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Endocrine Reviews 23 (4): 431-442
Copyright © 2002 by The Endocrine Society

Molecular Basis of Combined Pituitary Hormone Deficiencies

Laurie E. Cohen and Sally Radovick

Division of Endocrinology (L.E.C.), Children’s Hospital and Harvard Medical School, Boston, Massachusetts 02115; and Division of Pediatric Endocrinology (S.R.), University of Chicago, Chicago, Illinois 60637

Correspondence: Address all correspondence and requests for reprints to: Sally Radovick, M.D., Department of Pediatrics, University of Chicago, 5841 South Maryland Avenue, MC5053, Chicago, Illinois 60637. E-mail: sradovic{at}peds.bsd.uchicago.edu


   Abstract
Top
 Abstract
I. Introduction
 II. Pituitary Embryology
 III. Pituitary Transcription...
 IV. Summary
 References
 
Pituitary gland commitment from oral ectoderm occurs in response to inductive signals from the neuroepithelium of the ventral diencephalon. Invagination of the oral ectoderm leads to the creation of Rathke’s pouch. Intensified cell proliferation within Rathke’s pouch results in formation of the anterior pituitary lobe. Subsequently, highly differentiated cell types arise sequentially due to overlapping, but distinct, spatial and temporal patterns of signaling molecules and transcription factors. Mutations in some of the pituitary-specific transcription factors have been identified in patients with hypopituitarism, confirming the role of these factors in pituitary development.

I. Introduction

II. Pituitary Embryology

III. Pituitary Transcription Factors

A. Rpx

B. Pitx

C. Lhx3/Lhx4

D. Prop-1

E. Pit-1

IV. Summary


   I. Introduction
Top
 Abstract
 I. Introduction
II. Pituitary Embryology
 III. Pituitary Transcription...
 IV. Summary
 References
 
DURING EMBRYOGENESIS, signaling molecules and transcription factors are expressed in the region of the future anterior pituitary gland in overlapping, but distinct, spatial and temporal patterns. By interacting with each other, these factors control pituitary development and cell determination and specification. The primordium of the anterior pituitary, Rathke’s pouch, is first detected at embryonic day (e) 8.5 in the mouse (1, 2) and can be identified by the third week of pregnancy in humans (3). It forms as an upward invagination of a single cell thick layer of ectoderm that contacts the neuroectoderm of the primordium of the ventral hypothalamus (1, 2). Intensified cell proliferation within Rathke’s pouch results in the formation of the anterior pituitary lobe by e12.5 (2).

Eventually, the mature pituitary gland is populated by at least five highly differentiated cell types, with corticotrophs appearing to arise first. Thyrotrophs arise from two lineages, one related to somatotrophs and lactotrophs. Gonadotrophs also appear to be related to thyrotrophs, somatotrophs, and lactotrophs because of dependence on common transcription factors (4). Ultimately, transcription factors are also involved in the cell-specific expression and regulation of the gene products of these pituitary cells, with corticotrophs producing ACTH, thyrotrophs producing TSH, gonadotrophs producing gonadotropins (LH and FSH), somatotrophs producing GH, and lactotrophs producing PRL.

Hypopituitarism is the deficiency in varying degrees of any or multiple of these pituitary hormones, and patients with pituitary hormone deficiencies have been found to have mutations in several of the pituitary-specific transcription factors that play a role in the determination of the pituitary cell types. In this review, pituitary development will be discussed, as well as molecular defects in pituitary-specific developmental factors that result in GH deficiency in combination with other hormonal deficits.


   II. Pituitary Embryology
Top
 Abstract
 I. Introduction
 II. Pituitary Embryology
III. Pituitary Transcription...
 IV. Summary
 References
 
The initiation of anterior pituitary gland development depends on the competency of the oral ectoderm to respond to inducing factors from the neural epithelium of the ventral diencephalon (4). The bone morphogenic protein (BMP)-4 signal from the ventral diencephalon is the critical dorsal neuroepithelial signal required for organ commitment of the anterior pituitary gland. Wnt5a and fibroblast growth factor (FGF)-8 are also expressed in the diencephalon in distinct overlapping patterns with BMP-4. Subsequently, a BMP-2 signal arises from the boundary of a region of oral ectoderm in which Sonic hedgehog expression, initially expressed uniformly in the oral ectoderm, is selectively excluded from the developing Rathke’s pouch. The ventral->dorsal BMP-2 signal and the dorsal->ventral FGF-8 signal appear to create opposing activity gradients that are suggested to dictate overlapping patterns of specific transcription factors underlying cell lineage specification. The various extensions of these transcription factors in their fields are theorized to combinatorially determine the specific cell types. The FGF-8 gradient determines the dorsal cell phenotypes (4, 5), and dorsally expressed transcription factors include Nkx-3.1, sine oculis-like homeobox 3, Pax-6 (6), and Prophet of Pit-1 (Prop-1) (Ref. 7 and Fig. 1Go). Temporally specific attenuation of the BMP-2 signal is required for terminal differentiation of the ventral cell types, and ventrally expressed transcription factors include Islet-1 (Isl-1) (4), Brn-4 (7), P-Frk (4), and GATA2 (8).



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  		Figure 1. Pituitary development in the mouse (at e11). Dorsal signals (including BMP-4, FGF-8, Prop-1, Nkx3.1, and Pax6) and ventral signals (including BMP-2, Isl-1, and Brn-4) form gradients that appear to determine overlapping patterns of specific transcription factors. [Modified from C. Kioussi et al.: Mech Dev 81:23–35, 1999 (102 ).] Pax6 has a role in the sharp boundary of attenuation of the ventral signals that dictate thyrotroph and gonadotroph cell lineages. An additional population of thyrotrophs is found in the rostral tip and probably derives from Isl-1-expressing cells.

 
Before the appearance of the ventral cell types, Pit-1 is required for somatotroph, lactotroph, and thyrotroph development in the intermediate caudomedial field (9, 10), whereas the orphan nuclear receptor steroidogenic factor-1 (SF-1, also known as Ftx-F1) is selectively expressed in the gonadotrophs (11, 12). The ventral->dorsal gradient induces GATA2 in a corresponding gradient in presumptive gonadotrophs and thyrotrophs, and high levels of GATA2 in the most ventral aspect of the developing anterior pituitary directly or indirectly restricts Pit-1 gene expression out of the presumptive gonadotrophs. In the absence of Pit-1, GATA2 expression appears sufficient to induce the entire set of transcription factors that are typical of the gonadotroph cell type, including SF-1, P-Frk, and Isl-1. Conversely, the absence of GATA2 dorsally is critical for differentiation of Pit-1-positive cells to somatotroph/lactotroph fates. It is hypothesized that the level of GATA2 expression in the thyrotrophs is below the threshold required to inhibit activation of the Pit-1 gene early enhancer permitting the emergence of a Pit-1+, GATA2+ cell, which results in the thyrotroph fate (8). Pax6 has a role in the sharp boundary of attenuation of the ventral signals that dictate thyrotroph and gonadotroph cell lineages. In the absence of Pax6, the ventral lineages, particularly thyrotrophs, become dorsally extended at the expense of somatotroph and lactotroph cell types (6), and Pax6 mutant mice are GH and PRL deficient (13). After terminal differentiation, the gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs are located ventrally to dorsally, respectively. An additional population of thyrotrophs is found in the rostral tip and probably derives from Isl-1-expressing cells (4).


   III. Pituitary Transcription Factors
Top
 Abstract
 I. Introduction
 II. Pituitary Embryology
 III. Pituitary Transcription...
IV. Summary
 References
 
Several pituitary-specific transcription factors, Rpx (Rathke’s pouch homeobox), Pitx (pituitary homeobox), Lhx3 (LIM-homeobox-3), Prop-1, and Pit-1, play a role in the determination of the pituitary cell lineages (Fig. 2Go).



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  		Figure 2. Pituitary-specific transcription factors involved in anterior pituitary development. The homeodomain factors play a role in the determination of specific pituitary cell lineages. Rpx is the earliest known specific marker for the pituitary primordium. Pitx1 and 2 are both present in the fetal pituitary and in most cells of the adult pituitary, albeit at different levels. Lhx3 and Lhx4 are expressed in gonadotrophs, thyrotrophs, somatotrophs, and lactotrophs. Prop-1 determines Pit-1 cell lineages and may play a role in directing some of the precursors of the Pit-1 cell lineage into the gonadotroph lineage, before terminal differentiation events. Pit-1 has been shown to be essential for the development of somatotrophs, lactotrophs, and thyrotrophs. There is a Pit-1-independent population of thyrotrophs in the rostral tip of the developing anterior pituitary gland that phenotypically disappears by the day of birth.

 
A. Rpx
Rpx, also known as Hesx1 (homeobox gene expression in embryonic stem cells), is a member of the paired-like class of homeobox genes originally described in Drosophila melanogaster (14). It is the earliest known specific marker for the pituitary primordium, suggesting that it has a role in early determination or differentiation of the pituitary (15), although no target genes for Rpx have yet been identified (16). In the mouse embryo, Rpx is first expressed in a small patch of cells in anterior midline visceral endoderm as gastrulation commences (14). It is then found in the anterior midline ectoderm and prechordal plate precursor at e6.5–7, in the anterior neural plate at e7.5–8.5, and subsequently in the oral ectoderm that gives rise to Rathke’s pouch at e8.5. By e9.5, Rpx expression is entirely restricted to Rathke’s pouch (14, 15). From e11.5, Rpx is down-regulated, first in the rostral tip of the incipient anterior lobe, and then progressing dorsally. Rpx transcripts are undetectable by e15.5, coinciding with activation of Pit-1 (15). However, Pit-1 is not essential for Rpx regulation, as Rpx repression has normal kinetics in Pit-1-deficient mice. On the other hand, Lhx3 is required for maintenance of Rpx expression, as Rpx is extinguished early at e12.5 in Lhx3-knockout mice (17). Prop-1 is required for repression of Rpx transcription, as there is abnormal persistence of Rpx expression until e17.5 in the Ames dwarf mouse, which has a mutation in Prop-1 (18). As Rpx expression is transient, it cannot be involved in the maintenance of the anterior pituitary cell types (16).

Embryonic mice lacking Rpx have bifurcations in Rathke’s pouch and pituitary dysplasia, reduced prosencephalon, anophthalmia or microophthalmia, defective olfactory development, and ventral midline defects in the hypothalamus. Neonatal mice have abnormalities in the corpus callosum, anterior and hippocampal commisures, and septum pellucidum, similar to the defects seen in septo-optic dysplasia (SOD) in man (14).

SOD is a heterogeneous condition with any combination of optic nerve hypoplasia, pituitary gland hypoplasia, and midline abnormalities of the brain, such as absence of the corpus callosum and septum pellucidum. Two siblings with agenesis of the corpus callosum, optic nerve hypoplasia, and panhypopituitarism were found to have a homozygous mutation at codon 53 in the homeodomain of Hesx1, resulting in conversion of a highly conserved arginine to a cysteine. The result is a drastic reduction in DNA binding to a palindromic PIII sequence, to which mouse Hesx1 has been shown to bind (14).

Thomas et al. (19) scanned 228 patients with a wide spectrum of congenital hypopituitarism phenotypes: 85 with isolated pituitary hypoplasia [including isolated GH deficiency and combined pituitary hormone deficiency (CPHD)], 105 with SOD, and 38 with holoprosencephaly or related phenotypes. They identified three missense mutations: 1) A serine to leucine at codon 170, located immediately C terminal to the homeodomain, was identified in two affected brothers, both with GH deficiency and one with optic nerve hypoplasia. 2) A threonine to alanine at codon 181 (T181A) was identified in one patient with isolated GH deficiency. 3) A glutamine to histidine at codon 6 causing a nonconservative substitution in exon 1 was identified in an individual with multiple pituitary hormone deficiencies. All affected individuals had inherited the mutation from one of their parents, who were not affected; and the T181A mutation was identified in a nonaffected sibling (19). These sequence changes may represent polymorphisms that do not compromise Hesx1 function. However, 100 control sequences were identical with previously published wild-type data. Incomplete penetrance would not be surprising given that heterozygous Rpx+/- mice display low penetrance of a mild phenotype (19).

B. Pitx
Three Pitx transcription factors have been identified by several groups and have been giving the following names: Pitx1 (20), Ptx1 (21, 22), P-OTX (pituitary OTX) (23), and Otx1; Pitx2 (24), Ptx2 (18), Otx2 (25), and RIEG (24); and Pitx3 (26, 27). Pitx transcription factors are paired-like homeodomain transcription factors closely related to the mammalian Otx genes that are expressed in the rostral brain during development and are homologous to the D. orthodenticle (otd) gene, which is essential for the development of the head in D. melanogaster (21). Their homeobox is also highly homologous to that of Caenorhabditis elegans unc-30, which controls differentiation of GABAergic neurons (25).

Pitx1 and 2 are both present in the fetal pituitary and in most cells of the adult pituitary, albeit at different levels (28), suggesting complementary functions in development and function (18). They are 97% identical at the homeodomain and 67% identical at the C terminus (18). Their action on cell-specific target genes may result from physical interaction with lineage-restricted transcription factors (29). Pitx3 is not expressed in the anterior pituitary.

1. Pitx1.
Pitx1 mRNA is first detected in epithelial layers on e8. Pitx1 expression then appears in the first branchial arch mesenchyme beginning at e9 in a proximal-to-distal gradient, and it is restricted to a central stripe in the mandibular component (22). On e9.5, it is expressed throughout the oral epithelium lining the roof of the buccal cavity and in the Rathke’s pouch ectoderm. Expression continues throughout development in all regions of the anterior pituitary (23). In adults, Pitx1 is specifically expressed at higher levels in cells of the {alpha}-glycoprotein subunit (GSU) lineage (30). Although a fraction of proopiomelanocortin (POMC)-expressing cells appear to have high levels of Pitx1, most corticotrophs do not express Pitx1 at high levels (30). Pitx1 is also expressed in derivatives of the first branchial arch, the duodenum, and the hindlimbs (23). Pitx1 expression overlaps temporally and spatially with Lhx3 (23) and is required for sustained expression of Lhx3 (16).

Pitx1 directly activates the {alpha}-GSU promoter and is essential for its sustained expression (16, 23). In addition, Pitx1 is important for maintenance of cell-specific transcription in corticotrophs (express Pitx1 exclusively) and gonadotrophs (express Pitx1 predominantly) (16). A T box factor, Tpit, present only in POMC-expressing cells, is capable of initiating POMC cell differentiation and of activating POMC transcription synergistically with Pitx1 (29). POMC transcription is enhanced by other factors, including leukemia inhibitory factor (LIF) (31) and the basic helix-loop-helix heterodimer NeuroD1/PanI (also known as corticotroph upstream transcription factor, or CUTE) (21, 32, 33). Pitx1 appears to modulate SF-1 activity on the LH-ß promoter (16, 34). SF-1 is an orphan nuclear receptor transcription factor specifically expressed in gonadotroph cells and is required for the maintenance of the gonadotroph phenotype (11). Pitx1 is also synergistic with the immediate early response gene product Egr1 on the LH-ß promoter (35). Moreover, Pitx1 transactivates the GH promoter, and synergistically with Pit-1 activates the PRL promoter (18).

However, in vivo data do not support a role for Pitx1 in GH gene expression. In mice made deficient in Pitx1, GH expression in the somatotrophs appears unchanged (20). In these mice, all known early developmental events, including invagination of Rathke’s pouch, exclusion of Sonic hedgehog from the invaginating epithelium, and activation of FGF-8, BMP-2, Isl-1, Lhx3, {alpha}-GSU, Prop-1, and Pit-1 are normal. Evaluation of the pituitary gland from e15.5 through postpartum d 0 reveals a decrease in the number of gonadotrophs and thyrotrophs and diminished levels of LHß and TSHß transcripts and protein within individual cells. The TSHß transcripts are reduced most severely in the Pit-1-independent rostral tip thyrotroph population. Interestingly, there is an increase, rather than decrease, in the levels of ACTH transcripts and peptides in the corticotrophs (20). The role of Pitx1 in pituitary development is not clear from this model, however, as there may be redundancy between Pitx1 and Pitx2. The authors speculate that the altered phenotype may reflect the synergistic role of Pitx1 in target gene induction (20). These mice also have changes of skeletal structures within a specific region of the hindlimb, as well as severe defects in the development of the palate and its derivatives.

2. Pitx2.
Pitx2 transcripts are first detected at e8.5 in the mouse embryo in oral epithelium and oral ectoderm. At e9.5, there is expression of Pitx2 in the nascent Rathke’s pouch, as well as expression in the mesenchyme near the optic eminence, the basal plate of the central nervous system, the base of the forelimbs, and in domains of the abdominal cavity. At e16.5, there is a low level of expression in the intermediate lobe and the rostral tip of the anterior lobe of the pituitary gland in an apparently homogeneous pattern (25). The Pitx2a and Pitx2b mRNA isoforms are expressed in the adult pituitary gland in the thyrotrophs, gonadotrophs, somatotrophs, and lactotrophs, but not in the corticotrophs, where Pitx1 is highly expressed (18). The Ptx2c isoform is expressed in all five of these lineages (36). Pitx2 appears required for pituitary gland development shortly after formation of the committed pouch (36), and Pitx2 may be a determinant for one or more anterior pituitary cell types or may function by acting in concert with other transcription factors. Unlike Pitx1, it is also expressed in the adult in kidney, lung, testis, and tongue (18).

In the chick, Xenopus, and mouse, Pitx2 expression is on the left side of the embryo in the lateral plate mesoderm and then continues to be expressed asymmetrically in several organs that are asymmetric with respect to the left-right axis of the embryo. Ectopic expression of Pitx2 results in reversed looping of the heart and intestine and reversed body rotation in chick and Xenopus embryos, suggesting that Pitx2 may interpret and subsequently execute the left-right developmental program dictated by upstream signaling molecules (37).

Pitx2-knockout mice have been generated (36, 38, 39, 40). These animals have normal formation of Rathke’s pouch including induction of Rpx, Lhx3, and Pitx1. However, there is decreased cell content by e10.5. BMP-2 and {alpha}-GSU are initially expressed, as well as low levels of GATA2, Prop-1, and Nkx3.1. There is failure of organ progression with undetectable levels of Pit-1, TSH-ß, and Lhx4, and only a few POMC-positive cells. Thus, Pitx2 may fail to correctly activate target genes that require synergistic activation by Pitx2 and Lhx3 (38).

RIEG is the human homologue of Pitx2 (24). In individuals with Rieger syndrome, an autosomal dominant condition with variable manifestations including anomalies of the anterior chamber of the eye, dental hypoplasia, a protuberant umbilicus, mental retardation, and pituitary alterations, several mutations of RIEG have been found. All mutations described to date have been heterozygous. Eight mutations were found to affect the homeobox region: five were missense mutations in the homeodomain (24, 41, 42), two were splicing mutations in the intron dividing the homeobox sequence (43), and one was an in-frame duplication of 21 bp causing a seven-amino-acid duplication of threonine 44 to lysine 50 (residues 6–12 of the homeodomain) (41). Patients with Rieger syndrome do not have an alteration in organ situs, which may be due to the presence of a wild-type allele (37). There appears to be a differing sensitivity of various organs to Pitx2 deficiency, and mice that are heterozygous for hypomorphic or null alleles for Pitx2 mimic some aspects of Rieger syndrome (36).

Several of the mutations have been studied. One, a substitution of a glutamine for a highly conserved leucine at codon 54 (L54Q) in helix 1 of the homeodomain leads to an unstable protein. Another is a nonconserved threonine altered to a proline at codon 68 (T68P) in helix 2 of the homeodomain. T68P binds to a DNA consensus sequence, but with somewhat lower affinity than wild-type Pitx2, and does not allow for the Pit-1-enhanced binding of Pitx2. Unlike wild-type Pitx2, the T68P mutant does not transactivate the PRL promoter, nor does it yield synergistic activation with Pit-1 (43). A valine altered to a leucine in the third helix of the homeodomain has a slight decreased reduction in DNA binding but has a 200% increase in transactivational activity over wild-type Pitx2, and it is speculated that the increased transcriptional activity may also be disease causing (41). The seven-amino-acid duplication of residues 6–12 of the homeodomain has a greater than 100-fold reduction in DNA binding and no detectable transactivation of a target promoter construct (41). Another homeodomain mutation resulting in change of a lysine to a glutamic acid (K88E) also has reduced DNA binding and transactivation activities. Unlike the other mutations, however, the K88E mutant Pitx2 suppresses the Pit-1 synergism of wild-type Pitx2, suggesting a dominant-negative effect (42).

GH promoter activity was not evaluated, but because there is GH insufficiency in a subset of affected individuals with Rieger syndrome, Pitx2 may also have a role in activation of the GH gene (24). As Pitx1 has a low level expression in lineages other than {alpha}-GSU-expressing lineages (30), and Pitx1-deficient mice have a normal number of somatotrophs (20), it is possible that Pitx2 may be required for development of the somatotrophs. However, it remains unclear that heterozygosity for inactivating mutations of Pitx2 in humans result in pituitary hormone deficiencies.

C. Lhx3/Lhx4
Lhx3 is a LIM-type homeodomain protein, where the acronym LIM comes from the original members of the LIM homeobox genes, lin-11, isl-1, and mec-3 (44). Lhx3 is also known as LIM-3 (45) and P-Lim (pituitary LIM) (46). The LIM proteins contain two tandemly repeated unique cysteine/histidine LIM domains located between the N terminus and the homeodomain (the DNA binding domain) (44). The LIM domains do not bind to DNA (46) but may be involved in transcriptional regulation (44). There is weak expression of one isoform, Lhx3a, at e8.5 in the mouse embryo, whereas the other isoform, Lhx3b, does not appear until e9.5. At e9.5, Lhx3 is expressed in Rathke’s pouch and the closing neural tube. During subsequent development, there is Lhx3 expression in the anterior and intermediate lobes of the pituitary gland, the ventral hindbrain, and the spinal cord. Lhx3 expression persists in the adult pituitary, suggesting a maintenance function in one or more of the anterior pituitary cell types (44). Lhx3 is expressed at high levels before the initial detection of {alpha}-GSU transcripts and binds to and activates the {alpha}-GSU promoter. Lhx3 and Pit-1 are synergistic in transcriptional activation of the TSH-ß and PRL promoters and the Pit-1 enhancer (46).

In Lhx3-knockout mice, in which the two LIM domains and some of the homeodomain are deleted, Rathke’s pouch is initially formed but fails to grow. There are also changes in the expression of pituitary-specific markers. Rpx is detected at e10.5, suggesting that initial specification of Rathke’s pouch occurs and initial expression of Rpx is independent of Lhx3. However, Rpx expression ceases early at e12.5. {alpha}-GSU is undetectable at e12.5 and 15.5. TSH-ß and GH are undetectable at e16.5. There are no LH-positive cells at e18.5, but because there is late activation of LH-ß and small numbers of gonadotrophs in wild-type mice, it is unclear whether LH-ß is missing or decreased. There are also no Pit-1 transcripts, so Lhx3 must be required directly or indirectly for Pit-1 expression (17).

In these mice, POMC is detected in the floor of the diencephalon and in a small cohort of cells at the ventral base of the Rathke’s pouch remnant, with placement corresponding to the position of the first presumptive corticotroph cells to differentiate at e13. Specification of the corticotroph cell lineage occurs, confirming that the derivation of the corticotroph lineage must be distinct from that of the other anterior pituitary cell lineages. However, the POMC cells fail to proliferate, suggesting that there is an intrinsic feature common to all pituitary cells, or failure of more than one pituitary cell type to differentiate and produce trophic factors that indirectly affect proliferation of other neighboring cells, or failure to respond to factors produced by adjacent structures or proliferative factors (17). Corticotrophs appears to preferentially express the Lhx3b isoform (47).

Humans have been found to have mutations in the Lhx3 gene. These patients have complete deficits of GH, PRL, TSH, and gonadotropins and a rigid cervical spine leading to limited head rotation. A tyrosine to cysteine conversion at codon 116 (Y116C) in the LIM2 domain is associated with a hypoplastic anterior pituitary. An intragenic 23-amino-acid deletion predicting a severely truncated protein lacking the entire homeodomain is associated with an enlarged pituitary (48). Whereas the intragenic gene deletion mutant protein does not bind DNA, the Y116C mutant does. Both mutant Lhx3 proteins have a reduced gene activation capacity (49).

A closely related gene, Lhx4 (Gsh-4) is expressed in specific fields of the brain and spinal cord (50, 51) and, with Lhx3, regulates proliferation and differentiation of pituitary lineages (52). As in Lhx3-knockout mice, Rathke’s pouch forms in Lhx4-knockout mice, and therefore, Lhx4 is not required for induction of the pouch. Although both Lhx3 and Lhx4 are expressed throughout the invaginating pouch at e9.5, Lhx4 expression becomes restricted to the future anterior lobe of the pituitary at e12.5, whereas Lhx3 remains expressed in the whole pouch. Lhx4 expression diminishes at e15.5, whereas Lhx3 expression is maintained. Lhx3/Lhx4 double knockout mice display an early arrest of pituitary development that is more severe than either single mutant, suggesting a redundant function by these two factors. Lhx3 plays a more significant role than Lhx4 during formation of the mature pituitary structures, because at least one functional copy of Lhx3 is required (52). Unlike Lhx3-knockout mice, Lhx4-knockout mice have transcripts for {alpha}-GSU, Pit-1, GH, and TSH-ß. There are a few LH-positive cells, suggesting that Lhx4 may support, but is not required for, specification of gonadotroph cells. However, all five anterior pituitary cell lineages show reduced numbers (52).

Patients with deficiencies of GH, TSH, and ACTH (LH, FSH, and PRL were not evaluated), a small sella turcica, a persistent craniopharyngeal canal, a hypoplastic anterior hypophysis, an ectopic posterior hypophysis, and a deformation of the cerebellar tonsil into a pointed configuration have a heterozygous intronic point mutation of the splice acceptor site preceding exon 5. This human mutation was transmitted in a dominant manner, affecting only the maternal side. Transmission suggests that the gonadotropin axis is intact. The result is two mutant products from use of two cryptic splice-acceptor sites located within exon 5. Use of the first splice-acceptor site is predicted to lead to an in-frame deletion of four highly conserved amino acids in the third helix of the homeodomain. Use of the second splice-acceptor site should alter the reading frame at position 47 of the homeodomain, leading to a premature termination codon within exon 5 (53). However, the targeted disruption of Lhx4 in mice is asymptomatic in the heterozygous state, so the relationship of this mutation with pituitary hormone deficiencies is not confirmed.

D. Prop-1
Prop-1 is a paired-like homeodomain transcription factor (54). Prop-1 expression is restricted to the anterior pituitary. It is first detected at e10–10.5 in the mouse embryo, particularly over the dorsal portion of the gland, and there is maximal expression at e12 over the full caudomedial area in which Pit-1 is later expressed. Expression decreases after e14.5 (1).

The Ames dwarf mouse has a homozygous mutation that involves a serine-to-proline substitution at amino acid 83 (S83P) in the {alpha}-1 helix of the homeodomain (Fig. 3Go). These mice have deficiencies of GH, PRL, and TSH (7). There is normal initial pituitary development, with normal patterns and levels of expression of Pitx and Lhx3 (7). At e13, recently divided cells fail to accumulate in the caudomedial region where Pit-1 is normally expressed (55), and there is subsequent failure of determination of Pit-1 lineages, lack of Pit-1 gene activation, and absence of progression to mature cells (1). The size of the nascent pituitary gland is reduced by e14.5, with the adult pituitary size decreased by 85% (2). The adult Ames dwarf mouse has less than 1% of the normal complement of somatotrophs, a scarcity of lactotrophs and thyrotrophs (7), and reduced expression of gonadotropins (56). In contrast to the Ames dwarf mouse, there is normal expression of gonadotropins in Pit-1-defective Snell dwarf mice and humans. Hence, Prop-1 may play a role in directing some of the precursors of the Pit-1 cell lineage into the gonadotroph lineage before terminal differentiation events (56).



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  		Figure 3. Schematic representation of the Prop-1 gene. Exons are depicted as shaded areas, and the homeodomain is noted in black. Mutations are noted at their locations. The S109X mutation results from both a 2-bp deletion between nt 296–301, and from a 2-bp deletion at codon 50 (nt 149–150) that causes divergence of amino acid sequence from codon 52. The P164X mutation results from a 1-bp deletion at codon 50, which causes divergence of amino acid sequence from codon 53. The P160X mutations results from a 13-bp deletion between nt 112–124, which causes divergence of amino acid sequence from codon 38.

 
The mutant S83P Prop-1 has 8 times lower affinity DNA binding to two dimeric DNA sites that bind wild-type Prop-1 in the Pit-1 -5- to -10-kb early enhancer, an enhancer required for initial Pit-1 gene activation. There is reduction in transcriptional activation of these two dimeric DNA binding sites. However, wild-type Prop-1 alone fails to clearly activate the enhancer. Therefore, either additional factors or signals are required for a Prop-1 direct effect in the initial action of the Pit-1 gene early enhancer, or the effects of Prop-1 are indirect (7).

Several human mutations of Prop-1 resulting in combined pituitary hormone deficiency of GH, PRL, and TSH have also been described (Fig. 3Go). Some subjects do not produce LH and FSH at a sufficient level to enter puberty spontaneously (56), whereas others have a loss of gonadotropin secretion with age but enter puberty spontaneously (albeit delayed), suggesting that Prop-1 is not needed for gonadotroph determination but may have a role in gonadotroph differentiation (54). The mutant Prop-1 proteins studied have further decreases in binding to, and activation of, Prop-1 target genes relative to the Ames dwarf mouse mutant Prop-1 (56).

Several nonconsanguineous patients from eight different countries have a documented recurring homozygous autosomal recessive mutation of Prop-1, delA301,G302 (also known as 296delGA). This mutation involves a 2 bp GA or AG deletion among three tandem GA repeats (296-GAGAGAG-302) of exon II. The change in reading frame causes a divergence in amino acid sequence at codon 102, and the serine at codon 109 is changed to a stop codon (S109X). This results in a truncated gene product with only the N terminus and first helix of the homeodomain (57, 58, 59, 60, 61, 62, 63). This mutant lacks promoter binding and transcriptional activation (56). Interestingly, one family was noted to have progressive ACTH deficiency with age (63). Likewise, several patients in a large consanguineous Indian pedigree bearing a 112–124del mutation resulting in a premature stop codon at position 480 (the proline at codon 160 is altered to a stop codon, P160X) had an impaired pituitary-adrenal axis (64). These clinical findings suggest that signals from the other pituitary cell lineages may be important in maintaining corticotroph function (63). Additionally, pituitary enlargement with subsequent involution has been described, but the mechanism is unclear (60, 61).

delA301,G302 has been found in compound heterozygosity with 149delGA. This 2-bp frame shift deletion at codon 50 causes divergence from wild-type Prop-1 at amino acid 52 and results in the same S109X mutation as delA301,G302 (58, 62). A 1-bp deletion has also been found in codon 50, which predicts divergence of amino acid sequence at codon 53 and changes the proline at codon 164 to a stop codon (P164X) (62). Another truncated product is caused by a C-to-T transition at nucleotide (nt) 295 that converts an arginine at codon 99 to a stop codon (R99X) (62).

In one patient, the delA301,G302 mutation has also been found in compound heterozygosity with a phenylalanine converted to an isoleucine at amino acid 117 (F117I). This phenylalanine is part of the core DNA binding motif and is almost invariant within the homeodomain family (56). Several other families have a highly conserved arginine mutated to a cysteine at amino acid 120 (R120C), located in the third helix of the homeodomain (54, 56). Both the F117I and R120C substitutions bind DNA with greatly reduced affinity and activate target gene reporter constructs with significantly reduced efficiency (56). A homozygous replacement of a highly conserved phenylalanine in the hydrophobic core of the first helix of the homeodomain of Prop-1 by a serine at codon 88 (F88S) has been described in one patient. The mutant Prop-1 shows minimal DNA binding and significantly impairs transcriptional activation of a target gene (65). Another missense mutation, an arginine altered to a cysteine at codon 73 (R73C), involves a residue conserved in 95% of the more than 400 homeodomain proteins so far identified (66). An intronic point mutation (an A-to-T substitution) involving the last nucleotide of the splice acceptor site preceding exon III abolishes normal splicing. It generates a major transcript (1690 bp) retaining intron II, and a minor transcript (622 bp) that results from utilization of a cryptic splice site within exon III, causing loss of the first 12 nucleotides of this exon (66).

E. Pit-1
Pit-1 (official nomenclature now POU1F1), also known as GHF-1 (GH factor-1), is a member of a family of transcription factors, POU, responsible for mammalian development. POU is an acronym for Pit-1, Oct-1, which is widely expressed; Oct-2, which is expressed in B lymphocytes and in certain areas of the brain; and Unc-86, which functions in C. elegans neuronal cell development (67). Pit-1 expression is restricted to the anterior pituitary lobe (68) and was identified by its specific binding to AT-rich cell-specific elements in the rat PRL and GH genes (69).

Pit-1 contains two protein domains, termed POU-specific (POU-S) and POU-homeo (POU-H), which are both necessary for high-affinity DNA binding on the GH and PRL genes (70, 71). X-ray crystallographic evidence suggests there are four {alpha}-helices present in the POU-S and three {alpha}-helices present in the POU-H (72). Pit-1 usually binds to multiple sites on target genes, and dimerization of Pit-1 on DNA appears to be important for high-affinity DNA binding (73, 74, 75). The third {alpha}-helices of the POU-S and POU-H make the majority of contacts with the major grooves of DNA, and Pit-1 forms dimers on DNA by interactions between the POU-S domain of one molecule and the C terminus of the POU-H of the other (72). When bound to DNA, Pit-1 activates GH and PRL gene expression, in part, through an N-terminal transactivation domain rich in hydroxylated amino acid residues (serine- and threonine-rich) (69, 73, 74).

The level of Pit-1 in pituitary cell lines is sufficient to activate the minimal elements in the GH promoter necessary for cell-specific expression of this gene (71), but Pit-1 alone is not sufficient for regulated GH gene expression. For example, Lipkin et al. (76) cloned a zinc finger transcription factor, termed Zn-15, which is responsible with Pit-1 for synergistic activation of the GH gene. Pit-1 also appears necessary, along with the thyroid hormone receptor, for T3 regulation of rat GH promoter activation (77, 78). However, Suen and Chin (79) concluded that T3-stimulated rat GH promoter activity is independent of Pit-1 using an in vitro transcription system with a rat pituitary cell line (GH3) nuclear extract, so synergism between Pit-1 and the thyroid hormone receptor remains unclear.

In addition to its role in cell-specific gene expression and regulation, Pit-1 has been shown to be essential for the development of certain anterior pituitary cells. Pit-1 is first detected on e13.5 in the mouse in cells occupying a central position in the primordial pars distalis, and then 1 d later in the entire pars distalis (80). Pit-1 transcripts initially appear in cells of the caudomedial region of the anterior pituitary gland on e14.5 and are exclusively in these cell types by e15 (9). Pit-1 protein is detected in the somatotrophs and lactotrophs, preceding GH and PRL gene expression on e16 and 17, respectively, suggesting that Pit-1 is the major cell-specific activator of hormone expression from these cell types (9).

Pit-1 protein is also expressed in the thyrotrophs (9). Thyrotrophs appear to arise from two independent cell populations in mice. The first population is Pit-1 independent and transient; it appears on e12 in the rostral tip of the developing anterior pituitary gland before the first detectable expression of Pit-1 on e14.5 but phenotypically disappears by the day of birth. The second population is Pit-1 dependent and arises subsequently in the caudomedial portion of the developing pituitary gland on e15.5 after the initial expression of Pit-1 in this area. Pit-1 appears necessary for the appearance of these precursors of the mature thyrotroph cell type based on the following observations: 1) caudomedial thyrotroph cells are not present in the Pit-1-defective Snell dwarf mouse, and 2) Pit-1 can bind to and transactivate the TSH-ß promoter (81). A PAR-bZIP protein, thyrotroph embryonic factor, may be involved in the initial expression of the mouse TSH-ß promoter, as it is first selectively expressed in the rostral tip cells of the anterior pituitary concomitantly with the activation of TSH-ß gene expression. Thyrotroph embryonic factor binds to and can effectively transactivate the TSH-ß promoter (82).

In analysis of the Snell dwarf mouse, Pit-1 transcripts appear at the normal time in the expected region of the pituitary gland, but by e18.5 there is a significantly decreased level of Pit-1 compared with wild-type mice. Nevertheless, Pit-1 expression is detectable until postnatal d 0–5 (83). These findings suggest that once Pit-1 protein has reached a critical threshold, autoregulation is subsequently required to sustain Pit-1 gene expression. Additional transcriptional regulation likely maintains Pit-1 transcription as well. For example, retinoic acid induction of the Pit-1 gene requires both the retinoic acid receptor and Pit-1 (83).

Naturally occurring mutations in the Pit-1 gene have confirmed that Pit-1 is essential for the development of certain anterior pituitary cells. The Jackson dwarf mouse has a gross structural alteration of the Pit-1 gene with either an inversion or insertion of a greater-than-4-kb segment of DNA. These animals have hypoplastic anterior pituitaries; CPHD of GH, PRL, and TSH; and no Pit-1 gene expression (10). Snell dwarf mice also have hypoplastic anterior pituitaries and CPHD, but they have a low level of Pit-1 expression. These mice have a tryptophan altered to a cysteine in codon 261 (W261C) in the putative recognition helix of the POU-H. This mutant Pit-1 does not bind a high-affinity Pit-1 site in the PRL promoter, Prl-1P (10).

A number of humans with CPHD and Pit-1 gene mutations have also been described (Fig. 4Go). The inheritance pattern and phenotypic presentation is quite different among these patients, reflecting the location of the mutation in Pit-1. A C-to-T sporadic mutation, changing an arginine to a tryptophan in codon 271 (R271W) in one allele of the Pit-1 gene, is the most common mutation and has been described in several unrelated patients of different ethnic backgrounds (84, 85, 86, 87, 88, 89, 90, 91, 92). The tryptophan substitution reduces the positive charge in a basic amino acid region of Pit-1. Mutant R271W Pit-1 binds normally to DNA, but the mutant protein acts as a dominant inhibitor of transcription (84) and may impair dimerization (72). Thus, a mutation need only be present in one allele to cause CPHD.



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  		Figure 4. Schematic representation of the Pit-1 protein. The four {alpha}-helices of the POU-specific domain and the three {alpha}-helices of the POU-homeodomain are depicted as boxes, and mutations are noted at their locations. OH indicates the serine- and threonine-rich transactivation domain. The E250X mutation results from both a missense mutation at codon 250 and from a 1-bp deletion at nt 747, which also causes a missense mutation at codon 249. The Jackson dwarf mouse has a gross structural alteration of the Pit-1 gene, which is not depicted.

 
A patient with GH deficiency but dysregulation of PRL and TSH has an amino acid change from a lysine to a glutamic acid at codon 216 (K216E). The mutant Pit-1 binds to DNA and does not inhibit basal activation of the GH and PRL genes. However, the mutant Pit-1 is unable to support retinoic acid induction of the Pit-1 gene distal enhancer either alone or in combination with wild-type Pit-1. Thus, the ability to selectively impair interaction with the superfamily of nuclear hormone receptors is another mechanism responsible for CPHD (93).

Two Dutch kindreds have an alanine-to-proline conversion at codon 158 (A158P). One family has homozygous autosomal recessive inheritance, whereas members of the other family are compound heterozygotes with one absent Pit-1 allele. The A158P mutant Pit-1 has a minimal decrease in DNA binding. However, it has no to low transcriptional activation of the GH, PRL, and Pit-1 promoters (94). Seven Middle Eastern patients with hypopituitarism from three unrelated families have a homozygous alteration of a proline to a serine at codon 239 (P239S) at the beginning of the second {alpha}-helix of the POU-H. The P239S Pit-1 mutant binds to DNA but has decreased GH promoter activation (95). A phenylalanine-to-cysteine conversion at codon 135 (F135C) was found in four siblings with complete GH deficiency, born to consanguineous parents, who only later developed central hypothyroidism and were found to have undetectable PRL levels. This mutation is found within the hydrophobic core of the POU-S, near the dimer interface of the POU-S (96). Molecular modeling studies suggest that the F135 residue acts as an actual key in the assembling and stability of the first, third, and fourth {alpha}-helices of the POU-S, and the mutant POU-S is perturbed to such an extent that any interaction with other transcription cofactors might be prevented (97).

Other Pit-1 mutations affect DNA binding. One patient has compound heterozygosity of mutations lying within exon IV in a region that encodes the third {alpha}-helix of the POU-S. One mutation is a glutamate-to-glycine conversion at codon 174 (E174G) in the POU-H. The E174G Pit-1 mutant has 1% specific binding activity. The other mutation is a nonsense mutation of an arginine at codon 172 (R172X) resulting in a truncated protein (98). The R172X Pit-1 mutant should lose transcriptional activation activity, as well as DNA binding, as the third {alpha}-helix of the POU-S and the entire POU-H are lost. The R172X mutation is also homozygous in an unrelated patient (99). Another patient with severe deficiencies of GH, PRL, and TSH also has compound heterozygosity of Pit-1 gene mutations. Amino acid 193 is changed from a tryptophan to an arginine (W193R). The W193R mutant Pit-1 binds to DNA with approximately 500-fold reduced affinity and cannot activate transcription of target genes. A 1-bp deletion frame-shift mutation (747delA) leads to a nonfunctional truncated protein with a glutamic acid-to-asparagine conversion at codon 249 and a nonsense mutation at codon 250 (100). This latter mutation is similar to that described by Irie et al. (101): a nonsense mutation in codon 250 that alters a glutamate to a stop codon (E250X) with the complete loss of helix 3 of the POU-H and presumably loss of DNA binding.

Other Pit-1 gene mutations have been identified but not characterized. There is an autosomal recessive mutation with conversion of an arginine to a glutamine in codon 143 (R143Q) (86). This mutation might disrupt DNA binding by interfering with stabilization of Pit-1 on a negatively charged DNA backbone, as it is found in the first {alpha}-helix of the POU-S. Another patient has a proline converted to a leucine in codon 24 (P24L) in only one allele, which has been postulated to disrupt transactivation (86).

Thus, some Pit-1 mutations alter residues important for DNA-binding and/or alter the predicted {alpha}-helical nature of the Pit-1 protein [F135C, R143Q, K145X (our unpublished data), A158P, R172X, E174G, W193R, E250X, W261C]. Others have been shown to or postulated to impair transactivation of target genes (P24L, A158P, K216E, P239S, R271W).


   IV. Summary
Top
 Abstract
 I. Introduction
 II. Pituitary Embryology
 III. Pituitary Transcription...
 IV. Summary
References
 
Extrinsic and intrinsic signaling gradients determine expression patterns of pituitary-specific factors in the developing anterior pituitary gland (102). Initially, pituitary gland commitment from oral ectoderm occurs in response to inductive signals from the neuroepithelium of the ventral diencephalon (e6–8.5). Invagination of the oral ectoderm, still in contact with the developing diencephalon (e8.5–10.5), leads to the formation of Rathke’s pouch (e10.5–12.5), the primordium of the anterior pituitary gland. Ultimately, there is the sequential appearance of the terminally differentiated cell types (e12.5–birth), with the gonadotrophs, thyrotrophs, somatotrophs, lactotrophs, and corticotrophs located ventrally to dorsally, respectively.

Mutations in many of the pituitary-specific transcription factors have been identified in mice (Table 1Go) and in patients (Table 2Go) with hypopituitarism. Correlation of the genetics with the phenotypes of these individuals, as well as studies of mice with targeted disruption of these genes (Table 1Go), has yielded great insights into pituitary development. It is likely that many of the cases of CPHD that were previously defined as "idiopathic" actually have a molecular basis.


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  		Table 1. Pituitary transcription factor knockout and mutant animals

 

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  		Table 2. Human mutations of pituitary transcription factor genes

 


   Acknowledgments
 
This work was supported by NIH Grants DK-02329 (to L.E.C.) and DK-53977 (to S.R.), March of Dimes (to S.R.), and the Genentech, Inc. Foundation for Growth and Development (to L.E.C., S.R.).


   Footnotes
 
Abbreviations: BMP, Bone morphogenic protein; CPHD, combined pituitary hormone deficiency; e, embryonic day; FGF, fibroblast growth factor; GSU, glycoprotein subunit; Hesx, homeobox gene expression in embryonic stem cells; Isl-1, Islet-1; Lhx, LIM-homeobox; nt, nucleotide; Pitx, pituitary homeobox; POMC, proopiomelanocortin; POU-H, POU-homeo; POU-S, POU-specific; Prop-1, Prophet of Pit-1; Rpx, Rathke’s pouch homeobox; SF-1, steroidogenic factor-1; SOD, septo-optic dysplasia.


   References
Top
 Abstract
 I. Introduction
 II. Pituitary Embryology
 III. Pituitary Transcription...
 IV. Summary
 References
 

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