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Division of Endocrinology and Metabolism (B.A.), and Howard Hughes Medical Institute (M.G.R.), Department of Medicine, University of California, San Diego, La Jolla, California 92093-0648
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Abstract |
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 Top Abstract I. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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I. Introduction
II. Structure and Transcriptional Function of POU Domain Factors
A. DNA-binding sites for POU domain factors
B. Protein chemistry
C. Protein-protein interactions
D. Mechanisms of transactivation
III. Development of the Hypothalamo-Pituitary Region
IV. Expression and Function of POU Domain Factors in the Neuroendocrine System
A. Pit-1
B. Oct-1
C. Oct-2
D. Overview of expression of Brn-1, Brn-2, Brn-4, and Tst-1 in the neuroendocrine system
E. Brn-2
F. Brn-4
G. Tst-1
H. Oct-3
I. Brn-5 and RPF-1
V. Relevance of POU Domain Factors to Diseases of the Neuroendocrine System
A. Pit-1 mutations as a cause of combined pituitary hormone deficiency in humans
B. Brn-4 mutations in humans
C. Potential implications for other diseases.
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I. Introduction |
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TopAbstractI. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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).
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Although some general conclusions can be reached about POU domain factors, more importantly the various members serve a wide variety of functions during development. In fact, it is reasonable to predict that an individual POU factor may play multiple distinct roles in those organs in which it is expressed during development and in the adult. There is evidence for the role of POU domain factors in inhibition and promotion of cell proliferation and determination of cell lineages, as well as in regulation of cell migration, survival, and terminal differentiation. Because of their overlapping expression patterns and similar DNA-binding preferences, POU factors coexpressed in a single organ may be functionally redundant. In addition to a role in early and late development, members of this family play roles in physiological regulation of gene expression in the adult. While POU domain genes are expressed in all organ systems, this review focuses on the role of POU domain factors in the neuroendocrine system with special emphasis on the hypothalamo-pituitary axis.
A great deal of knowledge has accumulated about the biological function of POU domain factors since their initial identification in 1988. In addition to insightful in vitro experiments, progress in this field has been greatly facilitated by the ability to create targeted mutations of POU domain genes in the mouse. These studies, as well as the discovery of human disease mutations involving the POU domain factor Pit-1, have established a crucial role for POU domain factors in neuroendocrine development and function.
The 15 known mammalian POU domain genes have been classified into
six different groups based on homology in the POU domain region,
including the nonconserved linker region (Table 1). The class I POU factor, Pit-1
(GHF-1), is required for the generation and maintenance of three
different cell types in the anterior pituitary gland of humans and
other mammals (1, 2, 27). Expression analyses and in vitro
biochemical/molecular biology experiments have implicated the
octamer-binding proteins Oct-1 and Oct-2, which belong to class II POU
domain factors, in neuroendocrine function and development (28, 29, 30, 31).
The third class II factor, Skn-1a, is selectively expressed in
epidermis and has no role in the neuroendocrine system (32, 33, 34, 35). The
class III POU domain subgroup consists of four closely related factors,
Brn-1, Brn-2, Brn-4, and Tst-1/Oct-6/SCIP, which are all encoded by
intronless genes (36). For members of this subgroup, a role in
neuroendocrine development has been most clearly established for Brn-2,
which is required for the formation of hypothalamic nuclei and the
posterior pituitary gland (37, 38). The other class III POU factors may
also play a crucial role in neuroendocrine function and development.
Brn-3 and two other members of the class IV POU factors have been shown
to be important for various aspects of sensory development, but have no
known roles in the neuroendocrine system and will not be further
discussed in this review (39, 40, 41, 42, 43, 44, 45, 46, 47, 48). The class V POU factor Oct-3 (Oct-4)
is expressed in germ cells, embryonic stem cells, and in early
neurogenesis where its down-regulation may be crucial for proper neural
formation (49, 50, 51, 52, 53). Sprm-1, the second class V factor, is selectively
expressed in spermatocytes and has no role in the neuroendocrine system
(54, 55). The class VI POU factor Brn-5 is widely distributed and
highly expressed in hypothalamic regions (56, 57, 58). The other class VI
member, retina-derived POU-domain factor-1 (RPF-1), is expressed in the
retina, the medial habenula, and in dispersed populations of neurons in
the dorsal hypothalamus (59).
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II. Structure and Transcriptional Function of POU Domain Factors |
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TopAbstractI. IntroductionII. Structure and...III. Development of the...IV. Expression and Function...V. Relevance of POU...References |
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) (62, 63). This principle has been elegantly demonstrated for the
interaction of Oct-1 with an octamer site (ATGCAAAT) where the POU-S
domain and the POU-H domains associate with the ATGC- and AAAT-parts,
respectively (64, 65, 66). The spacing and orientation between the two
halves of a binding element can vary, and the ability of different POU
domain factors to recognize such sites depends in part on the length,
sequence, and flexibility of the linker tethering the POU-S and POU-H
domains (63, 67). In addition, there is evidence that diversity in the
POU DNA binding may stem, in part, from amino acid-base interactions
that are more flexible than those observed for classical homeodomains
(62). Often, high-affinity binding sites for POU factors are flanked by lower affinity binding sites that are capable of promoting cooperative DNA binding of POU factor dimers. These "second" sites are usually highly diverged from high-affinity binding sites and may contain isolated A/T-rich regions or the POU-S recognition sequence ATG (CAT on the opposite strand). In this section, we will first review the DNA binding characteristics of different POU domain factors, and then summarize the main conclusions from the collection of studies on this issue.
1. DNA binding sites for the class I factor Pit-1. Pit-1 was
originally identified based on its ability to bind to multiple sites in
the regulatory regions of its target genes GH and
PRL (1, 2). Similar Pit-1 binding sites have been described
in the regulatory regions of several other pituitary-expressed genes
that are likely targets of Pit-1. These include the Pit-1
gene itself (68, 69, 70), the TSH
gene (71, 72, 73), the rat and
human GHRH receptor gene-regulatory regions (74, 75), the rat
somatostatin receptor 1 gene-regulatory region (76, 77), and the human
TRH receptor gene-regulatory region (78). These sites, which vary
widely around the consensus sequence ATGNATA(A/T)(A/T)(A/T), tend to be
found in multiples in Pit-1 target genes (Table 2). On most sites, Pit-1 appears to bind
as a homodimer with one Pit-1 molecule contacting the high-affinity
octamer-related sequence, ATGNATA, and the second molecule binding
cooperatively to the 3' located lower affinity A/T-rich region
(79, 80, 81). On some sites Pit-1 can bind as a heterodimer with the POU
domain factor Oct-1, and on yet other sites it binds as a monomer (82).
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2. DNA binding sites for the class II factors Oct-1 and Oct-2.
The octamer DNA element, ATGCAAAT, frequently found within
gene-regulatory regions, is conserved and functionally important for
transcriptional regulation of several genes. These include the B
cell-specific immunoglobin genes (83), the ubiquitously expressed small
nuclear (sn)RNA genes (84, 85), the cell cycle-specific histone
H2B genes (86), and a host of other widely and selectively expressed
genes (Table 3). Early studies attempting
to identify the transcription factors binding to octamer elements led
to the isolation of Oct-1 and Oct-2 as the major components of
octamer-binding activity in eukaryotic cells. Review of the literature
indicates that the Oct-1 POU domain has a strong preference for the
core element, ATGCAAAT, but clearly several variants of the core
element are compatible with high-affinity DNA binding (Table 3).
Experiments have also indicated that the bases flanking the core
octamer provide additional binding specificity (60).
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). It is striking that alignment of
these sites results in a consensus sequence remarkably similar to that
identified in the two random site selection studies (Table 3). However,
this approach is unlikely to give a true representation of the type of
sites found in gene-regulatory regions because in their studies,
investigators are much more likely to identify and report on the well
known octamer site rather than more divergent sites.
Oct-1 can also bind to a distinct site, TAATGARAT (R is a purine),
found in the herpes simplex virus immediate early (HSV IE) promoters
(87, 88). The affinity of Oct-1 alone for this site is much lower than
for that of the octamer element (64), but in contrast to the classical
octamer site, this site can promote the formation of a complex of Oct-1
and VP16, which is required for viral transcription (Fig. 2). The critical feature of this site is
the 3'-GARAT motif, which is necessary for the recruitment of VP16,
possibly by a mechanism involving the recognition of specific
conformations of Oct-1 (62, 89) or direct nucleotide recognition (90).
On this site, the POU-S domain is located on the opposite side of the
POU-H domain as compared with Oct-1 bound to a classical octamer site
(62).
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Several divergent sites capable of binding Oct-1 have been identified
in diverse genes, including the GnRH gene-regulatory region
(28, 29) (Table 3). An unusual binding site has been described in the
human interleukin 5 (IL-5) promoter. In this site, inverted POU-S
binding sites seem to flank two POU-H domain binding sites (94, 95).
Yet other sites are similar to the TAATGARAT site in that they only
contain recognizable POU-H domain binding sites (Table 3). The binding
specificity of Oct-1 and Oct-2 has been found to be indistinguishable
in several studies, and other studies indicate that the third class II
POU factor, Skn-1a, has identical binding preferences (35, 96). These
observations suggest that within a POU class, binding preferences tend
to be similar.
3. DNA binding sites for class III factors. Studies have shown
that the class III POU factor Tst-1 binds to and activates octamer
site-dependent promoters (97, 98). Similar to Oct-1, different binding
affinities were observed with perfect octamer sites containing distinct
flanking sequences, indicating that the flanking sequences are also
important determinants of binding affinity for Tst-1 (98). Tst-1 also
binds to five distinct sites in the myelin-specific P0 promoter with a
consensus sequence, GA(A/T)T(T/A)ANA, which appears unrelated to the
octamer site (99). The rat GnRH promoter contains three Tst-1 binding
sites that appear to conform to the consensus derived from the Tst-1
binding sites in the P0 promoter (100) (Table 4). Tst-1 also binds with high affinity
to two sites in the JC virus promoter, but only one of the sites
confers transcriptional activation. While they are AT-rich, neither
site bears any similarity to octamer sites nor the P0 consensus site
(101).
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Brn-4 DNA binding preferences have also been evaluated by a random site
selection method where it was found that the optimal binding site,
CAATATGCTAAT, is related to the smaller octamer site, containing a TAAT
instead of the AAAT (105). Thus, the optimal binding sites selected
with Brn-2 and Brn-4 are different. It is quite possible that these
differences are due to methodological variables rather than bona fide
binding differences between these two class III POU factors. Since it
is likely that all POU domain factors can bind to DNA in different
conformations, it is not surprising that different studies have
identified distinct binding sites for class III POU factors. Nonoctamer
DNA-binding sites for Brn-4 have been found in the striatal D1 dopamine
receptor (106) and proglucagon genes (107). These sites seem to be most
similar to the Tst-1 sites in the P0 and rGnRH promoters (Table 4).
4. DNA binding sites for the class V factor Oct-3. Oct-3 binds
with high affinity to octamer sites (108, 109, 110), but also to so called
palindromic Oct factor recognition elements (PORE), which simulate a
pair of inverted homeodomain sites separated by 5 bp, ATTTG +5 CAAAT,
found in the osteopontin enhancer (Table 5). In addition to Oct-3, other Oct
factors can bind to this site, either as homodimers or heterodimers
(111). Oct-3 is reported to transactivate more robustly from this
element than a classical octamer site (111).
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), selectively recruits Oct-3 and Sox-2
and is incapable of binding complexes containing Oct-1 and Tst-1 (112).
5. DNA binding sites for the class VI factor Brn-5. In
experiments using a random site selection method, Brn-5 was found to
prefer the binding site GCATAA(T/A)TTAT, which is related to the class
IV factor Brn-3 optimal binding site (103). In different experiments
where Brn-5 site selection was performed with a fixed site, GCAT,
followed by eight random nucleotides, an octamer-like sequence was
selected: GCATATGATAAT (56). However, it is clear that Brn-5 binds
poorly to a classical octamer site (56). Alignment of three
high-affinity sites suggested a high-affinity consensus site
GCATN2–3TAAT (56) (Table 6).
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genes are bona fide response
elements. For most other sites identified in such studies, genetic
evidence for their importance is lacking. An alternative approach has
been to use a selection of high-affinity binding sites from a set of
random sites in vitro. While this approach may identify
high-affinity sites, those are not necessarily biologically important
sites. However, from these two approaches several general conclusions
about POU domain binding sites can be reached.
First, members of the same subclass of POU domain factors tend to have
similar DNA-binding preferences, most clearly demonstrated for class II
POU factors. Second, while distinct POU classes may have somewhat
distinct DNA-binding preferences, a single POU factor can recognize
several different DNA-binding sites. Third, POU factors of different
classes can often recognize the same element with similar affinity, and
all POU factors discovered so far seem to be capable of binding to
octamer sites with reasonably high affinity. Fourth, while the core
recognition sequence for monomeric binding is often 8 bp long, bases
outside the core region may be important for binding affinity and
transcriptional activity. Fifth, while POU domain factors can bind as
monomers, some factors, such as Pit-1 and class III factors, can
homodimerize, especially on appropriate symmetrical sites. Sixth, while
transcriptional activation may correlate with binding affinity on
monomeric sites, other features of the site, such as the ability to
allow dimerization and interaction with other factors, may be important
determinants of whether a particular site confers activation (Fig. 2).
Seventh, the DNA-binding site can determine the ability of a bound POU
factor to recruit associated proteins (Fig. 2). Finally, target genes
that are responsive to POU domain factors frequently have a cluster of
POU-binding sites, particularly when the regulatory regions are located
at a distance from the promoter.
B. Protein chemistry
The structure of the POU domain has been extensively studied for
both Oct-1 and Pit-1, using NMR (113, 114, 115, 116) and crystallographic (66, 117) methods. The similarity in structure between these two POU domains
strongly suggests that the basic observations from these studies can be
applied to other POU domains. The POU domain is composed of a 74- to
82-amino acid POU-S domain and a 60-amino acid POU-H domain with a 15-
to 56-amino acid flexible linker tethering the two domains (Fig. 1).
The two subdomains are structurally independent, and individually they
are capable of low-affinity DNA interactions (64). High-affinity DNA
binding, however, requires the intact POU domain, with each subdomain
making major groove DNA contacts. The structure of the POU-S domain is
characterized by four 
-helices surrounding a hydrophobic core
similar to the helix-turn-helix motif of bacteriophage 
and 434
repressor, and 434 Cro DNA-binding domains (113, 115). While somewhat
divergent, the POU-H domain, like classic homeodomains, exhibits a
helix-turn-helix structure with three -helices (118, 119, 120). For both
the POU-S and POU-H domains, the second and third helix form a
helix-turn-helix structure with the third helix docking to DNA in a
major groove where amino acids making direct base contacts tend to be
conserved among POU domain factors (121, 122).
The two subdomains do not contact each other, and the linker is not
visible in the cocrystals, suggesting that it may be unstructured,
perhaps providing flexibility to the POU domain (Fig. 1). This
flexibility would allow the two subdomains to acquire different
orientation and spacing relative to each other, presumably explaining
the versatility in DNA sites that can bind POU domain factors, and the
variability in surfaces available for interactions with coregulatory
proteins (63). This flexibility may depend, in part, on the length of
the linker as evidenced by results showing that the Brn-2 POU domain
can adopt two different orientations on DNA, while this was not
observed with Brn-3, which contains a shorter linker (62, 63). An
effect of the linker on DNA binding specificity was also found in
experiments comparing binding of chimeric molecules from the Oct-1 and
Pit-1 POU domains (123). It has been shown that optimal binding of
Oct-1 to an octamer site requires a minimum linker length between 10
and 14 amino acids (67). Oct-1 molecules with a shorter linker bind DNA
only if the POU-S recognition site is inverted in orientation,
apparently bringing it closer to the POU-H domain. In addition,
mutations of some conserved residues in the Oct-1 linker altered DNA
binding, indicating that there are both sequence and length
requirements for the function of the linker (67).
An example of the subdomain flexibility is provided by the binding of Oct-1 to the octamer element (ATGCAAAT) where the POU-S domain recognizes the 5'-ATGC-part and the POU-H domain recognizes the 3'-AAAT-part. On the TAATGARAT site from the herpes simplex virus (HSV) IE promoter, the Oct-1 POU-H domain recognizes the 5'-TAAT part, and the POU-S domain recognizes the 3'-GARAT part in a flipped orientation as compared with Oct-1 binding to an octamer site (62).
While the primary, secondary, and tertiary structures are similar,
crystallographic studies of Oct-1 and Pit-1 on two different DNA sites
have shown quite distinct structures as to the relationship between the
two POU subdomains (66, 117) (Fig. 1). The Oct-1 POU domain binds to
opposite faces of an octamer DNA-binding site (the histone H2B promoter
octamer). In contrast, the Pit-1 subdomains bind to perpendicular faces
of a palindromic DNA site (derived from the PRL 1P site), with the
POU-S domain exhibiting flipped orientation compared with Oct-1
structure (Fig. 1). In addition, while Oct-1 binds as a monomer to an
octamer site, Pit-1 forms homodimers on the palindromic PRL 1P-related
site via interactions between the C terminus of the DNA-recognition
helix of the homeodomain and helix 1 and the loop between helices
3 and 4 of the POU-S domain. Most likely, these differences in
structure between Oct-1 and Pit-1 are due to the different DNA-binding
sites used for cocrystallization, which is supported by data indicating
that the Pit-1 POU domain binds as a monomer to a classical octamer
site (124).
It has been shown that POU factors can be modified by phosphorylation.
Oct-1 and Pit-1, for example, are phosphorylated on homologous residues
in the POU domain, Ser-385 and Thr-220, respectively, and this
phosphorylation leads to decreased DNA binding to octamer and GH POU
sites (125, 126). On other sites, such as those found in the TSH and
PRL promoters, phosphorylation by protein kinase A or C enhances DNA
binding (127, 128). While cAMP-dependent kinase can phosphorylate these
sites in vitro, the identities of the relevant kinases
operating in vivo are unclear. For Oct-1, this
phosphorylation occurs in a cell cycle-specific manner, thus explaining
the S phase-specific activation of the histone H2B gene by Oct-1 (86, 125). Jun kinase (JNK)/stress- activated protein kinase can bind to the
activation domain of Brn-5 and phosphorylate its POU domain on sites
that are distinct from the aforementioned Oct-1/Pit-1 phosphorylation
sites. This phosphorylation leads to increased binding to DNA and
enhanced transactivation (129). Finally, it has been demonstrated that
Pit-1 can be acetylated by CREB-binding protein (CBP), but the
biological significance of this finding remains to be determined (130).
C. Protein-protein interactions
POU domain factors regulate transcription by interacting with
other proteins. POU interacting proteins can be classified into four
classes: DNA-binding transcriptional activators, coregulators, basal
factors, and replication factors (131). A prominent feature of POU
factors is their ability to form cooperative homodimers, and even
heterodimers with other POU factors, especially when bound to the
appropriate DNA site (80, 82, 132) (Fig. 2). POU factors also have a
striking ability to interact with a variety of divergent structural
domains in heterologous factors, including other DNA-binding proteins
and coregulators that are incapable of independent DNA binding. In this
chapter we will review protein-protein interactions involving those POU
factors expressed in the neuroendocrine system (Table 7). Many of these interactions may not be
directly relevant to regulation of gene expression in the
hypothalamo-pituitary axis, but the principles derived from these
studies are likely to apply to studies of POU domain factors in the
neuroendocrine system (Fig. 2).
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genes (80). This Pit-1
homodimerization has been shown in living cells with fluorescence
resonance energy transfer microscopy (FRET) (133). Functional
experiments indicate that the POU-S domain is important for Pit-1-Pit-1
interactions and that homodimerization is important for efficient
transactivation (80). The ability of some natural Pit-1 mutants to
inhibit Pit-1 action in a dominant negative fashion further supports
the importance of Pit-1 homodimerization in gene activation (see
Pit-1 mutations as a cause of combined pituitary hormone
deficiency in humans). In addition, Pit-1 can form
heterodimers with Oct-1, which results in synergistic transactivation
of the PRL promoter (82). Pit-1 can also interact to varying degree with several heterologous DNA-binding proteins which, when bound to the same promoter, often lead to synergistic transactivation. These interacting factors include the nuclear hormone receptors TR (thyroid hormone receptor), RAR (retinoic acid receptor), RXR (retinoid X receptor), ER (estrogen receptor), VDR (vitamin D receptor), and GR (glucocorticoid receptor). Pit-1, for example, interacts strongly with RXR and more weakly with TR and RAR, which may play a role in synergistic regulation of the GH promoter (134, 135, 136). Pit-1 can also interact with ER (137, 138, 139, 140) and VDR (141) in synergistic activation of the PRL gene. The human PRL promoter is inhibited by glucocorticoids, mediated by a gene regulatory region containing Pit-1 binding sites but no glucocorticoid response element (GRE). Studies show that GR and Pit-1 can interact in solution and that the inhibition does not require GR to bind to DNA (142).
Pitx1, a bicoid-like homeodomain factor, interacts with the N terminus of Pit-1 (143) and synergistically transactivates the PRL promoter with Pit-1 (144, 145). A direct interaction of Pit-1 with the related Pitx2 removes an inhibitory effect of a 39-amino acid C-terminal sequence of Pitx2, thus promoting DNA binding and transactivation (146). Pit-1 also interacts in solution with the LIM homeodomain protein P-Lim (Lhx3) and acts synergistically with P-Lim to regulate the PRL enhancer/promoter (147).
Synergistic interactions between Ets-1 and Pit-1 may be important in
regulation of the PRL promoter. The synergistic gene activation
requires direct Pit-1-Ets-1 protein-protein interaction and is
not observed with Ets-2 (148). This interaction, which has been shown
in living cells with FRET microscopy (133), maps to the POU domain
(149). Pit-1, an isoform of Pit-1 containing 26 additional amino
acids in the N terminus, is incapable of transcriptional synergism with
Ets-1, apparently because of additional interaction surface that maps
to the N-terminal sequence specific to this isoform (149).
Interactions between Pit-1 and the transcription factor GATA-2 are more
complex and have distinct features depending on whether both factors
bind to DNA (150, 151) (Fig. 2A). Pit-1 and GATA-2 can interact in
solution (150, 151), and the interaction interface maps to the POU-H
domain of Pit-1, specifically the N-terminal basic residues and the
surface of the second helix (P26, Q29) (151). In GATA2, the C-terminal
DNA-binding zinc finger and an adjacent cluster of basic residues seem
to be important. Pit-1 can inhibit binding of GATA-2 to cognate DNA
sites on promoters that do not have Pit-1 binding sites, such as the
gonadotrope-specific SF-1 promoter (151) (Fig. 2A). This seems to be
important for generation of the gonadotrope phenotype. In contrast,
Pit-1 leads to synergistic activation with GATA-2 on promoters
containing adjacent Pit-1 and GATA-2 sites, such as the
thyrotrope-specific TSH promoter (150, 151) (Fig. 2A). As described
later, these interactions may be important for the formation of both
thyrotrope and gonadotrope cell types.
b. Interactions with coregulator proteins.
In addition, Pit-1
has been demonstrated to interact with coregulators. The POU domain of
Pit-1 interacts with CREB binding protein (CBP) through two
cysteine-histidine rich domains (C/H1 and C/H3) (152, 153) (Fig. 3). Pit-1 can also, via its POU-H domain,
interact with the corepressor N-CoR (nuclear receptor and corepressor)
(153). As will be described later, these interactions may be important
for transactivation and the role of Pit-1 in signal transduction.
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Both Oct-1 and Oct-2 have been shown to interact with DNA-binding factors from other classes, including nuclear hormone receptors, such as RXR (138). The interaction interfaces seem to involve the Oct-1 POU-H domain, but not the POU-S domain, and the DNA-binding domain and hinge region of RXR. Functional studies suggest that Oct-1 may negatively regulate nuclear signaling pathways by altering the DNA-binding ability of nuclear receptor-RXR heterodimers. In contrast, on artificial promoters containing vitamin D response elements and octamer sites, Oct-1 and the vitamin D receptor exhibit cooperative binding and transcriptional synergism mediated by the DNA-binding domain of the vitamin D receptor (155).
In solution, the POU domains of Oct-1 and Oct-2 can bind to the DNA-binding domain of GR, including an interface that contains amino acids C500 and L501 (156). Again the POU-H domain of Oct-1 seems to be responsible for the interaction (156). Microinjection experiments in zebrafish embryos suggest that such DNA-independent effects of GR involving interactions with the Oct-1 and Oct-2 POU-H domains are biologically important (157). The GR-Oct-1 interaction can both modulate the transcriptional activity of Oct-1 on certain promoters, like the GnRH promoter as detailed later (158), and the activity of GR on its target genes (138). On the mouse mammary tumor virus (MMTV) promoter, it has been shown that Oct-1 and GR bind in a cooperative fashion and that this leads to a synergistic transcriptional regulation on naked DNA templates in vitro (159). Oct-2 can also bind cooperatively with GR, and this also results in synergistic transcriptional regulation (156, 160). In vivo results indicate that Oct-1 binding to the MMTV promoter is strictly glucocorticoid dependent, consistent with a role for GR in remodeling of chromatin to promote access of other factors to the MMTV promoter, such as nuclear factor 1 (NF-1) and Oct-1 (161, 162).
On templates containing GREs and octamer sites, Oct-2A and GR can synergize transcriptionally (156, 163). However, when GR sites are not present, GR strongly inhibits transactivation by Oct-2A, while the reciprocal is not true. This effect does not seem to depend on interference with Oct-2A DNA binding, but more likely is due to competition for rate-limiting coactivator molecules (163). A minimal promoter driven by Oct-1, binding to an octamer element, can also be inhibited by GR in a hormone-dependent manner. This interaction seems to be mediated by a strong solution interaction between GR and Oct-1, resulting in inhibition of Oct-1 DNA-binding (164). This interaction, which seems to involve the POU-H domain of Oct-1, can be inhibited by GR binding to a GRE (156). While the mechanism is unclear, functional analyses indicate that androgen-mediated repression of the dehydroepiandrosterone sulfotransferase gene promoter requires Oct-1 binding to the promoter. Since there seems to be no direct AR binding to this promoter, AR-Oct-1 interactions may be important (165).
Oct-1 can interact with several other DNA-binding factors, including
Sp1 (166, 167), AP-1 (168, 169, 170), CCAAT/enhancer binding protein-
(C/EBP) (171), NF-1 (172, 173), and MEF2 (174). Oct-1/2 interactions
with heterologous DNA-binding proteins can either result in
transcriptional synergism, as exemplified by the AP-1/Oct-1 interaction
(168, 169, 170), or interference as exemplified by C/EBP inhibition of
Oct-1 function (171).
Interaction of the high mobility group protein HMG2 with the POU domains of Oct-1 and Oct-2 leads to cooperative binding (175). The related HMG I(Y), a known octamer-binding protein, functions as a coactivator with Oct-2A but not Oct-1 for HLA-DRA transcription. Apparently HMG I(Y) facilitates the binding of both Oct-1 and Oct-2 to octamer sites (176) by a mechanism that involves interactions between HMG I(Y) and the POU domains of Oct-2 and Oct-1. However, synergistic transactivation of the HLA-DT gene is only found with Oct-2 and seems to depend on sequences within the C terminus of Oct-2 (177).
b. Interactions with coregulator proteins.
The B cell-specific
coactivator OCA-B (also referred to as OBF-1 and Bob-1), a 256-amino
acid, proline-rich protein with no close homologs in the database,
interacts specifically with the POU domains of Oct-1 and Oct-2 and is
required for B cell-specific activation of immunoglobin genes
(178, 179, 180, 181, 182, 183). By interacting with Oct-1 and Oct-2, OCA-B can be recruited
to a subset of octamer sites where it serves a coactivator function for
transcription (184) (Fig. 2B). In part, a strong transactivation domain
in the C terminus of OCA-B mediates this effect, while the N terminus
contains the domain interacting with POU domains (185). This
interaction is specific for Oct-1 and Oct-2 (Fig. 2B) and not observed
for other POU domain factors such as Oct-3, Oct-6, and Pit-1. The
specificity of the interaction of OCA-B with both the POU-S and the
POU-H domains is determined by residues in the first helix of the POU-S
domain (L6 and E7) and the end of the third POU-H domain helix (K155
and I159) (124, 184). Other residues in the POU domain, including L53
and N59 in the POU-S domain, have been shown to be important for the
Oct-OCA-B interactions. However, these residues are conserved in POU
domain factors that do not interact with OCA-B and are thus unlikely to
determine the specificity of the interaction.
Immunoglobin promoters are strongly activated by OCA-B, whereas the
histone H2B promoter is poorly activated, suggesting that there is some
promoter-specific restriction of the ability of OCA-B to transactivate.
Further specificity is observed on the immunoglobin promoters because
OCA-B only seems to activate on sites in the proximal promoter but not
on distally located enhancer sites (124). In addition, the
Oct-1/Oct-2-OCA-B complex can only occur on octamer sites that contain
an A in the fifth position of the octamer site, such as found in the
classical octamer site (Fig. 2B). Position +3, +4, and +6 also appear
to be important (124, 184). Apparently, OCA-B makes direct contacts
with the DNA backbone in the middle of the octamer site, via its N
terminus, in addition to POU domain contacts (186, 187). It has been
proposed that OCA-B clamps the two POU subdomains together and at the
same time bridges the DNA within the octamer motif at several
positions. According to this model, OCA-B may help organize the
conformation and stabilize the DNA binding of the Oct POU domain on DNA
(124, 184). There is also evidence that binding of OCA-B to the Oct-1
POU domain/octamer complex induces a partial folding of OCA-B (186).
Another extensively studied coregulator is VP16. In human cells
infected with the herpes simplex virus (HSV), viral gene expression is
initiated with the viral protein VP16, which regulates the HSV
immediate-early (IE) genes. VP16 does not bind DNA directly, but is
instead recruited to IE promoters by its association with Oct-1 (87, 188, 189, 190, 191, 192) (Fig. 2B). Another cellular protein, host cell factor (HCF),
is required for nuclear import of VP16 (193) and stabilization of the
VP16-Oct-1 interaction (194, 195, 196, 197). After infection, VP16 first forms a
complex with HCF, which then promotes its interaction with Oct-1 and
binding to the TAATGARAT motif in the HSV IE promoters (Fig. 2B). The
interaction of the Oct-1 POU domain and VP16 is selective and not
observed with the highly homologous Oct-2 POU domain (198) (Fig. 2B).
Selective residues, particularly a glutamic acid residue (E22) located
on the surface of the second helix of the POU-H domain, are critical
for the recognition by VP16 (199, 200). In the Oct-2 POU-H domain, the
analogous residue is an alanine, thus precluding interaction with VP16.
In addition, the interaction between Oct-1 and VP16 requires the 3'-end
GARAT of the TAATGARAT motif to be intact for ternary complex formation
(89, 90) (Fig. 2B). VP16 alters gene regulation by Oct-1 in two
distinct ways. First, it provides a transactivation domain that is
highly active on mRNA promoters where the Oct-1 transactivation domain
is inactive, and second, it stabilizes the Oct-1-binding to
VP16-responsive elements (184).
c. Interactions with basal transcription factors.
SNAPc (snRNA
activating protein complex; also referred to as PTF) is a basal
transcription factor, composed of several well defined peptides, that
binds to promoter elements called the proximal sequence element (PSEs)
in both RNA Pol II and Pol III snRNA promoters (84, 201, 202). Oct-1
binds to the enhancer or distal sequence elements (DSEs) in snRNA
promoters, which leads to cooperative binding with SNAPc via a
protein-protein interaction involving the POU-S domain of Oct-1,
especially residue E7, and the C-terminal region of SNAP190, a major
component of the SNAPc (203). The result is an enhanced transcriptional
activation by Oct-1-mediated recruitment of SNAPc to the PSE of snRNA
genes (204). The carboxy terminus of SNAP190 acts as an inhibitor of
SNAPc DNA binding, but the interaction with the Oct-1 POU domain
relieves the inhibition and promotes cooperative binding; SNAPc binds
with 8- to 10-fold higher affinity when Oct-1 is bound to the DSE (205, 206).
The POU domains of Oct-1 and Oct-2 have been shown to interact with the TATA binding protein TBP (207). Since this interaction was in solution and not on DNA, its significance is somewhat unclear. However, this finding is consistent with the observation that Oct-1 and Oct-2 can facilitate preinitiation complex formation (208). In addition, the binding of Oct-1 and Oct-2 to the lipoprotein lipase promoter octamer site was shown to be stimulated by transcription factor II B (TFIIB), apparently via protein-protein interactions (209).
A yeast two-hybrid screen with the Oct-1 POU domain identified MAT1 as a POU domain-binding protein capable of binding to Oct-1, Oct-2, and Oct-3 POU domains. MAT1 is required for the assembly of cyclin-dependent kinase (CDK)-activating kinase (CAK), which is functionally associated with the general transcription factor IIH (TFIIH). The interaction between POU domains and MAT1 can target CAK to octamer binding factors and promote their phosphorylation. This interaction may play a role in the recruitment of TFIIH to the preinitiation complex (210).
d. Interactions with replication factors.
Oct-1 is involved in
adenovirus replication where it binds to the highly conserved core
origin and interacts via its POU domain with one of the three viral
proteins required for replication, the precursor terminal protein (pTP)
(122, 211, 212). The interaction seems to involve several regions of
the pTP protein (213). The POU-H domain binds to the pTP-DNA polymerase
complex (213), but more weakly than the intact POU domain. No binding
is observed with the POU-S domain alone (214). Presumably, this
interaction facilitates the recruitment of the pTP-DNA polymerase
complex to the origin of replication. The stimulation of adenovirus DNA
replication seems to depend on a property of the POU domain that is
common to many POU domains because other POU domain factors, including
Pit-1 and Tst-1, can also stimulate DNA replication (215).
3. Class III POU interactions.
a. Interactions with DNA-binding proteins.
Based on
far-Western and mammalian two-hybrid experiments, all class III POU
domain factors can interact with each other via the POU homeodomain
(216). Consistent with this observation, Brn-2 exhibits highly
cooperative homodimerization on a consensus nonoctamer binding site
(103).
Sox/HMG proteins are transcription factors that contain Sry boxes that mediate binding to the minor groove of DNA. Such factors may frequently interact with POU domain factors that bind to the major groove of DNA. Tst-1 (Oct-6) can interact with HMG I(Y) in solution, which facilitates Tst-1-binding to an AT-rich site in the JC viral regulatory region (217). Tst-1 can also interact with the related HMG2 (218). Both Tst-1 and Brn-1 can cooperate with different Sox proteins in transcriptional activation. Specifically, Brn-1 can synergize with Sox-11 when both proteins are bound to adjacent elements; the synergism depends on the transactivation domains in both proteins. Such POU-Sox interactions may be important for a combinatorial code required for cell-specific transcriptional activation (219, 220).
b. Interactions with coregulator proteins.
Tst-1 can interact
with the large T antigen from the human papovavirus JC virus, and this
interaction seems to lead to transcriptional activation on the early
and late promoters from the JC virus (101, 221, 222). The
amino-terminal 82 residues of the large T antigen and the POU domain of
Tst-1 mediate this interaction, but for a functional effect on
transcription, the N-terminal transactivation of Tst-1 is also
required. This functional effect is specific for Tst-1, because while
Brn-1 is also capable of interacting with large T antigen, this
interaction is not transcriptionally productive, apparently because of
differences in the transactivation domains of Brn-1 and Tst-1 (223).
4. Oct-3 interactions.
a. Interactions with DNA-binding proteins.
Oct-3 can interact
with the HMG-box protein Sox2 (224), and these two factors have been
implicated in synergistic regulation of the fibroblast growth factor 4
(FGF-4) gene (49) and the embryonic stem cell coactivator gene UTF-1
(112). The synergism between Oct-3 and Sox2 on the FGF-4 promoter seems
to be due to cooperative binding, which can be disrupted by increasing
the distance between the two binding sites (109). In contrast, Sox2
functionally antagonizes Oct-3 on the ostepontin gene-regulatory region
where its binding site is adjacent to that of Oct-3 (111). Oct-3 and
Sox2 interact directly in solution, with the interaction interface
being the POU domain and the Sox2 HMG domain (109).
b. Interactions with co-regulator proteins.
In embryonic stem
cells Oct-3 transactivation is independent of the distance of the
octamer site from the promoter, but in differentiated cells, Oct-3
transactivation from a distance seems to require stem cell-specific
coactivators that bridge the remotely bound Oct-3 to the basal
transcriptional machinery (225). While these factors remain to be
identified, it is known that transactivation by Oct-3 is strongly
stimulated by E1A in 293 and HeLa cells and depends on both the intact
POU domain and the transactivation domain of Oct-3. This effect is
specific because it is not found with Oct-1 and minimally with Oct-2.
Direct binding of Oct-3 to E1A was also demonstrated (225). The Oct-3
POU domain can associate with two distinct domains on the E1A protein,
CR1 and CR2, and it can also associate with the HPV E7 protein (226).
These data suggest that viral oncoproteins have characteristics that
mimic a stem cell-specific activity (226).
5. Conclusions.
From studies on the protein-protein
interaction characteristics of POU domain factors several general
conclusions can be drawn. First, most factors that interact with POU
domain proteins have been found to interact with the POU domain rather
than other parts of the proteins. This finding may be due, in part, to
technical difficulties in identifying factors that interact with the
transactivation domains outside the POU domain. However, these results
indicate that the POU domain is not only a DNA-binding domain but also
an important interface for regulatory proteins. Second, POU domain
factors seem fairly promiscuous as to their ability to interact and
synergize with diverse DNA-binding proteins bound to the same promoter.
Most POU domain factors have been shown to interact in such a way with
nuclear hormone receptors and HMG proteins. Third, while the POU domain
is highly conserved, most POU domain interactions with coregulators are
remarkably selective; apparently proteins are able to discern small
differences in POU domain sequence (Fig. 2B). Thus, the E1A protein
interacts strongly with Oct-3 but not Oct-1 or Oct-2. VP16 interacts
strongly with Oct-1 but not Oct-2, Oct-3, or Tst-1 (Fig. 2B). OCA-B
interacts selectively with Oct-1 and Oct-2, but not with Oct-3 or Tst-1
(Fig. 2B). This selectivity for protein-protein interactions by POU
domains is in stark contrast to the lack of selectivity in DNA-binding
preferences. Fourth, the association of a coactivator with a POU domain
factor may be stabilized by a simultaneous direct interaction of the
coactivator with DNA, such as that found with Oct-1/2 and OCA-B (Fig. 2B). Fifth, some POU factor-coactivator complexes can only form on
selective DNA sites, perhaps in part due to direct DNA contacts of
coactivators or due to DNA-induced conformational changes of the POU
domain as has been described with OCA-B/Oct-1 and VP16/Oct-1
interactions (Fig. 2B). For example, the Oct-1/2-OCA-B interaction
requires an "A" in position 5 of an octamer site
(ATGCAAAT) (184), and the Oct-1-VP16 interaction requires
the GARAT part of a TAATGARAT site for ternary complex
formation (90). Sixth, additional functional specificity is provided by
sequences outside the POU domain, such that two distinct POU domain
factors may be capable of physical interaction with a coregulator, but
only one of the interactions will result in transcriptional activation,
apparently because of specific sequences in the transactivation domain.
Finally, while some protein-protein interactions involve only one of
the subdomains of the POU domain, e.g., the interactions of
VP16 with the POU-H domain and SNAPc with the POU-S domain, others such
as OCA-B interact simultaneously with the POU-S and the POU-H domains.
D. Mechanisms of transactivation
1. Transactivation domains.
While the N- and C-terminal
domains in POU domain factors are thought to act as transactivation
domains, little is known about their structures. These domains, which
frequently contain polymeric amino acid stretches, are poorly conserved
between different POU domain factors, consistent with the idea that
they confer specificity to the action of different factors, often
binding to the same DNA element. The N terminus is serine/threonine
rich in Pit-1, alanine/glycine rich in Tst-1, proline rich in Oct-3,
and glutamine rich in Brn-2. The N- and C-terminal transactivation
domains of Oct-2 have been most extensively characterized (227, 228, 229, 230).
The N terminus contains an 18-amino acid glutamine-rich motif, and the
C terminus contains a proline-rich domain (231). The function of these
motifs is enhanced when they are combined or reiterated independently
(229, 232), suggesting that they have a modular structure. The Oct-1
transactivation domain appears to be fairly specific for snRNA
promoters and has less effect on mRNA transcription, in contrast to the
Oct-2 transactivation domains that are active on mRNA promoters (184, 228).
Studies on Oct-3 suggest that transactivation domains may work in a cell-specific manner (233). The N-terminal sequence of Oct-3 fused to the GAL4 DNA-binding domain can transactivate in all cell types tested, whereas the C-terminal sequence shows cell-type specificity in the same assay. This cell type specificity of the C-terminal domain seems to be imposed by the Oct-3 POU domain because it functioned in all cell types tested when the C terminus was fused to Pit-1 POU domain. It is further possible that the transactivation specificity is regulated by phosphorylation as the variation in transactivating ability of the C-terminal domain correlates with differences in the phosphorylation status of Oct-3.
Studies on Pit-1 have defined additional specificity conferred by the N-terminal transactivation domain (234). Thus, synergistic activation by Pit-1 and the estrogen receptor on a PRL reporter construct requires an N-terminal 25-amino acid domain of Pit-1 that is not required for analogous synergism on the GH promoter. This synergy, which depends on two of three tyrosine residues, spaced by six amino acids, is preferentially used on sites where Pit-1 binds as a monomer instead of a dimer. These data suggest that the DNA-binding site, dictating whether Pit-1 binds in dimeric or monomeric form, can alter the segment of the N terminus used as a synergy domain.
The N terminus of Brn-2 contains a glutamine-rich region that is mainly encoded by CAG residues (36). Similar nucleotide triplets that can be expanded are found in disease genes causing fragile-X syndrome and myotonic dystrophy (235). It is unknown whether the same mutational mechanism might involve the Brn-2 gene. A polyglutamine tract-binding protein, PQBP-1, capable of binding to the polyglutamine tract of Brn-2 and other such tracts of triplet repeat disease, has been identified (236). While the function of this novel protein is unknown, it is known to be localized in the nucleus and capable of interfering with transactivation by Brn-2.
The N-terminal sequences of POU factors can also mediate repression, including regions from the N terminus of Oct-2 (237, 238, 239, 240, 241, 242). Oct-3 binds to the promoter of Rex-1, which encodes an acidic zinc finger protein expressed at high levels in embryonic stem cells (108). Depending on the cellular environment, Oct-3 can either activate or repress the promoter, with distinct domains within the N terminus being responsible for repression and activation.
2. Transactivation and chromatin.
Gene expression is
influenced by chromatin structure (243). Histones are regulated by
acetylation of lysines in their N-terminal tails, allowing changes in
histone conformation generally associated with increased access of
DNA-binding proteins to promoters. In contrast, histone deacetylation
is coupled to transcriptionally silent chromosomal domains. Many
transcriptional coactivators possess intrinsic histone acetylation
activity that is targeted to specific genes through interactions with
DNA-binding proteins. Repressors, on the other hand, can recruit
deacetylating enzymes to promoters, either directly or via the
corepressor mSin3 (244, 245).
N-CoR, originally identified as a nuclear receptor corepressor (246),
has been reported to bind to the homeodomain of Pit-1 and actively
suppresses transactivation by Pit-1. This suppression is dependent on
mSin3, SAP30, and histone deacetylase (153, 247). N-CoR may be limiting
under certain conditions, and its binding to other transcription
factors, such as nuclear hormone receptors, may indirectly activate
transcription by Pit-1. The Pit-1 POU domain also associates with the
coactivator complex consisting of CBP/p300 and P/CAF, both of which
possess histone acetylase activity (152, 153) (Fig. 3). Thus, the
transcriptional activity of Pit-1 may be mediated by the competing
binding of complexes responsible for either acetylation or
deacetylation, resulting in activation or repression (Fig. 3).
3. Conclusion.
Ultimately, it is likely that POU domain
factors mediate their transcriptional activation by a combinatorial
mechanism involving the N- and C-terminally located transactivation
domains and coactivators/corepressors that interact with the POU domain
(Fig. 3). Since the involved coregulators participate in gene
activation by many different transactivators, it is likely that the
unique transactivation domains of POU domain factors provide
specificity to the activation (228).
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III. Development of the Hypothalamo- Pituitary Region |
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) (139, 248, 249, 250): 1) the anterior lobe
with five major hormone-producing cell types that respond to
neuropeptides released from hypothalamic nuclei carried to the anterior
pituitary gland via the portal circulatory system; 2) the posterior
lobe containing specialized astroglia referred to as pituicytes, and
magnocellular neuronal axons projecting from hypothalamic nuclei,
releasing vasopressin and oxytocin (OT); and 3) the intermediate lobe
containing melanotropes and endorphin- secreting cells (Fig. 4).
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Most of the hypothalamus appears to arise from the neuroepithelium ventral to the hypothalamic sulcus while the preoptic region arises rostral to the optic sulcus (256). During development of the hypothalamus, between embryonic days 11 and 18 (e11 and e18) in the rat, periventricular neurosecretory cells become organized into several nuclei. These include the paraventricular (PVH), supraoptic (SO), arcuate, and periventricular nuclei (PVN). Development of the hypothalamus appears to progress in an outside-in gradient with the late-arising neurosecretory cell types occupying the most medial or periventricular zone of the hypothalamus. For descriptive purposes, four rostrocaudal levels of the hypothalamus can be described: preoptic, anterior, tuberal, and mammillary.
Within the hypothalamus, two distinct neurosecretory systems are
organized (Fig. 5) (256). One system is
composed of magnocellular neurons that project axons directly into the
posterior lobe of the pituitary gland (Fig. 5). The magnocellular
neuronal system includes neurons of the PVH and SON that synthesize OT
and vasopressin (VP) and release these peptides into the posterior lobe
of the pituitary gland. The second system is composed of parvocellular
neurons that synthesize neuropeptides released into the pituitary
portal circulation for regulation of the anterior pituitary gland (Fig. 5). In addition to the magnocellular neurons, the PVH also harbors
parvocellular neurons that synthesize, among other neuropeptides, CRH
and TRH for regulating the ACTH/adrenal axis and the TSH/thyroid axis,
respectively. In the anterior periventricular and arcuate nuclei are
parvocellular neurons that provide dopaminergic control of lactotrophs,
and somatostatin/GRH control of somatotrophs. GnRH neurons originate in
the olfactory placode and migrate during embryonic development to the
hypothalamus (257). GnRH is synthesized in scattered neurons throughout
the hypothalamus, especially in preoptic hypothalamic neurons. It is
involved in the initiation of puberty and regulation of FSH and LH
synthesis in the anterior pituitary gland and thereby regulates
reproduction.
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). When this oral ectoderm makes contact
with the overlying neuroectoderm, around e9 in the mouse, a cascade of
signaling events is initiated that ultimately lead to the generation of
the anterior and intermediate lobes of the pituitary gland containing
six distinct hormone-producing cells by e17.5 (139, 258). These cell
types are corticotropes secreting ACTH, melanotropes secreting MSH,
gonadotropes secreting LH and FSH, thyrotropes secreting TSH,
somatotropes secreting GH, and lactotropes secreting PRL.
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). Thyrotropes and gonadotropes are most
ventral and somatotropes and lactotropes reside in a dorsal position
(139, 258) (Fig. 6). During this stage of pituitary organogenesis, at
e15, corticotropes are distributed throughout the anterior pituitary
gland (258, 259, 260). The spatial restricted organization of thyrotropes,
gonadotropes, somatotropes, and lactotropes may be regulated by
gradients of signaling molecules, including sonic hedgehog, bone
morphogenic proteins (BMPs), FGFs, and Wnts, which in turn create
overlapping spatial gradients of transcription factors (Fig. 6). Thus,
the transcription factors Nkx3.1, Six3, and Prop1 are expressed
predominantly dorsally; Isl1, Brn-4, P-Frk, SF-1, and GATA-2 are
expressed predominantly ventrally; and Pit-1 is expressed in an
intermediary location, the site where lactotropes and somatotropes
initially appear (248, 249). BMP4 signaling from the ventral diencephalon seems to be important for the early formation of the Rathke’s pouch and seems to be linked to the induction of the transcription factor Isl1 (261). Later, FGF8 released from the ventral diencephalon and BMP2 released from mesenchymal cells ventral to the pouch and within the pouch seem to be important for patterning and cell proliferation (261, 262, 263). Embryological studies in the rat suggest that the programs required for specification of all hormone-producing cell types is completed as Rathke’s pouch closes (around e12 in the mouse).
Concurrent with the appearance of the anterior and intermediate lobes
of the pituitary gland, formed from the ventral and dorsal aspects of
Rathke’s pouch, respectively, the posterior gland develops from neural
ectoderm (Fig. 6).
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