Review
Molecular mechanisms of pituitary organogenesis: In search of novel regulatory genes

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Abstract

Defects in pituitary gland organogenesis are sometimes associated with congenital anomalies that affect head development. Lesions in transcription factors and signaling pathways explain some of these developmental syndromes. Basic research studies, including the characterization of genetically engineered mice, provide a mechanistic framework for understanding how mutations create the clinical characteristics observed in patients. Defects in BMP, WNT, Notch, and FGF signaling pathways affect induction and growth of the pituitary primordium and other organ systems partly by altering the balance between signaling pathways. The PITX and LHX transcription factor families influence pituitary and head development and are clinically relevant. A few later-acting transcription factors have pituitary-specific effects, including PROP1, POU1F1 (PIT1), and TPIT (TBX19), while others, such as NeuroD1 and NR5A1 (SF1), are syndromic, influencing development of other endocrine organs. We conducted a survey of genes transcribed in developing mouse pituitary to find candidates for cases of pituitary hormone deficiency of unknown etiology. We identified numerous transcription factors that are members of gene families with roles in syndromic or non-syndromic pituitary hormone deficiency. This collection is a rich source for future basic and clinical studies.

Introduction

Height of 2 or more standard deviations (SD) below the mean for age and sex is defined as short stature. Metabolic or endocrine disorders usually cause proportionate short stature, while skeletal defects often cause disproportionate short stature (Weedon and Frayling, 2008, Rimoin et al., 2007, Cha et al., 2004a, Jorge et al., 2007). The sitting height to standing height ratio can be used to distinguish proportionate and disproportionate short stature in cases where the distinction is not immediately obvious. Skeletal and hypothalamic–pituitary axis-based growth insufficiencies occur with similar frequencies. Genetic causes of growth hormone deficiency (GHD) are thought to occur in approximately 1/4000 to 1/10,000 births (Procter et al., 1998, Patel et al., 2006). These can be syndromic, including pituitary and head defects as well as defects in the development of other organs, or non-syndromic, with pituitary gland-specific effects (Table 1). Because the pituitary gland is critical for the development and function of many other organs, all defects in pituitary organogenesis cause secondary effects on target organs. Syndromic pituitary deficiencies include effects on non-pituitary tissues that are not within the expectations for secondary effects on target organs. Genetic defects in the GH gene itself and mutations in the growth hormone-releasing hormone receptor (GHRHR) cause isolated GHD (IGHD), but most cases of IGHD are idiopathic (reviewed in: Hernandez et al., 2007). Patients with mutations in the growth hormone receptor gene (Laron dwarfism, a growth hormone insensitivity syndrome) have a clinical presentation similar to patients with IGHD, but they have elevated levels of GH and are treated with insulin like growth factor because they are unable to respond to GH therapy (reviewed in: Savage et al., 2006).

The availability of recombinant growth hormone has generally led to effective correction of growth insufficiency in children with multiple or IGHD due to pituitary developmental defects, but it is not always efficacious for some idiopathic short stature patients or for other problems associated with syndromic pituitary hormone deficiency (i.e. septo-optic dysplasia and other severe craniofacial abnormalities) (Bryant et al., 2007, Hintz, 2005, Zucchini, 2008, Zucchini et al., 2008, Cohen et al., 2008). Thus, treatment of children with pituitary hormone deficiency can be challenging, as well as expensive.

Studies in genetically engineered and mutant mice have advanced the understanding of the mechanisms underlying pituitary organogenesis defects that lead to short stature (Zhu et al., 2007, Kelberman and Dattani, 2007). In many cases, genes discovered in the mouse led to the discovery of lesions in human patients and have revealed the mechanism of action and genetic hierarchy of control of pituitary cell specification and growth (Kelberman and Dattani, 2009). For example, the discovery of the etiology of the Snell and Ames dwarf mutations (Pou1f1 and Prop1, respectively), and characterization of the phenotypes of genetically engineered mice with mutations in Tpit (officially Tbx19), Hesx1, Lhx3 and Lhx4, paved the way for identification of the mutated human genes. Some genes necessary for normal growth in mice, i.e. Aes, have not yet been reported to have lesions in human patients, but there can be a considerable lag between discovery in mice and identification of rare human patients (Brinkmeier et al., 2003, Wang et al., 2004).

The ability of mouse mutants to predict the correct human patient characteristics for screening is remarkable, as evidenced by Pou1f1, Prop1, Tpit, Hesx1, Lhx3 and Lhx4, although the correspondence is imperfect. For example, LHX4 mutations cause similar hormone deficiencies in humans and mice, and while the mouse mutations are recessive and cause perinatal lethality, the human mutations are haploinsufficient and viable (Sheng et al., 1997, Castinetti et al., 2008, Pfaeffle et al., 2008, Rajab et al., 2008, Kristrom et al., 2009). PROP1 mutations are another example of an imperfect correspondence between the mouse and human features. In mice, lesions in Prop1 cause pituitary hypoplasia and congenital pituitary hormone deficiency, including GH, TSH, PRL, and gonadotropin deficiencies (Bartke et al., 1977, Tang et al., 1993). Both male and female mutant mice go through puberty and become fertile with GH, thyroid hormone and PRL supplements, suggesting that the gonadotropin deficiency is secondary to the lack of POU1F1 (Buckwalter et al., 1991, Soares et al., 1984). In contrast, humans have variable pituitary size and progressive hormone deficiency, usually with failure to undergo puberty and the additional involvement of evolving ACTH deficiency, which can be fatal if untreated (Pernasetti et al., 2000, Reynaud et al., 2006, Bottner et al., 2004, Riepe et al., 2001). The missense mutation (S83P) in the spontaneous Ames dwarf mutant, Prop1df/df, minimally transactivates an artificial paired homeodomain binding site in cell transfection assays, while the most common human PROP1 mutation creates a frame shift and likely complete loss of function (Sornson et al., 1996, Deladoey et al., 1999). This does not account for the differences in pituitary dysfunction between the species, as mice homozygous for genetically engineered Prop1 loss of function alleles have features similar to the missense mutation, and the S83P mutant appears to have no activity in culture on the Pou1f1 early enhancer, which is considered a bona fide target (Sornson et al., 1996, Nasonkin et al., 2004, Olson et al., 2006).

Dissimilarities in the features that characterize mouse mutants and human patients may be attributable to differences in the effect of the mutations (i.e. partial vs. complete loss of function), species differences in temporal or spatial expression, overlapping gene function amongst gene family members, and/or “genome variation.” Genome variation means different phenotypic manifestations of the same genetic defect due to the influence of other genes in the genome that have functional variant alleles segregating in the population. Analysis of mutant mice on different strain backgrounds can be exploited to uncover the influence of these modifier genes that magnify or minimize the manifestations of reduced function in other genes (Nadeau, 2003). For example, genetic background has a profound effect on the survival of Prop1 mutant mice, ranging from neonatal lethal, juvenile lethal to completely viable (Nasonkin et al., 2004). The utility of genetically engineered mice and well defined inbred strains may make it possible to tease out the genetic risk factors that could cause some human mutations to have mild effects in some individuals and more severe ones in other patients with the same mutation (Nadeau, 2003, Badano and Katsanis, 2002).

Classic embryology experiments involving tissue transplantation and recombination reveal that diffusible molecules produced by the neural tissue located dorsal to Rathke's pouch, the primordia for the intermediate and anterior lobes of the pituitary gland, are essential for pouch induction and growth (Couly and Le Douarin, 1985, ElAmraoui and Dubois, 1993, Hermesz et al., 2003, Ericson et al., 1998, Gleiberman et al., 1999, Takuma et al., 1998). Subsequently, members of the WNT, BMP, FGF, Notch, and hedgehog pathways were discovered to have profound effects on pituitary development (Olson et al., 2006, Ericson et al., 1998, Takuma et al., 1998, Potok et al., 2008, Cha et al., 2004b, Davis and Camper, 2007, Brinkmeier et al., 2007, Raetzman et al., 2004, Raetzman et al., 2007, Raetzman et al., 2006, Treier et al., 2001, Treier et al., 1998, Kita et al., 2007, Zhu et al., 2006, Ezzat et al., 2002, Ohuchi et al., 2000). Some essential signaling molecules are expressed in the infundibulum, but there are some in the mesenchyme surrounding the pituitary, i.e. Tgfbi, and some in the pouch itself (Brinkmeier et al., 2009). This suggests that the regulation of pituitary development by signaling molecules is complex.

Expression of NOGGIN, an antagonist of BMP signaling, TCF7L2, an effector of canonical WNT signaling, and WNT5A, typically acting in the non-canonical pathway, are critical for maintaining the balance of signaling pathways necessary for normal pituitary growth and morphology (Brinkmeier et al., 2003, Potok et al., 2008, Cha et al., 2004b, Davis and Camper, 2007, Brinkmeier et al., 2007). For example, excessive BMP signaling in noggin mutant mice is associated with reduced Fgf10 expression, alteration in the SHH signaling domain, and multiple invaginations of Rathke's pouch (Davis and Camper, 2007). Tcf7l2 deficient mice exhibit expansion of the Fgf10 and Bmp signaling domains and an abnormally large Rathke's pouch and subsequently oversized anterior lobe (Brinkmeier et al., 2007). Finally, Wnt5a deficient mice also have expanded Fgf and Bmp signaling domains, and the pouch is dysmorphic but not markedly oversized (Potok et al., 2008). In each of these cases, disruption of one signaling pathway has pleiotropic effects on other signaling pathways. This paradigm is emerging as a common theme for signaling pathway function in pituitary development.

Popular models suggest that signaling molecules influence the spatial patterns of pituitary transcription factor expression, leading to the emergence of specialized cell types that produce pituitary hormones, yet there is also compelling evidence that alterations in signaling pathways affect the morphology and size of the organ more than cell specification (Brinkmeier et al., 2003, Brinkmeier et al., 2007, Ericson et al., 1998, Potok et al., 2008, Cha et al., 2004b, Davis and Camper, 2007, Treier et al., 1998). The noggin, Wnt5a, and Tcf7l2 mutants are each able to generate the 5 major hormone-producing cells of the anterior lobe despite variations in size and shape of the organ. Rizzoti and Lovell-Badge (2005) recently reviewed the effects of various genetic lesions on pituitary growth and shape.

The developing pituitary transcriptome contains many members of the BMP, FGF, WNT, Notch and SHH signaling pathways (Brinkmeier et al., 2009). Using Genomatix software we identified 61 additional genes in these pathways that are expressed at a time when they could influence pituitary development. 17 genes may be involved in cross talk between the pathways. Gene ontology terms revealed an additional 72 genes that could contribute to cell signaling in the developing pituitary gland. RT-PCR surveys of WNT genes expressed in and around the developing organ have identified many different candidates for regulation of β-catenin activity, but little is known about the functional significance of many of these genes (Olson et al., 2006, Potok et al., 2008). Several pituitary transcription factors are regulated by β-catenin, including the Egr1, Nr5a1 complex, Pitx2, and the Hesx1, Prop1 complex (Olson et al., 2006, Salisbury et al., 2009, Salisbury et al., 2007, Garcia-Lavandeira et al., 2009, Kioussi et al., 2002). Because β-catenin is regulated by G-protein coupled receptors, some of the pituitary transcription factors that respond to β-catenin could be independent of WNT molecules themselves, which is an area for future study (Gardner et al., 2007). Many of the signaling pathways involved in pituitary development play important roles in ontogeny of other organs, leading to lethality in mice homozygous for complete loss of function alleles. Thus, it seems unlikely, but not impossible, that genes in these pathways will be responsible for hypopituitarism in humans.

Many transcription factors play important roles in pituitary development and hormone production (Table 1). The early-acting genes are not pituitary specific, and lesions in these genes cause defects in development of craniofacial or other structures. Some of these are homeobox genes with overlapping functions and multiple roles during ontogeny, i.e. Pitx1 and Pitx2, Lhx3 and Lhx4 (Sheng et al., 1997, Sheng et al., 1996, Charles et al., 2005, Suh et al., 2002, Gage et al., 1999a, Gage et al., 1999b, Ellsworth et al., 2008). Defects in some of these genes cause apoptosis, reduced cell proliferation, or both, which ultimately results in pituitary hypoplasia. The functions of genes like Nr5a1, Pitx2, and Gata2, a downstream target of Pitx2, with broad expression patterns and roles in the pituitary as well as other critical organs, can be dissected by tissue-specific disruption in mice (Zhao et al., 2001a, Zhao et al., 2001b). Such studies reveal roles for Gata2 in thyrotropin and gonadotropin production, and implicate Gata3 as a gene with potential for compensatory activity (Charles et al., 2006, Charles et al., 2008).

Prop1 and Pou1f1 are examples of homeodomain transcription factors critical for pituitary development, specifically. Mutations in the human ortholog of Prop1 are the most common known cause of multiple pituitary hormone deficiency in humans (Kelberman and Dattani, 2009, Deladoey et al., 1999, Mody et al., 2002, Cogan et al., 1998). There are dramatic differences in the effects of Prop1 and Pou1f1 mutations on fetal and neonatal pituitary development in mice. Prop1 mutants have poor pituitary vascularization and dysmorphology that appears to result, in part, from the failure of progenitors to migrate away from the proliferative zone and undergo differentiation (Ward et al., 2006, Ward et al., 2005). The defect may result from failure to undergo epithelial to mesenchymal transition, as Prop1 is required for normal N-cadherin (Cdh2) expression, and changes in cadherin gene expression are typically associated with epithelial to mesenchymal transition (Himes and Raetzman, 2009, Kikuchi et al., 2006, Kikuchi et al., 2007). In contrast, there are no obvious effects on pituitary vascularization or morphology in Pou1f1 mouse mutants.

Pou1f1 is generally accepted as a direct downstream target of Prop1. This is based on the ability of PROP1 to transactivate a DNA fragment of Pou1f1 that contains the early enhancer in cell culture and the occupancy of PROP1 at that site by chromatin immunoprecipitation in extracts of microdissected embryonic pituitary glands at e12.5 and e13.5 (Sornson et al., 1996, Olson et al., 2006). Careful review of the evidence suggests that the story may be more complicated. First, there is a profound temporal delay (approximately 4 days) between activation of Prop1 and Pou1f1 expression in mice, which is unusual for a direct downstream target (Sornson et al., 1996). Second, human newborns with loss of function alleles in PROP1 have low but biologically significant levels of TSH, GH and PRL initially, suggesting that PROP1 is not required for initial expression of POU1F1 in humans (Bottner et al., 2004). Similarly, mice with Prop1 mutations express limited amounts of Pou1f1 and its targets Tshb, Gh, and Prl (Gage et al., 1995, Gage et al., 1996). More work needs to be done to reconcile these apparently conflicting observations and clarify the role of PROP1 in humans and mice.

We hypothesize that the role of PROP1 is to generate precursor cells that are capable of becoming hormone-producing cells of the anterior lobe and promote the transition from proliferation to differentiation. It may also play a role in regulating the accessibility of the POU1F1 regulatory elements. POU1F1 is activated in some of the precursor cells to promote differentiation into somatotrophs, thyrotrophs and lactotrophs and to expand the proliferation of that lineage after birth. If this hypothesis is true, the progressive hormone deficiency in humans with PROP1 mutations could arise by depletion of the progenitor pool, and the more severe, congenital hormone deficiency in Prop1 mutant mice could be due to a stronger and/or earlier requirement of Prop1 for establishing the precursor pool in mice than humans. Investigation of genes expressed in the developing pituitary gland between peak Prop1 and Pou1f1 expression may uncover direct targets of Prop1 that are intermediates between Prop1 and Pou1f1. Neurod4 (also known as Math3) is a candidate for an intermediate, as it is activated at e13.5 before Pou1f1 is generally detected, although maintenance of Neurod4 expression requires Pou1f1 (Zhu et al., 2006). Novel genes expressed at these early developmental times will be candidates to explain pituitary deficiency diseases of unknown etiology.

Several of the known pituitary transcription factors were discovered using the approach of defining the key cis-acting sequences in hormone genes and the trans-acting factors that bind to them (Ingraham et al., 1988, Bodner et al., 1988, Lamonerie et al., 1996, Lamolet et al., 2001, Gordon et al., 2002, Gordon et al., 1997). Additional advances could be made by pursuing this strategy more extensively and/or by identifying the regulatory sequences for some of the early-acting pituitary-specific transcription factors and their binding factors, as well as the downstream targets of key transcription factors. For example, we used comparative genomics and bioinformatics to identify regulatory sequences in Prop1, and confirmed their relevance in cell culture and transgenic mice (Ward et al., 2007). A highly conserved intragenic enhancer that affects spatial expression of a Prop1 transgene is a target of Notch signaling (Zhu et al., 2006, Ward et al., 2007). This suggests that screening for PROP1 mutations in human patients should include a scan of the intronic enhancer that controls spatial expression of the gene in mice (Carvalho et al., 2007).

Another approach is to identify gene expression differences in the pituitary glands of normal and mutant mice to identify potential downstream targets of Prop1 and Pou1f1 (Brinkmeier et al., 2009, Douglas et al., 2001, Douglas and Camper, 2000, Carninci et al., 2003, Davis et al., 2009). This gene discovery approach has revealed new members of transcription factor families that are exciting candidates for regulating pituitary development and the basis of human hormone deficiency disease. Here we report the discovery of transcription factors expressed in the developing mouse pituitary gland that are members of several important gene families including basic helix-loop-helix, high mobility group, and T-box. These genes are intriguing candidates for future functional studies and evaluation in human patients. In addition, we present a summary of the clinical features associated with hypopituitarism caused by known transcription factors, with the purpose of streamlining molecular diagnostic studies.

Section snippets

Prioritizing genes for molecular studies in human patients

There are approximately a dozen different transcription factor genes that are mutated in cases of short stature and/or pituitary gland dysfunction (Table 1). These are classified based on the type of pituitary defect that they produce as well as any other clinical features. Several of these genes are expressed in the developing hypothalamus and are likely to affect anterior pituitary gland development by disrupting the normal balance of signaling pathways and inductive factors produced by the

Future directions

The developing pituitary cDNA libraries we made and analyzed reveal that the transcriptome has great depth at the time the cells are differentiating and the organ is undergoing substantial growth. There are already several compelling precedents for functional overlap of transcription factors within a particular gene family, i.e. PITX and LHX families. Thus, the discovery of many members of the Forkhead, HMG, bHLH and T-box families, suggests that there may be genes with essential functions that

Note added in proof

Recent analysis of Lhx2 by in situ hybridization shows a broader range of expression in the ventral diencephalon than we report by immunohistochemistry. This report also reveals a direct role for LHX2 in the development of the posterior lobe of the pituitary gland. It is required in the infundibulum to restrict growth factor expression and suppress proliferation, and it is necessary for vasopressin expression in the posterior lobe. LHX2 deficiency causes dysmorphology of the intermediate and

Acknowledgments

Funding: NIH R37HD030428, R01HD034283 (SAC); University of Michigan Center for Computational Medicine and Biology, Clinical Translational Science Award (SAC), Reproductive Sciences Training Grant (NIH T32 HD07048 (SWD & BSE), NIH NRSA F32-HD046300 (BSE), Endocrine Society, International Scholar's Program (LC), Novo-Nordisk, ADERM, and University of Michigan Center for Genetics in Health and Medicine (FC).

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