Origin

Phylogenetic studies in several vertebrate species led to the conclusion that the pituitary gland arises from oral epithelia. Fate-mapping experiments conducted in these animal species trace the origins of the pituitary gland back to the neural plate. In studies of grafting quail chick chimeras, the origin of the pituitary was localized to the midline of the anterior neural ridge. By means of surgical ablation performed in chick embryos, the rostral ridge of the neural plate was identified as the source of cells that give rise to pituitary tissue.4–6 In amphibians, tracing experiments have confirmed the neural origin of pituitary gland^7,8 and similar conclusions have been reached about zebrafish.^9,10 Additionally, by focalized application of a carbocyanin dye, DiI, into the rostral end of the neural plate at the open neurula stage (9.5 days postcoitus) in rats, labeled cells could be identified in Rathke’s pouch, and they could develop into the secretory cells of the adenohypophysis in 7 additional days.¹¹ Thus evidence indicates that the anterior neural ridge is the origin of Rathke’s pouch, which eventually gives rise to cells of the pituitary gland. Subsequent to the folding of the embryonic head, the anterior neural ridge is displaced ventrally to form the portion of the oral epithelium that later gives rise to the roof of the mouth and additional structures, including the pituitary gland. Consistent findings in many species make it apparent that the process of pituitary development is, for the most part, evolutionarily conserved from lower vertebrates to higher mammals.

Ontogeny

In humans, the anterior lobe of the pituitary gland originates from an invagination of the stomodeal epithelium termed Rathke’s pouch.¹² The stomodeal epithelium that contains the pituitary primordium is formed by the third fetal week, and the invagination of stomodeal epithelium occurs dorsally to form Rathke’s pouch by the fourth week. The formation of Rathke’s pouch is complete and disconnected from the oral epithelium by the end of the sixth week of fetal life.¹³ In parallel, the hypothalamus is the first region of the forebrain to differentiate. From 4 weeks, the hypothalamic sulcus, chiasmatic plate, and mammillary bodies are recognizable. These two organs, hypothalamus and pituitary, develop interdependently.¹⁴

Similar to the ontogeny observed in humans, Rathke’s pouch in mice is derived from an anlage that arises as an upgrowth from the lining of the oral cavity’s roof. At its earliest stage, the murine pituitary primordium is defined as an intimate point of contact between the neural ectoderm and the oral roof ectoderm on embryonic day 8.5 postcoitus (e8.5), which marks the first event in the pituitary’s development. Organogenesis of the adenohypophysis begins as the cells of the pituitary placode in the oral ectoderm thicken and invaginate to form the nascent pituitary. In the e9.5 mouse embryo, this anlage can be seen located rostrally to the oropharyngeal membrane. Dorsal movement of the epithelial layer from the roof of the mouth induces a cone-shaped intrusion dorsally as Rathke’s pouch, or the adenohypophyseal pouch. Before the formation, a developmentally important molecular marker, Sonic hedgehog (Shh), is expressed uniformly in the oral epithelial layer. The expression of Shh is excluded before the intrusion of pituitary anlagen can occur in the e9 mouse embryo.¹⁵ Rathke’s pouch thickens as development proceeds and elongates dorsally relative to the oral cavity by the stomodia-adenohypophyseal channel. By e10.5 in the mouse, Rathke’s pouch has formed as a rudimentary structure and separated from the ventral pharyngeal epithelium.

At the time Rathke’s pouch is pinched off at e11 in mice, the first round of accelerated mitotic activity is initiated in the anlagen.16,17 In the ensuing patterning period, mitotic activity is observed most prominently in the rostral part of Rathke’s pouch, with several buds emerging and enveloping areas of vascularized mesenchyme. Progenitors of the hormone-secreting cell types arise from the ventral proliferation of cells, and this region of rostral Rathke’s pouch eventually gives rise to the anterior lobe, or the pars distalis. The dorsal aspects of Rathke’s pouch, in contact with the descending infundibulum processes and rostroventrally with the hypophyseal cleft, remain thin and form the intermediate lobe, or the pars intermedia. Anterior pituitary cell types are positionally determined as they initially emerge from proliferation zones,^15,18 with the somatotrope/lactotrope cells arising caudomedially, gonadotrope cells more rostroventrally, corticotrope cells ventrally, and melanotrope cells dorsally. This pattern of pituitary development is generally similar in most mammals.

Cell Lineage Determination

Endocrine pituitary cell types in the adenohypophysis are derived from a single population of cells. The initial expression of pituitary hormone genes marking the terminal differentiation events of individual cell types occurs in a sequential manner. In mice, POMC gene expression emerges as the first pituitary marker at e11.5 and can be detected in the anterior pituitary by e13.5. However, the fate of cells that will give rise to those five different anterior pituitary cell types is determined prior to the initial pituitary POMC expression. In tissue-culture experiments where pituitary anlagen were taken and placed in a culture away from the influence of the diencephalons, pituitary anlagen taken at e11 were capable of generating cells expressing all five anterior pituitary hormone genes, while anlagen taken at e9.5 required additional growth factors, with the exception of corticotrope, which always differentiates regardless of the culture medium.¹⁹ Critical events occur at the time pituitary anlagen become committed to developing into pituitary precursors that will subsequently express pituitary genes that become regulated in a cell-autonomous fashion.²⁰ The timing of this commitment event is coincidental with the formation of Rathke’s pouch.

As an anlage, Rathke’s pouch is the source of all endocrine pituitary cell types. In mice, after the initial appearance of corticotrope, expression of GH gene can be detected by e15.5, followed by thyrotropins, gonadotropins, and PRL. Gene expressions of all anterior pituitary hormones are detectable by e17.5, with the exception of PRL, which can be consistently seen by the time of birth (e19 in the mouse). Another early marker of Rathke’s pouch is αGSU, and the transcripts are detected throughout Rathke’s pouch by e9,²¹ although they are confined to the rostral tip of the anterior lobe by e12.5 and ultimately restricted to thyrotrope and gonadotrope from late gestation through adulthood. Following proliferation and early organ expansion, a series of different cell types arise in a distinct spatial and temporal fashion. Table 1-1 provides a time line of the initial expression of pituitary hormone genes in several species.

Pit1 Gene

The Pit1 gene (POU domain, class 1, transcription factor 1 [POU1F1]) encodes a 33-kD, 291-amino acid transcriptional activator that is capable of DNA binding and transactivation, and it was initially isolated by its ability to bind to the responsive element of the GH gene promoter.22,23 Pit1 is expressed exclusively in the pituitary gland. In mice, the initial expression of the Pit1 gene transcripts can be detected by e13.5, exclusively in the anterior ventral pituitary (Fig. 1-1). The expression of Pit1 persists in adults and co-localizes with expression of GH, PRL, and TSHb genes. Further studies revealed that the product of the Pit1 is capable of binding to responsive elements in the promoters of the GH gene,²⁴ the growth hormone–releasing hormone receptor (GHRHR) gene,²⁵ the PRL gene,²⁶ and the TSHb gene. The Pit1 protein is also capable of binding to the responsive elements of the Pit1 gene itself and is required for the continued transcription of the Pit1 gene.²⁷ The structure of the Pit1 gene is evolutionarily conserved and is found in mouse, human, and all other vertebrate animals examined, although Pit1 may play diverse functional roles in different physiologic pathways in individual species.

Prop1 Gene

Prop1 (Prophet of Pit-1) is a homeodomain-containing transcription factor that is capable of binding to its cognate DNA site and activating its target genes. The expression pattern of the Prop1 gene has been examined in mice and is detected only in Rathke’s pouch. Prop1 expression is detected initially at e10 in the mouse, when the structure of Rathke’s pouch has been established. The expression initially is observed dorsally but subsequently involves most cells in Rathke’s pouch. Expression of Prop1 reaches a maximum level of intensity at e12 in Rathke’s pouch, with the signal diminishing by e14.5⁵² (see Fig. 1-1). It has been shown recently that Notch signaling is required for maintaining high levels of Prop1 expression at e12.5, which is mediated by Rbp-J protein bound to the evolutionary conserved site within the first intron of the Prop1 gene.⁵³ Expression of the Prop1 gene is required for activation of the downstream Pit1 gene.^54,55 The integrity of Prop1 is necessary for full-scale manifestation of pituitary gonadotrope cells, as well as the generation of somatotrope, lactotrope, and thyrotrope cells (see Table 1-2). Mutations in the Prop1 gene have been identified as the leading cause of familial CPHD, resulting in short stature as a consequence.

Hesx1 Gene

Hesx1 (homeodomain gene expressed in ES cells) is a paired-class homeodomain transcription factor that is capable of binding to its cognate DNA site and regulating its target genes. Mutations in the Hesx1 gene have been identified in septo-optic dysplasia and CPHD. In mice, the earliest expression of the Hesx1 gene can be detected at the embryonic stem cell stage. High levels of expression can be detected in the ectoderm, subsequently at the anterior extreme of the rostral neural folds, and finally restricted to the ventral diencephalon and to the thickened layer of oral ectoderm, which will give rise to Rathke’s pouch at e9.0 in the mouse.73,74 Hesx1 gene expression can be observed for 2 more days but only in Rathke’s pouch, with diminishing intensity at a time that coincides with the rise of Prop1 gene expression (Fig. 1-2; also see Fig. 1-1). In humans, strong expression of Hesx1 in Rathke’s pouch can be detected in a 7-week-old embryo. Hesx1 is the earliest molecular marker for the definitive pituitary primordium.

Lhx3 And Lhx4 Genes

Lhx3 (LIM homeo box gene 3) is a LIM-type homeodomain transcription factor. In addition to a C-terminus homeodomain, Lhx3 contains two tandem repeats of LIM zinc-binding motifs, each composed of 50 to 60 amino acids with a conserved pattern of cysteine and histidine residues that form a pair of zinc fingers, separated by a linker of 2 amino acids. Expression analysis revealed that mouse Lhx3 mRNA can be detected in the developing nervous system and accumulates in Rathke’s pouch beginning at e9.5 (see Fig. 1-1). Lhx3 remains expressed in the entire pouch, and its expression is maintained through e15.5; the expression is particularly strong in the anterior and intermediate lobes of the adult pituitary. In addition, Lhx3 is expressed bilaterally along the spinal cord and the hindbrain at early stages of development.⁸⁶

Structurally, Lhx4 (LIM homeodomain gene 4) is closely related to Lhx3. The Lhx gene family consists of at least 12 members; many of them are expressed in the pituitary during development, including Isl1, Isl2, Lhx2, Lhx3, and Lhx4. Lhx3 and Lhx4 have been genetically defined as required elements for both the early stages of pituitary determination and the later differentiation of pituitary cell types. By insitu hybridization, the Lhx4 gene is found to be expressed transiently in ventrolateral regions of the neural tube and the hindbrain of the developing mouse. During pituitary development, Lhx4 is expressed throughout the invaginating Rathke’s pouch at e9.5. At e12.5, Lhx4 expression becomes restricted to the future anterior lobe of the pituitary gland, and by e15.5, Lhx4 expression diminishes. In the adult pituitary, Lhx4 is found in the anterior and intermediate lobes at a much lower level than that of Lhx3.⁸⁷

Tbx19 Gene

Tbx19 is a T-box transcription factor family member (the T-box in the mouse T [Brachyury] gene) that encodes a 448-amino acid protein.⁹⁸ Functional identification of Tbx19 was established after the observation of elements in a critical cis-acting sequence in the POMC promoter. Transcripts of Tbx19 can be found only in the anterior and intermediate pituitary and brain (see Fig. 1-1); Tbx19 is specifically required for continued POMC transcription.^99,100

Pitx1 And Pitx2 Genes

Pitx1 and Pitx2 represent two of the bicoid-related Pitx homeodomain transcription factors. They display distinct but overlapping patterns of expression and are critical in the development of several organs, including the pituitary, with Pitx1 required for the gonadotrope, thyrotrope, and POMC gene expression107,108 and Pitx2 required for the earliest phases of pituitary development for the patterning and proliferation events within Rathke’s pouch.^109–111 Genetic studies have shown that they are required for cell proliferation, survival, and differentiation in a dosage-sensitive manner, with Pitx2 playing a more prominent role than Pitx1. They both function redundantly in controlling Lhx3 expression.^111–113

Other Transcription Factors

Transcription factors act as activators and repressors and often are expressed in a coordinated fashion, mediating organogenesis and cell type specification in the pituitary gland (see Figs 1-1 and 1-2). During the early commitment stage, expression of Pitx1/2 and Hesx1 are found in the anterior neural plate stages and in the invagination of Rathke’s pouch. Lhx3 is expressed on e9.5 in the nascent RP and is required for initial organ commitment and growth⁸⁸ and for cell proliferation, together with Lhx4.⁸⁹ Prop1 appears on e10.5 and is required for determination of four ventral cell types, including the Pit1-dependent lineages (somatotrope, lactotrope, and thyrotrope) and the gonadotrope.^52,59 Sequential expression of this cascade of homeodomain genes represents a model system of transcription control of organogenesis and cell type determination and differentiation in mammals. Phenotypic comparisons in these pituitary loci in both mice and humans suggest that the developmental pathways in determination of the pituitary gland are highly conserved.

Some of these factors express transiently in Rathke’s pouch, and their reduced expression is likely to be required for the progression of specific cell types, as evident in the Prop1^Ames mutant, where the temporal patterns of Hesx1, Prop1, and Brn4 gene expression are extended. As the lineage-determining transcription factor Pit1 appears, certain transcription factors that characterize earlier stages of development are gradually eliminated, including Hesx1, Pfrk,¹⁵ GATA-2,⁵¹ Pax6,¹²⁸ and Brn4.¹²⁹

The list of pituitary-expressed transcription factors implicated in the developing pituitary gland is constantly expanding. Several families of factors, including homeodomain factors (Isl1, Isl2, Oct1, Otx2, Pax6, Pitx3, Six1, Six3, and Six6), zinc-finger (Krox24, Gli1, GATA2, Nzf1, Sp1, Sp3, Zfhep, and Zn16), nuclear receptor (T3R, SF1, ERa, ERb, and Dax), basic HLH domain (AP2, NeuroD, Mash1, Nhlh2, and Math3) and other (Gli2, AP1, Ets1, Foxl2, CBf, Cp1, Rb, Men1, Preb, and Tef) (see reviews [130,131]). Multiple members of the Six family (Six1, Six6, Six2, and Six3) are also expressed in the pituitary. Six6, acting as a strong tissue-specific repressor in association with dachshund (Dach) corepressors, directly represses cyclin-dependent kinase inhibitors, including the p27^Kip1 promoter, and regulates early progenitor cell proliferation in retinogenesis and pituitary development.¹³² Six1 exhibits synergistic genetic interactions with the eyes absent (Eya) family of protein phosphatases and is required to regulate genes that encode growth control and modulate precursor cell proliferation. The phosphatase activity of Eya converts the function of Six1-Dach from repression to activation, causing transcriptional activation through recruitment of coactivators.¹³³ Evidence suggests that Six3 acts upstream of the Wnt pathway, and deletion of Six3 in mice resulted in failure of development of the ventral diencephalon and, consequently, development of the pituitary.¹³⁴ GATA2 is involved in establishing molecular memory of signaling gradients during pituitary development in conjunction with Pit1, and loss of GATA2 is associated with failure to differentiate into gonadotrope.^51,111 The orphan nuclear receptor steroidogenic factor 1, Sf1, is essential for pituitary gonadotrope.¹³⁵ Mice with simultaneous inactivation of both Gli1 and Gli2 have very severe defects in pituitary gland development; about half of these mutants completely lack a Rathke’s pouch at e12.5. In these mutants, the domains of expression of Shh and Nkx2.1 are abnormal, and the loss of Shh signaling boundary in the oral ectoderm could be a cause of this defect.¹³⁶ Gli2 is an upstream regulator of Lhx3 (see Fig. 1-2). One consequence of the Gli2 defect in yot-mutant Zebrafish embryos was the absence of Lhx3 expression in the anterior part of the adenohypophysis anlage.¹³⁷ In the mouse, absence of Lhx3 results in failure of development of Rathke’s pouch into the adenohypophysis.

Otx1 is expressed in the postnatal pituitary gland. Cell culture experiments have shown that Otx1 may activate transcription of GH, FSHβ, βLH, and αGSU. Analysis of Otx1-null mice indicates that at the prepubescent stage, they exhibit transient dwarfism and hypogonadism due to low levels of pituitary GH, FSHβ and LHβ hormones, which in turn dramatically affect downstream molecular and organ targets. Nevertheless, Otx1–/– mice gradually recover from most of these abnormalities, showing normal levels of pituitary hormones with restored growth and gonadal functions at 4 months of age. Since the patterns of expression of hypothalamic hormone-releasing hormones (GHRH, GnRH) and their pituitary receptors (GHRHR, GnRHR) are unchanged, it seems that ability to synthesize GH, FSHβ, and LHβ, rather than the number of cells producing these hormones, is affected.¹³⁸

Because Otx2-null mice exhibit early embryonic lethality,¹³⁹ Otx2 protein has been only recently shown to play a role in pituitary development. Clinical study of one patient has revealed a new heterozygous Otx2 mutation that is affecting the transactivation domain of Otx2 and subsequent lack of activation of Hesx1 and Pit1 promoters in cell culture transfection assay.¹⁴⁰ Heterozygous Otx2 knockout mice have highly variable brain and ocular phenotypes, and although pituitary structure and function have not been studied, this recent study might implicate more attention for the role of Otx2 protein in the pituitary.

Although pituitary cell types in the adult anterior lobe do not appear to be stratified, initial appearance of these cell types follows a ventral-dorsal pattern. With the Rathke’s pouch cleft as the dorsal reference, GH, PRL, and TSHβ of Pit1 lineages are located dorsally, whereas gonadotrope cells appear ventrally. Several transcription factors display vertical gradients, including Pax6, which exhibits a dorsal-to-ventral expression gradient; Pax6-mutant mice exhibit increased numbers of ventral thyrotrope and gonadotrope, at the expense of the more dorsal somatotrope and lactotrope cell types.¹²⁸ Thus Pax6 may functionally oppose Shh signaling to specify a dorsal rather than ventral cell fate. Another pituitary transcription factor displaying an initial dorsal-ventral gradient is Prop1 (see Fig. 1-1). Prop1-mutant mice lose dorsal cell types of the Pit1 lineage, while ventral cell types of corticotrope and gonadotrope are less affected.

The induction of expression of transcription factors in spatially overlapping patterns in the developing pituitary may act as a molecular memory of prior signals in the positional determination of specific cell types. The signaling pathways that dictate expression patterns of transcription factor are the focus of current research application, with several classes of early morphogenic gradients of broadly expressed signaling molecules likely to be critical elements of pituitary cell type determination and differentiation.

Sonic Hedgehogs

Shh, one of three vertebrate homologues of the Drosophila-secreted protein Hh, plays an important role in embryo patterning, as well as in the specification of different cell types and in the control of proliferation of numerous cell types.¹⁴⁵ In the early embryo, it is expressed in Hensen’s node, the floor plate of the neural tube, the posterior of the limb buds, and throughout the notochord. The mouse, zebrafish, and human Shh homologues are highly conserved,¹⁴⁶ suggesting conserved functional properties. The human Shh gene encodes a predicted protein that is 92.4% identical to its mouse homologue and is mapped to chromosome 7q. Many Shh mutations, including nonsense and missense, deletions, and insertion mutations, are identified throughout the gene in patients with holoprosencephaly, the most common forebrain defect in humans.

Mouse mutants homozygous for a disrupted Shh gene, similarly to the mutations in human patients, revealed defects in the establishment of maintenance of midline structures such as the notochord, floor plate, and cyclopia.¹⁴⁷ In mice, Shh is expressed in ventral diencephalons and throughout the oral ectoderm on e8 but is excluded from the invaginating Rathke’s pouch. The Hh receptor Patched1 is expressed in the developing pituitary, indicating that pituitary progenitors respond to the Hh signaling. Transgenic overexpression of hedgehog-interacting protein (HIP), which acts to attenuate Hh function, specifically blocks Hh signaling in the oral ectoderm and Rathke’s pouch within the head region, affecting both proliferation and cell type determination, and this results in an absence of ventral cell type markers in Rathke’s pouch.¹⁴⁴ In contrast, a gain-of-function transgenic approach to overexpress Shh in Rathke’s pouch results in a phenotype of the expansion of ventral cell types, with modified levels of Lhx3 gene expression. This phenotype is consistent with results derived from an animal cap explant culture with banded Hh in Xenopus laevis, in which the expression domains of pituitary-restricted factor Hesx1 are expanded,¹⁴⁸ supporting a role for Shh signaling in control of proliferative events in pituitary development.⁹

Three related zinc finger transcription factors, Gli1, Gli2, and Gli3, acting downstream of the Shh pathway, are expressed in the developing Rathke’s pouch.¹⁴⁹ In zebrafish, Shh produced by neuroectoderm instead of notochord or oral ectoderm is crucial for the initial patterning of the pituitary placode.¹⁵⁰ Mutations that disrupt Hh signaling, like smoothened^smu and gli2^yot, result in the development of lens tissue from the presumptive pituitary. In addition, in gli2^yot mutants, the rostral expression domains (analogous to the ventral domains in mice) of pituitary-specific transcription factors such as LIM3 (Lhx3) and Six3 are lost, and other pituitary-restricted factors such as nk2.2 are absent.¹³⁷ This observation is consistent with the sequential and cooperative interaction Bmps and Hh exert in limb and neural-tube development,¹⁵¹ in which Shh acts to induce the expression of Bmps. Overexpression of Shh in zebrafish results in expanded adenohypophyseal expression of lhx3, expansion of nk2.2 into the posterior adenohypophysis, and an increase in PRL- and somatolactin-secreting cells. In addition, Hh signaling is necessary between 10 and 15 hours post fertilization for induction of the zebrafish adenohypophysis, a time when Shh is expressed only in adjacent neural tissue. These results suggest multiple and distinct roles for Hh signaling in the formation of the vertebrate pituitary gland and also suggest that Hh signaling from neural ectoderm of ventral diencephalons is necessary for induction and functional patterning of the vertebrate pituitary gland.^9,150

Fibroblast Growth Factors

Fibroblast growth factors represent a large family of secreted molecules. Upon binding to their cognate receptors, FGFs activate signal transduction pathways which are required for multiple developmental processes.152,153 Functions of FGFs are mediated by four distinct FGF-receptor tyrosine kinase molecules. FGF activity and specificity are further regulated by heparan sulfate oligosaccharides with tissue-specific modifications, in the form of a trimolecular complex with receptors.^154,155 The FGF system plays significant roles in many biologic events, including pattern formation in many tissues during vertebrate embryogenesis. Several members of the FGF family are expressed in the infundibulum and provide proliferative and positional cues to Rathke’s pouch. FGF8 and FGF10 are expressed in a temporally and spatially overlapping manner within the infundibulum as an evagination of ventral diencephalon makes direct contact with the dorsal portion of Rathke’s pouch following Bmp4 induction.

In mice null for FGF10 or the FGFR2 (IIIb) isoform, which presumably would abolish FGF signaling including that of FGF10,156,157 Rathke’s pouch forms but rapidly undergoes apoptosis, with the pituitary becoming completely absent by e14.5, suggesting a critical role in FGF10 signaling for the continued proliferation of the pouch ectoderm. A similar observation has been made in transgenic mice expressing a dominant negative form of FGFR2(IIIb), suggesting that FGF10 signaling is essential for cell survival and proliferation.

FGF8 is expressed in the primitive streak of the gastrulating mouse embryo, as well as in the visceral endoderm. Mice null for FGF8 lack all embryonic mesoderm- and endoderm-derived structures and do not survive beyond e9.5.158,159 Therefore, function of FGF8 in pituitary development is largely drawn from studies of transgenic animals and in-vitro organ culture. In mice null for a homeodomain gene Nkx2.1, which is normally expressed in the ventral diencephalons but not in Rathke’s pouch, FGF8 fails to be expressed in the ventral diencephalons, leading to a loss of the infundibulum and consequently a loss of Lhx3 expression in Rathke’s pouch and the loss of all three lobes of the pituitary gland.¹⁶⁰ In transgenic mice misexpressing FGF8 in the ventral regions of the pituitary under control of the regulatory sequences for the aGSU gene, most ventral and intermediate cell types are absent, with dysmorphogenesis of Rathke’s pouch and hyperplasia of corticotrope and melanotrope observed, consistent with a role in the positional determination of dorsally arising pituitary cell types and pituitary progenitor cells.¹⁵ In mice null for Hesx1, the most severely affected embryos exhibit a complete arrest of pituitary development after the initial induction of Lhx3 on e9.5, with FGF8 and FGF10 ectopically expressed in the oral ectoderm to mirror the normal expression in the overlying neural ectoderm. In Hesx1 mutants with less severe pituitary defects, FGF expression is abnormally extended rostrally, causing formation of multiple Rathke’s pouches. This is potentially significant because transgenic misexpression of FGF8 in the oral ectoderm well before the initial invagination of Rathke’s pouch produces an identical blockage of pouch formation, and Hesx1 fails to be expressed in the Lhx3-positive rudiment that does form in the transgenic embryos. Thus the dynamic interplay between boundaries of Hesx and FGF8/10 expression⁷⁶ could suggest a model of reciprocal feedback regulation. This is in keeping with the role of FGFs in committing oral ectoderm to the Rathke’s pouch fate.¹⁶⁰ These genetic data, in conjunction with tissue co-culture evidence, where the infundibulum is both required and sufficient for the induction of Lhx3 gene expression in cultured pouch and infundibulum activity, can be replaced with FGF8 or FGF2 and inhibited by the FGF receptor antagonist, suggest an instructive role for FGF8 signaling in pituitary development.^2,18,161

TRANSFORMING GROWTH FACTORS AND BMPs

The transforming growth factor-beta superfamily of secreted signaling molecules, which includes several Bmps, has been demonstrated to play critical roles in patterning and cell type specification in several species.¹⁶² At least two members of the Bmp family, Bmp2 and Bmp4, participate in the development of the anterior pituitary. During the early stages of pituitary development, Bmp4 is expressed in the ventral diencephalons as the infundibulum makes direct contact with Rathke’s pouch at e9.0. Functional evidence with dual explants culture of embryonic diencephalon and Rathke’s pouch suggests that Bmp4 is one of the early signaling factors required for the initial commitment of a subpopulation of oral ectodermal cells to form the pituitary gland.^15,18 Deletion of the Bmp4 gene causes embryonic death at about e10, in which the initial invagination of Rathke’s pouch fails to occur.¹⁶⁰ Similarly, driven by the regulatory sequences of the Pitx1 gene to target the Bmp2/4 antagonist Noggin expression to the oral ectoderm, including Rathke’s pouch, pituitary development is arrested at e10, with a failure of the ventral proliferation of cells from the pouch beginning at e11.5 and an absence of pituitary cell types.¹⁵ The phenotype observed in Pitx1-Noggin transgenic mice is similar to the phenotype observed in mice with a targeted disruption of the Lhx3 gene critical for the determination of most pituitary cell types,⁸⁸ suggesting a requirement of Bmp4 signaling for the continued organ development after pouch formation. Together with the ventral diencephalic FGFs, Bmp4 is required for initial pituitary commitment and for continued cell proliferation and progression.

Expression of Bmp2 is initially detected at the ventral boundary between Rathke’s pouch and Shh, intrinsic to the pouch in the most ventral aspect of the invaginating gland at e9.5, and in a ventral-dorsal gradient at e10.5.¹⁵ Bmp2 expression expands throughout the pouch by e12.5. Bmp2 expression is also detected in the ventral juxtapituitary mesenchyme, along with Bmp2/4 antagonist Chordin in the caudal mesenchyme, potentially serving to maintain a ventrodorsal Bmp2 gradient. After the closure of Rathke’s pouch, Bmp2 is expressed in mesenchyme adjacent to the pituitary cells expressing ventrally the transcription factors GATA2, Isl1, and the hormone subunit αGSU. Similarly, cultivation of Rathke’s pouch explants in Bmp2 is sufficient for the induction of expression of ventral markers such as Isl1 and αGSU.¹⁸ In the developing pituitary expression of Bmp2 decreases dramatically after e14-e15. Overexpression of Bmp2/4 under the control of αGSU regulatory elements in ventral mouse pituitary initially leads to a dorsal expansion of the ventral lineage markers Isl1 and Msx1 with induction of GATA2 gene expression. Proper expression of Bmp2 is therefore critical for the initiation of the cell fate determination process, however, Bmp2 signaling has to be attenuated to achieve terminal differentiation.¹⁵ These studies suggest that pouch-intrinsic and ventral signals, including Bmp2, contribute to the establishment of the positional identity of ventral pituitary cell types of thyrotrope and gonadotrope marked by αGSU expression.

Another way to study Bmp signaling is to address the role of Bmp antagonists during development. In a recent study, three antagonists—follistatin-like 1, Nbl1, and noggin–expression pattern—have implied a possible role of these proteins during pituitary development.¹⁶³ Out of three, noggin-/- embryos have pituitary defects that range from a lack of a morphologic Rathke’s pouch to the formation of secondary pituitary tissue. Noggin attenuates the Bmp4 signal emanating from the ventral diencephalon during the induction and early patterning of Rathke’s pouch, but it does not play a role in cell specification in the anterior lobe.¹⁶³

Notchs

The Notch signaling pathway is an evolutionarily conserved mechanism that controls cellular differentiation, proliferation, and death in a broad spectrum of developmental systems.164,165 Multiple ligands and effectors of the Notch signaling pathway were shown to be expressed in the developing pituitary, and a series of recent studies have shown the importance of the pathway in pituitary development.^{53,65,166,167} First, Notch2 expression is almost entirely absent in the Prop1 mutant mice pituitary, suggesting that the Notch signaling pathway may play a role in the commitment and lineage-specific differentiation of progenitor cells in the embryonic pituitary—in particular, Prop1-dependent cell lineages.⁶⁵ Accordingly, conditional inactivation of Rbp-J, central mediator of the Notch signaling, using the transgenic Cre line under the control of Pitx1 regulatory sequences, leads to premature differentiation of progenitor cells, as well as a conversion of the Pit1 lineage into the coricotrope lineage. Premature progenitor differentiation is phenocopied in the mice deleted for the Hes1 gene, while the later phenotype is largely due to the significant down-regulation of Prop1 at e12.5. It has been shown that Rbp-J directly binds to the evolutionary conserved recognition site in the first intron of Prop1 gene, and it is recruited to this region during pituitary development. Hence Notch signaling directly regulates Prop1 transcription.⁵³

The function of the Notch signaling pathway in pituitary development has also been investigated in Hes1-deficient mice and in mice conditionally deleted for both Hes1 and Hes5 in Rathke’s pouch and the ventral diencephalon, using the Emx1-Cre mouse line.53,166,168 In addition to premature corticotrope differentiation, which is consistently observed in Rbp-J conditional KO,⁵³ these mutant embryos lack both the intermediate and posterior lobes of the pituitary gland, which is in sharp contrast to the enhanced intermediate-lobe melanotrope differentiation detected in Rbp-J-conditional KO. The discrepancy might be explained by the different targeting approaches of these studies. Signals emanating from the ventral diencephalon are probably a key aspect of this discrepancy: in mice with Rbp-J conditionally inactivated using the Pitx1-Cre transgene, the ventral diencephalon remains intact, whereas in Hes mutants, it is also targeted because both Hes1 and the Emx1-Cre are both expressed in the diencephalon.

Two independent studies have shown that down-regulation of Notch signaling is necessary for terminal differentiation of distinct cell lineages in the later phases of pituitary development.53,167 Consistently, persistent expression of Hes1 in pregonadotropes and prethyrotropes prevents their differentiation.¹⁶⁶ Since there is a feedback loop between Notch signaling and Prop1, it would be very interesting to find out what factor down-regulates the Notch pathway during cell differentiation in the developing pituitary gland.

Wnts

The Wnt proto-oncogene family contains at least 19 known members.¹⁶⁹ As classical morphogens, the Wnt family of signaling molecules induces various cellular responses from proliferation to cell fate determination and differentiation. The canonic Wnt pathway stated that Wnt ligands bind to the Frizzled family of seven-transmembrane domain receptors, leading to the stabilization and accumulation of β-catenin, which interacts with members of the TCF/LEF family of DNA-binding transcription factors and changes them from repressors to activators of transcription, primarily by displacing the Groucho/Tle corepressor to influence target gene expression.^170,171

Several Wnt-signaling molecules are expressed during the development of pituitary.15,69,172 So far, two of them, Wnt4 and Wnt5a, have been reported to be specifically associated with the developmental events in the anterior pituitary. Wnt4 is expressed in the ventral diencephalon, and Wnt5a is expressed in the cells of Rathke’s pouch. In Wnt4-mutant mice, the pituitary is mildly hypocellular, with the ventral cell types showing normal differentiation but incomplete expansion. Additionally, cultivation of Rathke’s pouch with Wnt5a and Bmp4 can induce expression of the early cell type marker αGSU.¹⁵ Wnt5a mutants have expanded domains of FGF10 and Bmp expression in the ventral diencephalon and a reduced domain of LHX3 expression in Rathke’s pouch. As a consequence, Wnt5a-/- mice have a morphologically distorted pituitary with an enlarged intermediate lobe and increased numbers of POMC cells.^172,173 Double Wnt4/Wnt5s knockout mice display an additive pituitary phenotype of dysmorphology and mild hypoplasia of the anterior lobe and hyperplasia of the intermediate lobe.¹⁷² The phenotype suggests independent roles of those two factors. Wnt6 is expressed near the pituitary gland during critical times in development; however, examination of embryos deficient in Wnt6 showed no obvious pituitary malformation.¹⁷² The effects of deficiencies of Wnt4, 5a, or 6 seem unlikely to account for the consequences of deficiencies in the known, critical, β-catenin-regulated transcription factors in the pituitary gland. Rather that Wnt signaling affects the pituitary gland via effects on ventral diencephalon signaling. Other Wnt molecules (Wnt2b, Wnt11, Wnt16)¹⁷² are also expressed during pituitary development, although their role in this process awaits further investigation.

In Pitx2-deficient mice, mutant embryos fail to survive to term and exhibit developmental arrest of early determination events in the anterior pituitary gland.109,113,115,174 Pitx2a has been demonstrated to be acting downstream of the Wnt signal, and Lef1 and β-catenin have been demonstrated to physically occupy the Pitx2a promoter in the context of a pituitary cell line. Pitx2-mutant pituitary glands contained decreased numbers of proliferating cells, whereas transgenic overexpression of Pitx2 in the anterior pituitary led to increased cell numbers.¹¹³ Also, Pitx2c isoform has been shown to be regulated directly by Wnt signaling.¹⁷⁵ In a subtraction expression profiling analysis of Prop1^Ames-mutant pituitary, several members of the Wnt signaling pathway are identified, including the frizzled2 receptor, APC, β-catenin, Groucho, and Tcf7l2.⁶⁴ This genetic evidence suggests critical roles for the Wnt pathway in pituitary cellular proliferation and in cell type determination and differentiation.

Other components of the Wnt/β-catenin signaling pathway, including frizzled1-6, frizzled8, Lef1, Tcf3, Tcf4, and others have been reported to be expressed in the developing pituitary.64,69,172,176 Tcf4 is detectable in the early pituitary, as well as in surrounding tissues; it is markedly down-regulated by e13.5.^64,177,178 Targeted inactivation of Tcf4 results in hyperplasia of the anterior lobe, probably caused by the expansion of FGF and BMP expression in the ventral diencephalon, with a concomitant increase in the number of progenitor cells that form Rathke’s pouch. Thus Tcf4 may play a role as a repressor to regulate growth of Rathke’s pouch by influencing Bmp and FGF signaling.¹⁷⁸ Lef1 exhibits biphasic expression, initially transiently at e9.0 in Rathke’s pouch and later appearing at e13.5 in the anterior and intermediate lobes of the gland. Targeted deletion of Lef1 leads to elevated expression of Pit1, as well as GH and TSHβ, consistent with the role of Lef1 in inhibiting Prop1/β-catenin-mediated Pit1 activation.⁶⁹ Those recent studies have shown yet another role of β-catenin and Wnt signaling during pituitary development. Targeted inactivation of β-catenin in pituitary cells using a Pitx1-Cre transgenic line results in a smaller gland with no Pit1 expression, absence of all three Pit1 lineages, and reduced numbers of gonadotropes. Surprisingly β-catenin does not exert its role through binding with its canonical Tcf/Lef partners. Induction of Pit1 is mediated by direct interaction between β-catenin and Prop1, through evolutionary conserved Pit1 early enhancer.^52,69,179 Moreover, Prop1/β-catenin complex is also acting as a transcriptional repressor for Hesx1, ensuring differentiation of the progenitors. Genetic studies have demonstrated that proper spatio-temporal activation of Wnt/β-catenin signaling is essential for proper pituitary development, because premature activation of β-catenin leads to Hesx1 repression and pituitary agenesis.⁶⁹

Interactions Between Different Morphogenic Factors

Other Potential Morphogenetic Factors

Extrinsically derived signals that possibly affect Rathke’s pouch, arising from ventral mesenchyme beneath the developing pituitary gland, include Indian Hedgehog (IHh), Wnt4, and Bmp2; whereas caudal mesenchyme is a source of a Chordin signal capable of opposing the function of Bmp2.¹⁸² Recently it has been found that blocking EGF/TGFa signaling, by the expression of a dominant negative form of EGF receptor, has a profound stage-specific effect. Expression of mutated EGF receptor in GH-producing cells of embryonic pituitary results in dwarfism and pituitary hypoplasia, with reduced numbers of lactotropes and somatotropes.¹⁸³ In addition, little is known about the contribution of cytokines, despite the potent ability of cytokines such as leukemia inhibitory factor (LIF) to maintain mouse embryonic stem cells in an undifferentiated state. A potential role for LIF in pituitary development investigated in pituitary-derived cell lines is that LIF can activate synthesis of ACTH in combination with the hypothalamic peptide corticotropin-releasing hormone.¹⁸⁴ In transgenic animals that express LIF under control of αGSU regulatory information, most cell types fail to properly differentiate, and the pituitary is characterized by the formation of ciliated cysts of Rathke’s pouch and corticotrope hyperplasia,¹⁸⁵ suggesting that LIF may contribute to the identity establishment of dorsal-cell phenotypes.

In addition to the dorsal and ventral structures, another organizer signaling center, the notochord, is located just posterior to the developing pituitary gland. The proximity of these two structures, albeit from different origins, suggests a role for the notochord in the initial invagination of Rathke’s pouch, as indicated by tissue explant experiments.¹⁹

Pit1 LOCUS—Multiple Enhancers Coordinate Expression of The Gene

Transgenic studies revealed that 14.8 kb of 5′-flanking sequence of the Pit1 gene was sufficient to direct the robust expression of a reporter in an identical spatial and temporal pattern as endogenous Pit1, whereas its minimal promoter (−327 bp to −13 bp) was insufficient to drive detectable reporter expression in transgenic mice.³¹ A distal enhancer located at −10.2 kb from the Pit1 transcription start site and containing multiple functional Pit1-binding sites was shown to be involved in autoregulation. This element also contains a vitamin-D receptor binding site and a retinoic acid response element that confers Pit1-dependent RA induction.³¹ However, the early activation of the Pit1 gene at e13.5 in the anterior pituitary requires the cooperation of Prop1 and Wnt/β-catenin signaling and is mediated by early enhancer located between at −7.8 kb upstream of the transcription start site⁶⁹ in cooperation with ATBF1—giant, multiple homeodomain and zinc finger family factor—to the −5.8 kb enhancer element.¹⁸⁷ Thus proper spatio-temporal expression of the Pit1 gene is assured through the cooperation of different enhancer elements and require Pit1 protein itself. In the dwarf mice, Pit1 expression is activated normally, but because Pit1 protein is defective, Pit1 expression declines and becomes extinguished in the perinatal period.³¹

Biochemical approaches such as chromatin immunoprecipitation (ChIP) on chromatin isolated from developing gland or pituitary cell lines allow detection of changes in chromatin status of regulatory regions of the Pit1 gene as the pituitary gland develops.⁶⁹ At e11.5, the 2mH3K4, 3mH3K4, and AcH3K9 marks of activation are absent, but 2mH3K9 is present on Pit1 regulatory elements, consistent with an active repression of the Pit1 gene at this time. By e12.5, 2mH3K4 mark is selectively present at early enhancer. At e13.5, marks associated with active promoters¹⁸⁸ are present on Pit1 promoter (2mH3K4, 3mH3K4, and AcH3K9) and −7.8 kb enhancer (2mH3K4). In the adult, the Pit1 gene promoter harbors the histone marks of gene activation (3mH3K4, and AcH3K9), with 2mH3K4 now present on both the late and early enhancers, showing the temporal progression of histone modifications on regulatory regions of the Pit1 gene.

GROWTH HORMONE LOCUS—LCR, BOUNDARY, AND HISTONE CODE

The growth hormone (GH) gene provides a well-studied transcription unit that is highly suited for defining how specific chromatin modifications might be responsible for the spatial and temporal order of lineage specification events in the developing pituitary gland. This region is well studied in human and mouse subjects, but important differences can be noted between the species.

The human GH locus is represented by a cluster of five GH-related genes and their cell type–specific expression is regulated by a Pit-1-dependent locus control region (LCR). Expression of pituitary-specific GH gene (hGH-N) is ensured by −500 bp promoter that contains binding sites for Pit1, Sp1, and zinc finger proteins and is required for efficient expression of the gene. The LCR is necessary for proper spatio-temporal expression of hGH-N transgene.¹⁸⁹ Dissection of the hGH-N LCR revealed the existence of two Pit1-dependent DNaseI hypersensitive sites (HSI and HSII) that are sufficient to activate hGH transgene expression.^190,191 The hGH LCR and the hGH-N promoter are encompassed within a 32-kb domain of acetylated histones H3 and H4 in pituitary chromatin, with highest levels of acetylation at HSI,II.¹⁹² Although the 3mH3K4 modifications at the active hGH transgene locus in the mouse pituitary paralleled PolII distribution, they extended through the LCR and adjacent CD79b region and were separated from the hGH-N promoter by an intervening gap of unmodified chromatin.¹⁹³ In mouse transgenic models, selective deletion of the pituitary-specific HSI results in the loss of both histone acetylation and methylation throughout the LCR and a marked decrease in hGH-N transcription.^193,194 This recent study revealed also that HSI plays an essential role in the establishment of a complex domain of intergenic transcription 5′ to the hGH cluster.¹⁹⁴ Insertion of an exogenous transcriptional terminator within this domain selectively blocked a subset of downstream LCR transcripts and repressed hGH-N transcription without markedly affecting histone acetylation and methylation within the locus.^193,194 More recent studies showed pituitary-specific interactions between the HSI,II region and the hGH-N promoter, suggesting that noncoding transcripts in the LCR region are essential and a probable prerequisite to looping and gene activation.¹⁹³

Studies of the cis-acting elements of the rat growth hormone gene have shown that the minimal information required for the specific expression in somatotropes but not lactotropes resides in the proximal 320 bp of the promoter, with the proximal 180 bp capable of targeting the reporter in vivo.¹⁹⁵ This region contains binding sites for Pit1, thyroid hormone receptor (TR), Sp1, a zinc finger protein (Zn-15), and a recently identified zinc finger–homeodomain transcriptional repressor (Zeb1).¹⁹⁶ Extensive analyses of transgenic mice have revealed that multiple factors are essential to mediate GH gene activation in somatotropes (Sp1), others for repression in lactotropes (Zeb1), while TR and Pit1 are required for both activation and repression. Interestingly, mutational studies have shown that replacing GH-specific Pit1 binding sites with the PRL-specific Pit1 site results in a loss of restriction from the lactotropes without affecting expression in somatotropes.⁴⁴ Comparative analysis of the cocrystal structure of the Pit1 POU domain dimer bound to either GH-specific or PRL-specific sites has revealed that the spacing between the DNA contacts made by the POU-specific domain and the POU homeodomain of each monomer is increased by 2 bp on the GH-specific site. Deletion of these 2 bp in this promoter leads to a lack of the effective restriction of the reporter gene expression from lactotropes. These data suggest that the Pit1 protein conformation is critical for its specific activity. The transcriptional repressor Zeb1 is bound to the GH promoter only in the lactotropes in the sumoylation-dependent manner, and it is not recruited to the GH promoter in somatotropes. Together with the other two components of the CtBP-CoREST-LSD1 complex, Zeb1 assembles an LSD1-containing corepressor complex on the GH promoter in lactotropes. Earlier in development, LSD1 is present at the GH promoter in the MLL1-containing complex in the somatotropes, which is required for activation of the gene.¹⁹⁶ Thus machinery controlling chromatin status (e.g., methylation of histones) is involved in developmental processes such as differentiation.

Another level of complexity of GH regulation in somatotropes comes from studies performed in mice. The murine GH genomic locus is found on mouse chromosome 11 and encompasses five genes. In contrast to the human locus, the mouse locus does not contain tandem duplications of the GH gene, and there is no known murine LCR. The proximal regulatory sequences are very well conserved between mouse and rat. However, specific activation of the endogenous murine GH gene also appears to require a boundary element imposed by an upstream SINE B2 repeat.¹⁹⁷ This SINE B2 repeat is able to produce short, overlapping Pol II and Pol III-driven transcripts that are both necessary and sufficient for its enhancer-blocking activity in cultured cells enhancer-blocking assay. Interestingly, the Pol II-driven transcript appears in a temporally regulated manner during pituitary development, concomitantly with translocation of the GH locus from condensed heterochromatin territory (marked by H3K9me3) to a euchromatic area (marked by H3K9me2). These studies suggest that active transcription of repetitive sequences may represent one of the strategies for the establishment of functional chromatin domains to control gene expression.

Many mechanisms such as long-range interactions (hGH, Pit1), use of multiple enhancers (Pit1, GH) and histone modifications have been shown to be implicated in regulation of the expression of genes during pituitary development. DNA methylation represents another layer of complexity in regulation of the gene expression. To date, only the POMC promoter has been described as having the particular DNA methylation code that allows expression of the gene in tissues where its promoter is not methylated and prevents gene expression when the promoter DNA is methylated. The regulatory mechanism of this process, however, is not yet understood.¹⁹⁸

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