR was performed to identify mice that carried the transgene. A forward primer located in the {alpha}GSU promoter (5' GCA ATG TGA TAT GAT CAA TTG ATG T 3') and a reverse primer located in the homeodomain of Pitx2b (5' ACC CGG ACT CGG GCT TCC GTA AG 3') were used for this PCR.

Histology and Immunohistochemistry
Tissues were fixed in 4% paraformaldehyde in PBS (pH 7.2) on ice. Embryos at day 10.5, 11.5, 12.5, 14.5, 18.5, and isolated adult pituitaries were fixed for 1.5, 2, 2, and 2.5 h, overnight, and 30 min, respectively. The fixed tissues were washed in PBS and dehydrated through successively more concentrated ethanol solutions and embedded in paraffin. Tissue sections of 6 µm thickness were prepared for fluorescent immunohistochemistry.

Slides were dewaxed and rehydrated before staining. Epitopes were exposed with a 10 min boil in 10 mM citric acid (pH 6.0). PITX1 antibody was used at 1:1500 (41). We confirmed the specificity of the PITX1 antibody by recapitulating esophageal and ocular staining patterns established by in situ (data not shown). SF1 antibody (K. Morohashi, National Institute for Basic Biology, Myodaiji-cho, Okazaki, Japan) was used at 1:1500. The LHX3 monoclonal antibody developed by Thomas Jessell (Columbia University, New York, NY) was obtained from the Developmental Studies Hybridoma bank developed under the auspices of the NICHD and maintained by the University of Iowa. The PITX2 antibody was previously described (38). Antibodies to the hormones were obtained from The National Hormone and Pituitary Program, NIDDK (Bethesda, MD) and included rat LHß, rat TSHß, rat FSHß, human ACTH, rat GH, and rat PRL. The antibody to rat PIT1 was generated by Jeff Voss and Simon Rhodes (IUPUI, Indianapolis, IN) and used at 1:500. Anti-FLAG M5 monoclonal antibody (Sigma, St. Louis, MO) was used at 1:200. Sections were incubated with biotin-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA), and signals were amplified using the tyramide signal amplification tetramethylrhodamine kit (PerkinElmer Life Sciences, Foster City, CA) or MOM kit (Vector Laboratories, Burlingame, CA). Diaminobenzidine (Sigma), which produces a brown precipitate, was used as the chromogen for some stainings. Programmed cell death in the embryos was detected by the TUNEL method using the FragEL kit (Calbiochem, San Diego, CA) according to the manufacturer’s protocol. Sections were counterstained with methyl green (Vector Labs), and fluorescent slides were counterstained with 4',6-diamidino-2-phenylindole (Molecular Probes, Eugene, OR). Selected slides were stained with hemotoxylin and eosin to show morphology.

The fraction of differentiated cells that express significant levels of PITX1 or PITX2 was quantified by counting the number of cells that expressed both a cytoplasmic hormone marker and the nuclear transcription factor and dividing this number by the total number of cells that stained positive for the hormone marker. We counted approximately 100-1000 hormone marker-positive cells in multiple, random fields from multiple wild-type CD-1 mice and scored the number of cells also expressing the nuclear transcription factors.


   FOOTNOTES
 
This work was supported by National Institutes of Health (NIH) Grants T32 GM07863, R01 HD34283, and 56-T32-BM07544 and the Michigan Diabetes Research and Training Center. In addition, we thank the Transgenic Animal Model Core Staff and their financial support including: NIH Grants CA46592, AR20557, 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).

Current address for P.J.G.: Department of Ophthalmology & Visual Sciences, 350 Kellogg Eye Center, University of Michigan, Ann Arbor, Michigan 48109-0714.

Current address for H.S.: Laboratory of Genetics, The Salk Institute, La Jolla, CA 92037.

First Published Online March 10, 2005

1 M.A.C. and H.S. are co-first authors. Back

Abbreviations: E, Embryonic day; {alpha}GSU, glycoprotein hormone {alpha}-subunit; POMC, proopiomelanocortin; PRL, prolactin; SF1, steroidogenic factor 1; TUNEL, terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling.

Received for publication January 20, 2005. Accepted for publication March 1, 2005.


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 MATERIALS AND METHODS
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