The pan-Pituitary Activator of Transcription, Ptx1 (Pituitary Homeobox 1), Acts in Synergy with SF-1 and Pit1 and Is an Upstream Regulator of the Lim-Homeodomain Gene Lim3/Lhx3
Jacques J. Tremblay,
Christian Lanctôt and
Jacques Drouin
Laboratoire de Génétique Moléculaire Institut
de Recherches Cliniques de Montréal Montréal
Québec Canada H2W 1R7
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ABSTRACT
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The Ptx1 (pituitary homeobox 1) homeobox
transcription factor was isolated as a transcription factor of the
pituitary POMC gene. In corticotrope cells that express POMC,
cell-specific transcription is conferred in part by the synergistic
action of Ptx1 with the basic helix-loop-helix factor NeuroD1. Since
Ptx1 expression precedes pituitary development and differentiation, we
investigated its expression and function in other pituitary lineages.
Ptx1 is expressed in most pituitary-derived cell lines and as is the
related Ptx2 (Rieger) gene. However, Ptx1 appears to be the only Ptx
protein in corticotropes and the predominant one in gonadotrope cells.
Most pituitary hormone-coding gene promoters are activated by Ptx1.
Thus, Ptx1 appears to be a general regulator of pituitary-specific
transcription. In addition, Ptx1 action is synergized by
cell-restricted transcription factors to confer promoter-specific
expression. Indeed, in the somatolactotrope lineage, synergism between
Ptx1 and Pit1 is observed on the PRL promoter, and strong synergism
between Ptx1 and SF-1 is observed in gonadotrope cells on the ßLH
promoter but not on the
GSU (glycoprotein hormone
-subunit gene)
and ßFSH promoters. Synergism between these two classes of factors is
reminiscent of the interaction between the products of the
Drosophila genes Ftz (fushi tarazu) and Ftz-F1. Antisense
RNA experiments performed in
T31 cells that express the
GSU
gene showed that expression of endogenous
GSU is highly dependent on
Ptx1 whereas many other genes are not affected. Interestingly, the only
other gene found to be highly dependent on Ptx1 for expression was the
gene for the Lim3/Lhx3 transcription factor. Thus, these experiments
place Ptx1 upstream of Lim3/Lhx3 in a cascade of regulators that appear
to work in a combinatorial code to direct pituitary-, lineage-, and
promoter-specific transcription.
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INTRODUCTION
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The pituitary gland has been a fruitful model with which to
identify factors involved in cell-specific transcription and in the
regulation of cell fate during development. The mature pituitary is
composed of six different cell types, five in the anterior lobe and one
in the intermediate lobe, that arise sequentially during development
and are easily distinguishable by the hormone they secrete (1). The
glycoprotein hormone
-subunit gene (
GSU) is the first hormone
subunit to be expressed in the developing mouse pituitary on embryonic
day 11.5 (e11.5), followed by anterior pituitary POMC on e12, TSH on
e14, intermediate lobe POMC for MSH synthesis on e14.5, GH and PRL on
e15.5, LH on e16.5, and FSH on e17.5 (2, 3, 4, 5).
During early development, the pituitary anlage, Rathkes pouch,
develops from a placode of the stomodeum, which itself is derived from
the cephalic ectoderm of the anterior neural ridge (6, 7). Rathkes
pouch is first identified in mouse at e8.5 as an invagination of the
oral epithelium that is in contact with the floor of the diencephalon
(8). The posterior lobe of the pituitary arises simultaneously from a
downward evagination of the diencephalic neuroectoderm, the
infundibulum (8). Contact between Rathkes pouch and the ventral
diencephalon is crucial for further pituitary development (9, 10, 11, 12). For
example, the TTF-1 (Nkx2.1,T/ebp) gene is essential for pituitary
development although not expressed in Rathkes pouch (12). Rather,
TTF-1 is expressed in the neuroepithelium that will later give rise to
the hypothalamus and to the infundibulum (12). Mice lacking this gene
not only fail to develop the posterior lobe but also the anterior and
intermediate pituitary lobes (12), confirming the importance of the
interaction between diencephalon and Rathkes pouch for proper
pituitary development. Around e12.5, Rathkes pouch pinches off from
the oral ectoderm, and intense cell proliferation (e12.5-e14) triggers
the formation of the anterior pituitary gland (8, 13).
The factors involved in the early events of pituitary development are
just beginning to be identified. We previously cloned a homeoprotein,
Ptx1 (pituitary homeobox 1), through its ability to bind and activate
the POMC gene (14). Ptx1 expression precedes Rathkes pouch formation
as it is expressed in the stomodeum from its first appearance (15) and
later maintained in all stomodeal derivatives, including Rathkes
pouch and the pituitary. Another recently reported Ptx family member,
Ptx2 (Rieg) (16, 17, 18) is also expressed at this early stage of pituitary
development (18). Thus, Ptx1 and Ptx2 represent the earliest known
genetic markers for pituitary development.
The homeobox gene Rpx (Hesx1) is transiently expressed in the
developing pituitary from e9 to e14.5 (19, 20). The precise function of
Rpx remains unknown, since no target gene has yet been identified (19).
However, Rpx can heterodimerize with Prop-1 (see below) and thus
interfere with Prop-1-dependent activation of the Pit1 gene (21).
Transgenic mice continuously expressing Rpx have hypoplasic pituitaries
suggesting that the extinction of Rpx is essential for proper pituitary
development (K. Mahon, personal communication). Lim3/Lhx3, a
lim-homeodomain protein, which is expressed from e9.5 onward (22, 23)
has recently been shown to be required for normal pituitary development
since targeted ablation of its gene results in blockade of cell
proliferation or survival at the Rathkes pouch stage and prevents
subsequent lineage specification (24). In these animals, Rpx gene
expression is prematurely decreased implying that Lim3/Lhx3 is required
for maintenance of Rpx gene expression (24). The Lim3/Lhx3
transcription factor may also take part in expression of pituitary
hormone-coding genes (22).
Prop-1 is a recently identified homeoprotein that is transiently
expressed during pituitary development (e1010.5 to e14.5) where it
stimulates the Pit1 gene, a member of the POU family of transcription
factors (21). Insufficient Pit1 gene expression, caused by a mutation
in the Prop-1 gene, is responsible for the Ames dwarf phenotype in
which there is severe depletion of three Pit1-dependent lineages: the
somatotropes, lactotropes, and thyrotropes (25). Moreover, Prop-1 seems
to be required for extinction of the Rpx gene since Rpx expression
persists through e18.5 in Prop-1-deficient mice (21, 26). Pit1 is first
detected at e14 in the developing mouse pituitary (27). As indicated
above, it is required for differentiation and maintenance of
thyrotrope, somatotrope, and lactotrope cell lineages (28, 29, 30). Pit1 is
an important transcription factor required for the expression of the
GH, ßTSH, and PRL genes, and it also activates its own expression
(29, 31, 32, 33, 34).
In the present study, we have defined the role of Ptx1 in
pituitary-specific transcription and its position in the regulatory
cascade of genes that direct pituitary development. Indeed, we show
that Ptx1 is essential for expression of the
GSU and Lim3/Lhx3
genes, thus identifying Ptx1 as the earliest regulator of pituitary
transcription.
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RESULTS
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Ptx1 Is Expressed in All Pituitary Cell Types
We showed previously that Ptx1 is expressed in corticotrope cells
of the pituitary where it activates the POMC gene. In situ
hybridization analysis had suggested that Ptx1 mRNA was also present in
other pituitary cell lineages (14). To investigate the expression
pattern of Ptx1, we performed Northern blot analysis on RNA obtained
from a panel of cell lines. As shown in Fig. 1
, a single Ptx1 RNA band of about 2.5 kb
was revealed in AtT-20 cells, a corticotrope cell line that expresses
POMC and from which we had cloned Ptx1 (14). Ptx1 mRNA was also
detected in several pituitary-derived cell lines including
T31
(gonadotrope precursor),
TSH (thyrotrope precursor), GHFT1.5
(somatolactotrope precursor), and GH3 and
GH4C1 (somatolactotropes), as well as in the
thyrotrope tumor TtT-97 and in adult mouse pituitary. Interestingly,
Ptx1 mRNA levels were much higher in
T31,
TSH, and GHFT1.5 than
in AtT-20 cells. These results confirm the presence of Ptx1 mRNA in
cells other than corticotropes.

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Figure 1. Ptx1 and Ptx2, but Not Ptx3, Are Expressed in
Several Pituitary-Derived Cell Lines
Northern blot analysis of 20 µg total RNA from multiple
pituitary-derived cell lines and mouse pituitary was used to determine
the expression pattern of the three Ptx family members. The blots were
subsequently probed with an 18S ribosomal RNA probe to ensure integrity
and loading of the RNA. Note the difference in exposure time for the
three blots.
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Two Ptx1-related cDNAs have been identified recently: they are Ptx2
(RIEG) (16, 17, 18), hereafter referred to as Ptx2, and Ptx3 (35). Ptx2 was
shown to be expressed in the pituitary by RT-PCR and in situ
hybridization (16, 18). However, no data are presently available
concerning its expression relative to Ptx1. Thus, we compared the
expression of Ptx1, 2, and 3 in pituitary-derived cell lines by
Northern blot analysis using gene-specific probes. As shown in Fig. 1
, the Ptx1 and Ptx2 genes are abundantly transcribed in pituitary cells
whereas Ptx3 is not. In some cell lines, such as AtT-20,
T31, and
GHFT1.5, Ptx1 is the major mRNA species, whereas in others such as GC
and MMQ, Ptx2 mRNA predominates (Fig. 1
).
The presence of Ptx1 mRNA does not necessarily imply synthesis of Ptx1
protein, as Pit1 mRNA, for example, is detectable in more pituitary
cells than those that express the protein (3, 27). To determine whether
Ptx1 protein was present in all pituitary-derived cell lines, we
performed Western blot analysis with a specific antiserum raised
against Ptx1 amino acids 2456. As shown in Fig. 2
, all cell lines tested, as well as the
adult mouse pituitary, contain Ptx1 protein. Overall, there is a good
correlation between the level of mRNA and amount of protein (Figs. 1
and 2
), although some discrepancies are noted and discussed below.

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Figure 2. The Ptx1 Protein Is Present in Several
Pituitary-Derived Cell Lines
The level of Ptx1 protein was assessed by Western blot analysis.
Aliquots of 40 µg whole cell extracts (overexpressing cells, lanes 1
and 2) or 80 µg nuclear extracts (cell lines and adult mouse
pituitary) were subjected to immunoblotting using a Ptx1-specific
antiserum as described in Materials and Methods. Protein
molecular size standards are indicated on the left.
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Ptx1 Activates Several Pituitary-Specific Promoters
We previously showed that Ptx1 is an important determinant for
expression of the POMC gene in AtT-20 cells (14). Ptx1 may play a
similar role for other pituitary genes. As shown in Table 1
, several pituitary-specific promoters
or enhancers contain at least one putative Ptx1-binding site. These
sites were identified by comparison to the Ptx1-binding site of the CE3
element of the POMC promoter and by comparison to binding studies with
bicoid-related homeoproteins (36, 37, 38). To test the ability
of Ptx1 to activate pituitary-specific promoters, a Ptx1 expression
vector was transfected along with various reporter constructs in CV-1
cells. As shown in Fig. 3
, Ptx1
significantly activates several pituitary promoters, including those
for
GSU and the ß-subunits of LH (ßLH), FSH (ßFSH), and TSH
(ßTSH), GnRH receptor (GnRH-R), GH, the Pit1 enhancer but not its
promoter, as well as the POMC promoter (14). The rous sarcoma virus
(RSV) promoter, which was used as a negative control, was not activated
by Ptx1. Similarly, the thymidine kinase promoter was insensitive to
Ptx1 (data not shown).

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Figure 3. Ptx1 Transactivates Several Pituitary-Specific
Promoters
The effect of Ptx1 was tested on various pituitary promoters including
-1700 bp GSU, -776 bp ßLH, -2400 bp ßFSH, -1200 bp GnRH-R,
-320 bp GH, -422 bp PRL, -220 bp Pit1, -220 bp Pit1+700 bp
enhancer, -6000 bp ßTSH, and -480 bp POMC. Each construct was
cotransfected in CV-1 cells with a control plasmid (empty RSV
expression vector, open bars) or a RSV-Ptx1 expression
(14) vector (solid bars). A viral promoter, RSV, was
used as negative control. Results are shown as fold activation (±
SEM).
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Mapping of Ptx1-Responsive Elements in the
GSU and ßLH
Promoters
In view of the large effect of Ptx1 on the
GSU and ßLH
promoters and of its high expression in
GSU cells (Fig. 2
and
below), we performed deletion analyses on the
GSU as well as on the
ßLH promoters to identify Ptx1-responsive sequences. As shown in
Table 1
, the
GSU and ßLH promoters contain several putative
Ptx1-binding sites. A short (-120 bp)
GSU promoter that contains
only one putative Ptx1-binding site was still activated by Ptx1,
whereas a deletion to -65 bp, which removes this site, was no longer
significantly activated (Fig. 4
). Similar
results were obtained with the ßLH promoter (Fig. 4
) where removal of
the most proximal putative Ptx1-binding site led to a loss of Ptx1
activation (Fig. 4
). Taken together, these data suggest that
transactivation of both the
GSU and ßLH promoters by Ptx1 is
likely to be mediated by the most proximal Ptx1-binding site. These
sites are conserved across many species (Table 1
). We cannot exclude
the possibility that more distal sites may also contribute to promoter
activity in an in vivo context or in association with other
transcription factors.

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Figure 4. Mapping of Ptx1-Responsive Elements in the GSU
and ßLH Promoters
CV-1 cells were cotransfected with various 5'-deletion constructs of
the GSU (left panel) and ßLH (right
panel) promoters with either a control vector (open
bars) or a Ptx1 expression vector (solid bars).
Results are shown as fold activation (± SEM). The sequence
of the putative Ptx1-responsive element is shown under the
graph for each promoter.
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Ptx1 Acts in Synergy with SF-1 and Pit1
To define the role of Ptx1 in lineage- and/or promoter-specific
expression, we tested its ability to stimulate promoter activity in
synergy with cell-type restricted transcription factors. Consistent
with this hypothesis, we have shown that Ptx1 specifically synergizes
with basic helix-loop-helix (bHLH) heterodimers containing NeuroD1 for
activation of POMC transcription (39).
Previous studies have reported the role of the orphan nuclear receptor
SF-1 in activation of the ßLH promoter (40, 41). In the pituitary,
this nuclear receptor is only expressed in gonadotrope cells (Refs. 42
and 43 and data not shown). As shown in Fig. 5A
, SF-1 and Ptx1 can each individually
activate the ßLH promoter. Coexpression of both factors resulted in a
strong synergistic activation of the ßLH (-776 bp) promoter (Fig. 5A
). The SF-1/Ptx1 synergism was lost when the SF-1-binding site
(located at -120 bp) was deleted from the promoter as in the -104-bp
ßLH promoter construct (Fig. 5A
); the SF-1- and Ptx1-binding sites
are 20 bp apart in the promoter. It was also suggested that SF-1 might
be implicated in expression of the
GSU and ßFSH genes (42, 44) and
Ptx1 can activate both promoters (Fig. 3
). No synergy, however, was
observed between Ptx1 and SF-1 on these two promoters (Fig. 5A
). The
combination of Ptx1, SF-1, and Lim3/Lhx3 did not result in a stronger
synergy on the ßLH promoter (data not shown).

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Figure 5. Ptx1 Can Synergize with SF-1 and Pit1
A, Synergistic transactivation by Ptx1 and SF-1. The effect of Ptx1 and
SF-1 alone or in combination was tested on the -776 bp and -104 bp
ßLH, -2400 bp ßFSH, and -341 bp GSU promoters. The -104 bp
ßLH promoter no longer contains the SF-1-binding site present at
-120 bp (40, 41). B, Ptx1 synergizes with Pit1. Transactivation by
either Ptx1, Pit1, or both was tested on four Pit1-dependent promoters:
-422 bp PRL, -320 bp GH, -6000 bp ßTSH, and the Pit1
promoter/enhancer. Promoter constructs were cotransfected with the
indicated expression plasmids in CV-1 cells. Results are shown as fold
activation (± SEM).
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Ptx1 can also synergistically activate transcription with another
cell-specific factor, Pit1 (45). The PRL promoter, which was only
slightly activated by Ptx1 (Fig. 3
), can be synergistically activated
by Ptx1 and Pit1 (Fig. 5B
). The same was true for the GH promoter
although to a lesser extent (Fig. 5B
). This interaction between Ptx1
and Pit1 was not observed on all Pit1-dependent promoters since the
ßTSH promoter and Pit1 promoter/enhancer were not synergistically
activated by the two factors (Fig. 5B
). Taken together, these
results indicate that Ptx1 exerts promoter-specific effects by
synergism with cell type-restricted transcription factors.
Ptx1 Protein Is Expressed at High Level in
GSU-Expressing
Cells
To correlate Ptx1 expression in cell lines derived from various
pituitary lineages (Figs. 1
and 2
) with normal pituitary cells, we used
double-labeling immunohistochemistry to analyze Ptx1 expression in the
adult pituitary gland. As shown in Fig. 6
, the Ptx1 protein can be detected in
the nuclei of all pituitary cells. The nuclear signal was not detected
with preimmune serum, and it was competed by addition of
maltose-binding protein (MBP)-Ptx1 but not with MBP-ßGal (data not
shown). Interestingly, all cells do not express Ptx1 at the same level,
as was observed previously for Ptx1 mRNA (14). Many strongly positive
cells for Ptx1 were identified as
GSU-expressing cells by
double-labeling immunohistochemistry (Fig. 6
). This result correlates
with expression in pituitary-derived cell lines (Fig. 2
). In
addition, high Ptx1 expression colocalized with
GSU-expressing
cells from the onset of
GSU expression during pituitary development
(C. Lanctôt and J. Drouin, in preparation).
Ptx1 Is the Major Ptx Protein Expressed in
GSU-Expressing
T31 Cells
As shown in the present study (Figs. 2
and 6
),
T31 cells as
well as mouse pituitary
GSU-positive cells contain high levels of
Ptx1 protein. Ptx2 mRNA was also detected in the developing pituitary
and in some pituitary-derived cell lines including
T31 cells (Fig. 1
and Ref.16). We do not yet know whether these cells contain Ptx2
protein. To determine the relative importance of Ptx1 protein in
T31 cells, we performed supershift experiments using nuclear
extracts from
T31 cells. The Ptx1 antibody used in our experiments
(Fig. 2
) is specific for Ptx1 since it did not recognize Ptx2 in
Western blot (Fig. 7A
, lane 3) or in gel
shift assays (Fig. 7B
, lane 4). The Ptx-binding activity present in
T31 nuclear extracts (Fig. 7C
, lane 2) was almost completely
supershifted by saturating amounts of the Ptx1-specific antibody (Fig. 7C
, lane 3). Taken together, these data clearly demonstrate that Ptx1
is by far the most abundant member of the Ptx family in
T31
cells.
Ptx1 Is Essential for
GSU and Lim3/Lhx3 Gene Expression
T31 cells have been considered as a model of
gonadotrope precursors because they express the
GSU and GnRH-R genes
but none of the ß-subunit genes (46). This cell line contains the
highest level of Ptx1 mRNA and protein (Figs. 1
, 2
, and 7
). Moreover,
Ptx1 strongly activated the
GSU promoter (Figs. 4
and 5
). To further
confirm the role of Ptx1 in
GSU gene expression, we generated Ptx1
knockdown cell lines by stably transfecting a Ptx1 antisense RNA
expression vector in
T31 cells. Three independent
neomycin-resistant clones expressing the Ptx1 antisense RNA were
analyzed. As control, clones stably transfected with the same vector
without the Ptx1 cDNA (empty vector) were generated, and one was chosen
as negative control (clone Ctl) along with the wild-type
T31 cells
(WT). In the three Ptx1 antisense clones (8, 9, 13), endogenous
Ptx1 mRNA (Fig. 8A
, upper
panel) and protein as assessed by DNA-binding assay (Fig. 8A
, lower panel) were markedly decreased, whereas other
transcription factors such as Pan1 and Oct1 were not significantly
affected, neither at the mRNA (Fig. 8B
, upper panel) nor at
the protein (DNA binding in electrophoretic mobility shift assay)
levels (Fig. 8B
, lower panel). GATA DNA-binding activity
might be slightly decreased (Fig. 8B
). The fact that Ptx DNA-binding
activity was almost undetectable in the antisense clones (Fig. 8A
) also
suggests that the Ptx2 gene product was not up-regulated in response to
the decrease in Ptx1. These Ptx1 null cell lines were then used to
analyze the level of
GSU gene expression. As shown in Fig. 9A
(top panel), no or little
GSU mRNA was detected in the antisense clones. This clearly
indicates that the endogenous
GSU gene was almost silent in these
cells. Further, the activity of a transfected
GSU-luciferase
reporter is considerably lower in the antisense clones than in control
cells (data not shown). Thus, Ptx1 is essential for
GSU gene
expression.

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Figure 8. Endogenous Ptx1 Activity Is Decreased in Ptx1
Antisense Clones
T31 cells were stably transfected with a Ptx1 antisense RNA
expression vector as described in Materials and Methods.
A, Levels of Ptx1 mRNA (upper panel) and protein
(lower panel) in wild-type T31 cells (WT), one
control clone (Ctl), and three independent antisense clones (8, 9, and
13) were monitored by Northern blot analysis and gel retardation assay,
respectively. B, Other transcription factors are not affected in the
Ptx1 antisense clones. The Northern blot used in panel A was
successively rehybridized with Pan1 and Oct1a probes (upper
panel), and the quality of the nuclear extracts was tested by
gel retardation assay for Oct1- and GATA-binding activity (lower
panel).
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Figure 9. Ptx1 Knockdown Cells Fail to Express the GSU and
Lim3/Lhx3 Genes
A, Northern blot analysis of several genes normally expressed in
T31 cells using RNA from the control clone (Ctl) and three
independent Ptx1-antisense clones (8, 9, and 13). Blots were
successively hybridized with probes for GSU, Lim3/Lhx3, LH-2, SF-1,
GnRH-R, and Six3. A ribosomal 18S RNA probe was used to verify RNA
loading. B, Similar blot hybridized with GSU, Ptx2, Pax6, and 18S
RNA probes.
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Using the Ptx1 knockdown cell lines, we investigated expression
of other genes normally expressed in
T31 cells. The GnRH-R,
another differentiation marker of the gonadotrope lineage, was also
decreased in these cells, although much less so than
GSU. There
might be a very small decrease of Six3 mRNA levels in the antisense
clones compared with the control, but the fold reduction was even less
than for the GnRH-R. The gonadotrope-restricted transcription factor
SF-1 and the lim factor LH-2 were not affected in the Ptx1-antisense
clones (Fig. 9A
). Similarly, neither the low-level Ptx2 expression nor
Pax6 (47) mRNA levels were altered in the antisense clones (Fig. 9B
).
These data do not support any cross-regulation between Ptx1 and Ptx2
gene expression. The Rathkes pouch marker Rpx was not detected in the
antisense or control cells (data not shown). Strikingly, the Lim3/Lhx3
mRNA was basically undetectable in the antisense clones, suggesting
that, as for the
GSU gene, Ptx1 is essential for Lim3/Lhx3 gene
expression. These experiments clearly indicate an essential role of
Ptx1 in control of Lim3/Lhx3 transcription and place Ptx1 upstream of
Lim3/Lhx3 in the regulatory cascade for pituitary development (Fig. 10
).

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Figure 10. Regulatory Factors during Pituitary Development
Summary of expression patterns for transcription factors involved in
pituitary gene expression and development. Top,
Representation of putative cellular intermediates during pituitary
differentiation indicating transcription factors expressed at each
stage. For terminally differentiated cells, factors shown previously or
in the present work to activate transcription synergistically with Ptx1
are shown in bold. Bottom, Timing of
onset and extinction (where appropriate) for pituitary transcription
factors. References for the data are provided in the text.
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DISCUSSION
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The present work supports the model that Ptx1 is a pan-pituitary
regulator of transcription and that it contributes to promoter-specific
transcription by synergism with cell-restricted factors such as
NeuroD1, SF-1, and Pit1. This broad regulatory function is consistent
with the early activation of Ptx1 in the stomodeum, the ectodermal
epithelium from which Rathkes pouch develops. Further, Ptx1 appears
to be an upstream regulator in a cascade of transcription factors
that control pituitary-specific transcription. Indeed, Ptx1 is required
for Lim3/Lhx3 gene expression in at least one pituitary cell model.
This regulatory hierarchy may also operate in vivo during
pituitary organogenesis.
Differential Expression of Ptx1 and Ptx2 in Pituitary Lineages
The Ptx1 and Ptx2 genes have an overlapping pattern of expression
in the stomodeum and in some of its derivatives with differences of
expression in craniofacial mesenchyme (15, 17, 18). Both are also
expressed in the pituitary primordium, Rathkes pouch, and we have
shown in the present work that both are expressed in the adult mouse
pituitary gland as well as in a panel of pituitary-derived cell lines.
Taken together, these lines are representative of many pituitary
lineages captured at specific moments of their differentiation pathway
(48). We have shown that all these cell lines but one, the
POMC-expressing AtT-20 cell, expressed both Ptx1 and Ptx2 mRNA (Figs. 1
and 2
). Although, in general, there is good correlation between Ptx1
mRNA and protein levels, one exception is GHFT1.5 cells in which Ptx1
mRNA levels are similar to those of
T31 cells, whereas protein
levels are much lower (compare Figs. 1
and 2
). Another discrepancy may
exist in
T31 cells that have both Ptx1 and Ptx2 mRNA but the bulk
of Ptx protein appears to be Ptx1 (Fig. 7
). The remainder of the
Ptx-related DNA-binding activity in those cells may be a N-terminal
variant of Ptx1, Ptx1b, that is not recognized by the antiserum used in
these experiments (J. J. Tremblay and J. Drouin, in preparation).
This discrepancy in mRNA and protein levels may be explained by
posttranscriptional regulation. At present, we do not know of any other
Ptx family member expressed in the pituitary and, as shown, the Ptx3
gene (35), which has an almost identical homeodomain, is not expressed
in this tissue (Fig. 1
and data not shown). Thus, Ptx1 appears to be
the only Ptx family member expressed in corticotropes and the
predominant one in gonadotropes.
A pan-Pituitary Regulator of Transcription
As a marker of the stomodeum (15), the most anterior segment of
the body plan, Ptx1 may be recruited as a tissue-specific regulator of
transcription in many epithelial derivatives of the stomodeum, as has
been shown in the present work for the pituitary. This recruitment
would be consistent with a combinatorial model for cell-specific gene
expression (Fig. 10
) in which genes encoding transcription factors are
activated at specific times and places during development to control
organogenesis, cell differentiation, and gene transcription. All the
pituitary-specific promoters found to be activated by Ptx1 in the
present work (Fig. 3
) have putative Ptx1-binding sites (Table 1
) except
the Pit1 gene, which does not have a site in its promoter but does in
its enhancer. Only one Ptx1-binding site appears to be necessary for
transcriptional activation as we have shown for POMC (14), ßTSH
(Table 1
and Fig. 3
), ßLH, and
GSU (Fig. 4
). These sites appear to
bind Ptx1 monomers (14), and their sequence is consistent with the
documented DNA-binding specificity of bicoid-related
homeoproteins (36, 37, 38).
Despite their great conservation in DNA-binding specificity, the
various bicoid-related homeoproteins have different
transcriptional properties (35). The homeobox transcription factors
most closely related to Ptx1 are the Otx1 and Otx2, which are
specifically expressed in the brain (49), but not at all in the
pituitary (Otx2) or at very low levels postnatally (Otx1) (D. Acampora,
S. Mazan, F. Tuorto, V. Avantaggiato, J. J. Tremblay, D. Lazzaro,
A. di Carlo, A. Mariano, P. E. Macchia, V. Macchia, J. Drouin, P.
Brûlet, and A. Simeone, in preparation). In striking contrast to
Ptx1, Otx1 has no effect on POMC, and it does not synergize with SF-1
on the ßLH promoter (data not shown). Thus, in addition to their
complementary expression patterns during development of head
structures, the ability of these homeobox factors to synergize with
specific partners for control of transcription may account for the
specificity of their roles during development.
Promoter-Specific Synergism
While Ptx1 may contribute to mechanisms for
pituitary-specific transcription as we had originally shown for POMC
(14), it is clearly not the sole determinant for lineage-specific
transcription of either POMC or any other pituitary hormone-coding
gene. For this reason, the transcriptional interaction with other
transcription factors for cell-specific activity is of great
significance. Prior work has shown the importance of the bHLH factor
NeuroD1 for corticotrope-specific transcription of POMC (39, 50), thus
defining one partner of Ptx1 in a code for cell- and promoter-specific
control of transcription. Another Ptx1 partner is Pit1, which
specifically acts in synergy with Ptx1 to stimulate PRL gene
expression, and less so on the GH promoter (Fig. 5
and Ref.45).
The current work has extended the model by showing marked synergism
between Ptx1 and SF-1, an orphan nuclear receptor transcription factor
(Fig. 5
). This synergism is specifically exerted on the ßLH promoter
but not on the promoters of other genes specific to the gonadotrope
lineage such as
GSU, ßFSH, or GnRH-R (Fig. 5
and data not shown).
Both ßLH (40, 41) and
GSU (44) promoters contain SF-1-binding
sites. The SF-1-binding site of the ßLH promoter was shown to be
essential for promoter activity (Refs. 40 and 41) and Fig. 5
) but less
data support the role of SF-1 in
GSU promoter activity. The only
supporting data rested on the activity of oligomerized synthetic
GSU
SF-1- binding sites inserted upstream of the thymidine kinase promoter
(44). Inactivation of the SF-1 gene also suggested a predominant role
in ßLH expression. Indeed, both ßLH and ßFSH transcripts were
undetectable in SF-1-/- mice while
GSU transcripts
were only decreased (42), and expression of both ß-subunit genes was
restored by injection of GnRH (51). However, in their discussion, Ikeda
et al. (51) indicate that one third of the
SF-1-/- mice had detectable ßFSH mRNA by in
situ hybridization but never ßLH or GnRH-R, and they suggested
that transcription of ßFSH may be under more indirect SF-1 control
than ßLH. Our observation (Fig. 5
) of SF-1 synergism on the ßLH,
but not on the ßFSH, promoter is entirely consistent with their
hypothesis. It is noteworthy that
T31 cells that express Ptx1 and
SF-1 (Fig. 9
) do not express the ßLH gene (46): it is therefore
likely that other factor(s) are involved in further differentiation of
the gonadotrope lineage and activation or derepression of the ßLH
gene.
The Drosophila homolog of SF-1, Ftz-F1, was recently shown
to interact directly with an homeodomain transcription factor, fushi
tarazu (Ftz) to activate transcription synergistically (52, 53). Our
observations (Fig. 5
) constitute the first example of similar synergism
between a mammalian nuclear receptor and a homeobox factor. The domain
of Ftz that interacts with Ftz-F1 (53) is not conserved in Ptx1 such
that it is not possible, at the molecular level, to extend the
comparison with the synergism between Ptx1 and SF-1. Nonetheless, it
appears that synergism between these classes of transcription factor
may be a conserved mechanism for tissue specificity during
development.
The promoter-specific action of factors that synergize with Ptx1
correlates with their cell-restricted pattern of expression. Indeed,
NeuroD1 appears to be predominantly expressed in corticotrope cells
(39). Pit1 is expressed in GH, PRL, and TSH cells and its synergism
with Ptx1 is observed on the PRL and less so on the GH promoters.
Similarly, SF-1 is specifically expressed in the gonadotrope lineage
(42, 43), and its Ptx1 synergism is restricted to only one promoter
which is specific to this lineage. Taken together, these data are
consistent with a model in which progressive differentiation of the
different pituitary lineages is accomplished by the sequential
activation of regulatory genes during organogenesis (Fig. 10
).
Regulatory Cascade during Pituitary Development
The hierarchy of action of different factors involved in pituitary
development could be inferred from the timing of their initial
expression during development. As summarized in Fig. 10
, Ptx1 appears
to be the earliest factor in this cascade as it is already expressed in
the stomodeum before development of Rathkes pouch (15). It is
followed by Rpx at the early pouch stage (19) and by Lim3/Lhx3 soon
after (22, 23). As Rpx is expressed transiently in the pituitary, it
may be involved in activation of downstream genes but certainly not in
their maintenance (19, 24). In contrast, Ptx1 and Lim3/Lhx3 expression
is maintained throughout development in adult pituitary and in
pituitary-derived cell lines. The availability of these cells has
allowed us to demonstrate a strict dependence on Ptx1 for Lim3/Lhx3
expression (Fig. 9
) in at least one cellular model: this contrasts with
other regulators of the gonadotrope function such as LH-2 and SF-1. If
extrapolated to development in vivo, this dependence would
be consistent with a model in which activation of the Lim3/Lhx3 gene by
Ptx1 is required for differentiation of all pituitary lineages, except
for corticotropes, as indicated in Fig. 10
. The absence of Lim3/Lhx3
expression in AtT-20 cells is consistent with this as is the presence
of POMC-positive cells in the Lim3/Lhx3-/- mice (24).
The results of our knockdown experiments suggest that Ptx1 is essential
for the sustained expression of Lim3/Lhx3 and
GSU (Fig. 9A
).
Although Ptx1 directly activates the
GSU promoter (Figs. 3
and 4
),
we cannot exclude that part of the Ptx1 knockdown effect on
GSU
might also be mediated through depletion of Lim3/Lhx3. Indeed, it was
suggested that Lim3/Lhx3 might stimulate
GSU promoter activity
directly (22), but we have been unable to reproduce this finding (data
not shown). Ptx1, and consequently Lim3/Lhx3, appears to be dispensable
for expression of other gonadotrope marker genes such as SF-1 and,
thus, not all gonadotrope-specific functions require the continued
expression of Ptx1 and/or Lim3/Lhx3.
In summary, we have shown the importance of Ptx1 expression for
the maintenance of cell-specific transcription in two pituitary
lineages that either express Ptx1 exclusively (corticotropes) or
predominantly (gonadotropes). Indeed, we have previously documented the
importance of Ptx1 for POMC expression (14), and in the current work,
we show the importance of Ptx1 for Lim3/Lhx3 and
GSU expression
(Fig. 9
). Thus, Ptx1 may be the most upstream factor in the cascade of
regulators for pituitary gene expression. Its recruitment for
pituitary-specific transcription of most hormone-coding genes is
consistent with this role. Toward the establishment of a lineage- and
promoter-specific code for transcription, Ptx1 synergizes with
cell-restricted factors such as NeuroD1 in corticotropes (POMC) (39),
Pit1 in somatolactotropes (PRL, GH) (Fig. 5
and Ref.45) and with SF-1
in gonadotropes (ßLH, Fig. 5
).
 |
MATERIALS AND METHODS
|
---|
RNA Extraction and Analysis
Total RNA was extracted by the guanidium
thiocyanate-phenol-chloroform method (54) and analyzed by Northern blot
analysis (55). Ten or 20 µg of RNA were separated by
agarose-formaldehyde gel electrophoresis and then transferred to nylon
membrane (Hybond-N, Amersham Canada, Oakville, Ontario, Canada).
Membrane hybridizations with 32P-labeled cDNA probes were
done in 1 M NaCl, 1% SDS, 10% dextran sulfate, and 200
µg/ml of denatured salmon sperm DNA at 65 C. Blots were washed under
stringent conditions: 1x saline sodium citrate, 0.1% SDS for 30 min
at 65 C and 0.1x saline sodium citrate, 0.1% SDS for 30 min at 65 C.
DNA probes were cDNA fragments specific for Ptx1 (14), Six3 (56), Ptx2
(16), Rpx (19), Ptx3 (35), Pax6 (57), Pan1 (58), LH-2 (59), SF-1 (60),
Lim3/Lhx3 (23), GnRH-R (61), Oct1a (62), and
GSU (63). As a loading
control, all Northern blots were stripped and rehybridized with a
32P-labeled oligonucleotide
(5'-ACGGTATCTGATCGTCTTCGAACC-3') specific for 18S ribosomal RNA.
Nuclear Extracts and Gel Retardation Assays
Nuclear microextracts from cell lines used in the present study
were prepared as described previously (50). Ptx1 gel retardation assays
and supershift experiments were performed as outlined by Lamonerie
et al. (14) whereas GATA gel retardation assays were done
according to Grépin et al. (64).
Cell Culture and Transfection Assays
Murine
T31,
TSH, AtT-20, GHFT1.5, MMQ, L, and rat
GH4C1, GH3, GC, and African green
monkey kidney CV-1 cells were grown in DMEM supplemented with 10% FCS.
CV-1 and L cells were transfected by the calcium phosphate method as
described previously (14).
T31 cells were transfected using the
LipofectAMINE Reagent, as described (65). Data are presented as the
means ± SEM of three to eight experiments each
performed in duplicate.
For stably transfected
T31 cells, the evening before transfection,
cells were seeded at 400,000/90-mm petri dish and transfected the next
morning with 10 µg of control vector (pCDNA3, Invitrogen, San Diego,
CA) or antisense Ptx1 vector (containing the full-length Ptx1 cDNA in
reverse orientation) mixed with 600 µl of serum-free DMEM and added
to a solution containing 27 µl of LipofectAMINE Reagent and 600 µl
of serum-free DMEM, incubated for 3045 min, and applied on cells for
5 h before rinsing with FCS-supplemented DMEM. Stable
transfectants were selected 24 h later for resistance to neomycin
(300 µg/ml), and individual clones were picked and subsequently
cultured in medium containing 50 µg/ml neomycin.
Western Blot Analysis
Thirty microgram whole cell extracts from transfected CV-1 cells
and 60 µg nuclear extracts from AtT-20,
T31,
TSH, GHFT1.5,
GH3, GH4C1 cells and adult mouse
pituitary were denatured before electrophoresis by boiling the samples
for 3 min in loading buffer containing 1% SDS, 1%
ß-mercaptoethanol, and 100 mM dithiothreitol. Samples
were loaded on denaturing 12% polyacrylamide gel containing 0,1% SDS.
The gel was migrated at 200 V for 75 min at room temperature using the
Bio-Rad Mini-Protean II electrophoresis apparatus (Bio-Rad, Richmond,
CA). Proteins were transferred to polyvinylidene fluoride membranes
(Amersham Canada) by electroblotting at 100 mA for 2 h at 4 C in
transfer buffer [25 mM Tris-HCl, 192 mM
glycine, 20% methanol (vol/vol), pH 8.4] using the Bio-Rad Mini
Trans-Blot apparatus. Polyvinylidene fluoride membranes were blocked
for 16 h at 4 C and then for 30 min at room temperature in 20
mM Tris-HCl, 0.9% NaCl (wt/vol) (TBS) and 15% powdered
milk (wt/vol). Membranes were incubated in TBS containing 0.2%
Tween-20 (vol/vol) (TBST) and 5% powdered milk (wt/vol) and a 1:20
dilution of an affinity-purified Ptx1-specific antiserum for 90 min at
RT. The rabbit antiserum was raised against a MBP-Ptx1 fusion protein
containing amino acids 2456 of Ptx1. After the incubation, membranes
were washed three times for 5 min each in TBST at room temperature and
then incubated for 1 h at room temperature in TBST containing a
1:2000 dilution of a biotinylated anti-rabbit IgG (Vector Laboratories,
Burlingame, CA). Meanwhile, an avidin-biotin complex was prepared using
a 1:500 dilution of Avidin-D and a 1:1000 dilution of
biotinylated-horseradish peroxidase (Vector Laboratories) and kept on
ice for 1 h. The membranes were washed as described above and
incubated with the avidin-biotin complex for 1 h at RT. Finally,
the membranes were washed and immune complexes were visualized using
0.8 mM diaminobenzamine as substrate in the presence of 0.3
mM nickel chloride and 0.009% hydrogen peroxide at RT for
10 min (66).
Plasmids and Oligonucleotides
The SF-1 expression vector was generously provided by Dr. Keith
L. Parker. Mouse -6 kb ßTSH-luciferase and
GSU-luciferase (-1.7
kb, -0.48 kb, -0.381 kb, and -0.297 kb) reporter plasmids were
kindly provided by Dr. David F. Gordon.
GSU promoter deletions to
-0.212 kb, -0.113 kb, and -0.065 kb were generated by PCR. Bovine
-0.776 kb ßLH-luciferase was kindly provided by Dr. John Nilson.
Deletion to -0.104 kb ßLH-luciferase was obtained by cutting the
-0.776 kb plasmid with SmaI and religating, and to -0.033
kb by cutting the -0.776 kb plasmid with XhoI and
PstI, blunting both extremities with T4 DNA
polymerase, and religating. Ptx1 expression vector was constructed by
cloning a NcoI-KpnI fragment of Ptx1 cDNA in the
corresponding sites of a RSV-driven expression vector. This vector was
derived from RSV-Luc reporter by replacing the
HindIII-KpnI luciferase fragment by the multiple
cloning site of Bluescript KS- and by changing the pBR322 backbone to
Bluescript SK+ to increase copy number in bacteria.
The Ptx2 cDNA was obtained by RT-PCR from mouse pituitary first-strand
cDNA using forward (5'-TCCTCTAGACGATAACCGGGAATGGAG-3') and reverse
(5'-CAGGATCCTCAGTCTTTCTGGGGCAGA-3') primers and subsequently subcloned
in Bluescript KS- and the RSV expression vector. WT and
mutant (M1) Ptx1 oligonucleotides, as well as DE2A and GATA probes used
in the gel retardation assays, were described previously (14, 64).
Oligonucleotides were synthesized with an Applied Biosystem (Foster
City, CA) synthesizer.
 |
ACKNOWLEDGMENTS
|
---|
We are grateful to Pamela Mellon for her pituitary-derived cell
lines and for the GnRH-R-Luc reporter, to David Gordon (
GSU,
ßTSH), Michael Karin (GH), Kathy Mahon (Pit1 promoter and enhancer),
Richard Maurer (ßFSH), John Nilson (ßLH), and Michael Rosenfeld
(PRL) for their reporter constructs. We also thank Keith Parker and
Michael Karin for the SF-1 and Pit1 expression vectors, respectively.
Pan-1, Oct1, Lim3, Rpx, LH-2, GnRH-R, Pax6, and Six3 probes were
provided by Chris Nelson, Hans Schöler, Nabil Seidah, Kathy
Mahon, Richard Maurer, Kevin Catt, Tom Glaser, and Peter Gruss,
respectively. We thank Michel Chamberland for oligonucleotide
synthesis. The efficient secretarial assistance of Lise Laroche was
much appreciated.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. Jacques Drouin, Institut de Recherches Cliniques de Montréal, 110 des Pins Ouest, Montréal Québec Canada H2W 1R7. e-mail: drouinj@ircm.umontreal.ca.
J. J. Tremblay was recipient of a studentship from the Cancer Research
Society Inc. and C. L. Lanctôt was a Research Student of the
National Cancer Institute of Canada. This work was funded by the
National Cancer Institute of Canada supported with funds provided by
the Canadian Cancer Society.
Received for publication October 2, 1997.
Revision received November 14, 1997.
Accepted for publication December 8, 1997.
 |
REFERENCES
|
---|
-
Voss JW, Rosenfeld MG 1992 Anterior pituitary
development: short tales from dwarf mice. Cell 70:527530[Medline]
-
Elkabes S, Loh YP, Nieburgs A, Wray S 1989 Prenatal
ontogenesis of pro-opiomelanocortin in the mouse central nervous system
and pituitary gland: an in situ hybridization and
immunocytochemical study. Dev Brain Res 46:8595[Medline]
-
Simmons DM, Voss JW, Ingraham HA, Holloway JM, Broide RS,
Rosenfeld MG, Swanson LW 1990 Pituitary cell phenotypes involve
cell-specific Pit-1 mRNA translation and synergistic interactions with
other classes of transcription factors. Genes Dev 4:695711[Abstract]
-
Japon MA, Rubinstein M, Low MJ 1994 In situ
hybridization analysis of anterior pituitary hormone gene expression
during fetal mouse development. J Histochem Cytochem 42:11171125[Abstract/Free Full Text]
-
Jacobson L, Drouin J 1994 Regulation of proopiomelanocortin
gene transcription. In: Imura H (ed) The Pituitary Gland. Raven Press,
Ltd, New York, pp 117138
-
Couly GF, Le Douarin NM 1985 Mapping of the early neural
primordium in quail-chick chimeras. I. Developmental relationships
between placodes, facial ectoderm, and prosencephalon. Dev Biol 110:422439[Medline]
-
Couly GF, Le Douarin NM 1987 Mapping of the early neural
primordium in quail-chick chimeras. II. The prosencephalic neural plate
and neural folds: implications for the genesis of cephalic human
congenital abnormalities. Dev Biol 120:198214[Medline]
-
Schwind J 1928 The development of the hypophysis cerebri of
the albino rat. Am J Anat 41:295319
-
Kawamura K, Kikuyama S 1995 Induction from posterior
hypothalamus is essential for the development of the pituitary
proopiomelacortin (POMC) cells of the toad (Bufo japonicus).
Cell Tissue Res 279:233239[CrossRef][Medline]
-
Le Douarin NM, Ferrand R, Le Douarin G 1967 La
différenciation de lébauche épithéliale de
lhypophyse séparée du plancher encéphalique et
placée dans les mésenchymes hétérologues. C R
Acad Sci 264:30273029
-
Le Douarin NM, Ferrand R, Le Douarin G 1967 Evolution de
lébauche de ladénohypophyse isolée du plancher
encéphalique aux jeunes stades du développement. C R Soc
Biol 161:18071811
-
Kimura S, Hara Y, Pineau T, Fernandez-Salguero P, Fox CH, Ward
JM, Gonzalez FJ 1996 The T/ebp null mouse: thyroid-specific
enhancer-binding protein is essential for the organogenesis of the
thyroid, lung, ventral forebrain, and pituitary. Genes Dev 10:6069[Abstract]
-
Ikeda H, Yoshimoto T 1991 Developmental changes in
proliferative activity of cells of the murine rathkes pouch. Cell
Tissue Res 263:4147[Medline]
-
Lamonerie T, Tremblay JJ, Lanctôt C, Therrien M,
Gauthier Y, Drouin J 1996 PTX1, a bicoid-related homeobox
transcription factor involved in transcription of pro-opiomelanocortin
(POMC) gene. Genes Dev 10:12841295[Abstract]
-
Lanctôt C, Lamolet B, Drouin J 1997 The
bicoid-related homeoprotein Ptx1 defines the most anterior
domain of the embryo and differentiates posterior from anterior lateral
mesoderm. Development 124:28072817[Abstract/Free Full Text]
-
Gage PJ, Camper SA 1997 Pituitary homeobox 2, a novel member
of the bicoid-related family of homeobox genes, is a
potential regulator of anterior structure formation. Hum Mol Genet 6:457464[Abstract/Free Full Text]
-
Semina EV, Reiter R, Leysens NJ, Alward WL, Small KW, Datson
NA, Siegel-Bartelt J, Bierke-Nelson D, Bitoun P, Zabel BU, Carey JC,
Murray JC 1996 Cloning and characterization of a novel bicoid-related
homeobox transcription factor gene, RIEG, involved in Rieger syndrome.
Nature Genet 14:392399[Medline]
-
Muccielli ML, Martinez S, Pattyn A, Goridis C, Brunet JF 1996 Otlx2, an Otx-related homeobox gene expressed in
the pituitary gland and in a restricted pattern in the forebrain. Mol
Cell Neurosci 8:258271[CrossRef][Medline]
-
Hermesz E, Mackem S, Mahon KA 1996 Rpx: a novel
anterior-restricted homeobox gene progressively activated in the
prechordal plate, anterior neural plate and Rathkes pouch of the
mouse embryo. Development 122:4152[Abstract/Free Full Text]
-
Thomas PQ, Johnson BV, Rathjen J, Rathjen PD 1995 Sequence,
genomic organization, and expression of the novel homeobox gene
hesx1. J Biol Chem 270:38693875[Abstract/Free Full Text]
-
Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, OConnell
SM, Gukovsky I, Carriere C, Ryan AK, Miller AP, Zuo L, Gleiberman AS,
Andersen B, Beamer WG, Rosenfeld MG 1996 Pituitary lineage
determination by the Prophet of Pit-1 homeodomain factor defective in
Ames dwarfism. Nature 384:327333[CrossRef][Medline]
-
Bach I, Rhodes SJ, Pearse RV, 2nd, Heinzel T, Gloss B, Scully
KM, Sawchenko PE, Rosenfeld MG 1995 P-lim, a lim homeodomain factor, is
expressed during pituitary organ and cell commitment and synergizes
with pit-1. Proc Natl Acad Sci USA 92:27202724[Abstract]
-
Seidah NG, Barale JC, Marcinkiewicz M, Mattei MG, Day R,
Chretien M 1994 The mouse homeoprotein mlim-3 is expressed early
in cells derived from the neuroepithelium and persists in adult
pituitary. DNA Cell Biol 13:11631180[Medline]
-
Sheng HZ, Zhadanov AB, Mosinger B, Fujii T, Bertuzzi S,
Grinberg A, Lee EJ, Huang SP, Mahon KA, Westphal H 1996 Specification
of pituitary cell lineages by the LIM homeobox gene LHX3. Science 272:10041007[Abstract]
-
Gage PJ, Roller ML, Saunders TL, Scarlett LM, Camper SA 1996 Anterior pituitary cells defective in the cell-autonomous factor, df,
undergo cell lineage specification but not expansion. Development 122:151160[Abstract/Free Full Text]
-
Gage PJ, Brinkmeier ML, Scarlett LM, Knapp LT, Camper SA,
Mahon KA 1996 The ames dwarf gene, df, is required early in pituitary
ontogeny for the extinction of rpx transcription and initiation of
lineage-specific cell proliferation. Mol Endocrinol 10:15701581[Abstract]
-
Dollé P, Castrillo JL, Theill LE, Deerinck T, Ellisman
M, Karin M 1990 Expression of GHF-1 protein in mouse pituitaries
correlates both temporally and spatially with the onset of growth
hormone gene activity. Cell 60:809820[Medline]
-
Bodner M, Castrillo JL, Theill LE, Deerinck T, Ellisman M,
Karin M 1988 The pituitary-specific transcription factor GHF-1 is
a homeobox-containing protein. Cell 55:505518[Medline]
-
Ingraham HA, Chen R, Mangalam HJ, Elsholtz HP, Flynn SE, Lin
CR, Simmons DM, Swanson L, Rosenfeld MG 1988 A tissue-specific
transcription factor containing a homeodomain specifies a pituitary
phenotype. Cell 55:519529[Medline]
-
Li S, Crenshaw EBI, Rawson EJ, Simmons DM, Swanson LW,
Rosenfeld MG 1990 Dwarf locus mutants lacking three pituitary cell
types result from mutations in the POU-domain gene pit-1. Nature 347:528533[CrossRef][Medline]
-
Mangalam HJ, Albert VR, Ingraham HA, Kapiloff M, W Ilson L,
Nelson C, Elsholtz H, Rosenfeld MG 1989 A pituitary POU domain protein,
Pit-1, activates both growth hormone and prolactin promoters
transcriptionally. Genes Dev 3:946958[Abstract]
-
Nelson C, Albert VR, Elsholtz HP, Lu LIW, Rosenfeld MG 1988 Activation of cell-specific expression of rat growth hormone and
prolactin genes by a common transcription factor. Science 239:14001405[Medline]
-
Rhodes SJ, Chen R, DiMattia GE, Scully KM, Kalla KA, Lin SC,
Yu VC, Rosenfeld MG 1993 A tissue-specific enhancer confers
Pit-1-dependent morphogen inducibility and autoregulation on the pit-1
gene. Genes Dev 7:913932[Abstract]
-
Steinfelder HJ, Hauser P, Nakayama Y, Radovick S, McClaskey
JH, Taylor T, Weintraub BD, Wondisford FE 1991 Thyrotropin-releasing
hormone regulation of human TSHB expression: role of a
pituitary-specific transcription factor (Pit-1/GHF-1) and potential
interaction with a thyroid hormone-inhibitory element. Proc Natl Acad
Sci USA 88:31303134[Abstract]
-
Smidt M, van Schaick HSA, Lanctôt C, Tremblay JJ, Cox
JJ, van der Kleij AAM, Wolterink G, Drouin J, Burbach PH 1997 A
homeodomain gene PTX3 has highly restricted brain expression
in mesencephalic dopaminergic neurons. Proc Natl Acad Sci USA 94:1330513310[Abstract/Free Full Text]
-
Wilson DS, Sheng G, Jun S, Desplan C 1996 Conservation and
diversification in homeodomain-dna interactions: a comparative genetic
analysis. Proc Natl Acad Sci USA 93:68866891[Abstract/Free Full Text]
-
Driever W, Nusslein-Volhard C 1989 The bicoid protein is a
positive regulator of hunchback transcription in the early
Drosophila embryo. Nature 337:138143[CrossRef][Medline]
-
Ma X, Yuan D, Diepold K, Scarborough T, Ma J 1996 The
Drosophila morphogenetic protein bicoid binds DNA
cooperatively. Development 122:11951206[Abstract/Free Full Text]
-
Poulin G, Turgeon B, Drouin J 1997 NeuroD1/BETA2 contributes
to cell-specific transcription of the POMC gene. Mol Cell Biol 17:66736682[Abstract]
-
Keri RA, Nilson JH 1996 A steroidogenic factor-1 binding site
is required for activity of the luteinizing hormone beta subunit
promoter in gonadotropes of transgenic mice. J Biol Chem 271:1078210785[Abstract/Free Full Text]
-
Halvorson LM, Kaiser UB, Chin WW 1996 Stimulation of
luteinizing hormone beta gene promoter activity by the orphan nuclear
receptor, steroidogenic factor-1. J Biol Chem 271:664550:2[Abstract/Free Full Text]
-
Ingraham HA, Lala DS, Ikeda Y, Luo X, Shen WH, Nachtigal MW,
Abbud R, Nilson JH, Parker KL 1994 The nuclear receptor steroidogenic
factor 1 acts at multiple levels of the reproductive axis. Genes Dev 8:23022312[Abstract]
-
Asa SL, Bamberger AM, Cao B, Wong M, Parker KL, Ezzat S 1996 The transcription activator steroidogenic factor-1 is preferentially
expressed in the human pituitary gonadotroph. J Clin Endocrinol
Metab 81:21652170[Abstract]
-
Barnhart KM, Mellon PL 1994 The orphan nuclear receptor,
steroidogenic factor-1, regulates the glycoprotein hormone
alpha-subunit gene in pituitary gonadotropes. Mol Endocrinol 8:878885[Abstract]
-
Szeto DP, Ryan AK, OConnell SM, Rosenfeld MG 1996 P-OTX: a
PIT-1-interacting homeodomain factor expressed during anterior
pituitary gland development. Proc Natl Acad Sci USA 93:77067710[Abstract/Free Full Text]
-
Windle JJ, Weiner RI, Mellon PL 1990 Cell lines of the
pituitary gonadotrope lineage derived by targeted oncogenesis in
transgenic mice. Mol Endocrinol 4:597603[Abstract]
-
Walther C, Gruss P 1991 Pax-6, a murine paired box gene, is
expressed in the developing CNS. Development 113:14351449[Abstract]
-
Alarid ET, Windle JJ, Whyte DB, Mellon PL 1996 Immortalization
of pituitary cells at discrete stages of development by directed
oncogenesis in transgenic mice. Development 122:33193329[Abstract/Free Full Text]
-
Simeone A, Acampora D, Gulisano M, Stornaiuolo A, Boncinelli E 1992 Nested expression domains of four homeobox genes in developing
rostral brain. Nature 358:687690[CrossRef][Medline]
-
Therrien M, Drouin J 1993 Cell-specific helix-loop-helix
factor required for pituitary expression of the pro-opiomelanocortin
gene. Mol Cell Biol 13:23422353[Abstract]
-
Ikeda Y, Luo X, Abbud R, Nilson JH, Parker KL 1995 The nuclear
receptor steroidogenic factor 1 is essential for the formation of the
ventromedial hypothalamic nucleus. Mol Endocrinol 9:478486[Abstract]
-
Yu Y, Li W, Su K, Yussa M, Han W, Perrimon N, Pick L 1997 The
nuclear hormone receptor Ftz-F1 is a cofactor for the
Drosophila homeodomain protein Ftz. Nature 385:552555[CrossRef][Medline]
-
Guichet A, Copeland JW, Erdélyl M, Hlousek D,
Zàvorszky P, Ho J, Brown S, Percival-Smith A, Krause HM, Ephrussi
A 1997 The nuclear receptor homologue Ftz-F1 and the homeodomain
protein Ftz are mutually dependent cofactors. Nature 385:548552[CrossRef][Medline]
-
Chomczynski P, Sacchi N 1987 Single-step method of RNA
isolation by acid guanidium thyocyanate-phenol-chloroform-extraction.
Anal Biochem 162:156159[CrossRef][Medline]
-
Maniatis T, Fritsch EF, Sambrook J 1982 Molecular Cloning: A
Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor,
NY
-
Oliver G, Mailhos A, Wehr R, Copeland NG, Jenkins NA, Gruss P 1995 Six3, a murine homologue of the sine oculis gene, demarcates the
most anterior border of the developing neural plate and is expressed
during eye development. Development 121:40454055[Abstract/Free Full Text]
-
Glaser T, Walton DS, Maas RL 1992 Genomic structure,
evolutionary conservation and aniridia mutations in the human pax6
gene. Nature Genet 2:232239[Medline]
-
Nelson C, Shen LP, Meister A, Fodor E, Rutter WJ 1990 Pan: a
transcriptional regulator that binds chymotrypsin, insulin, and AP-4
enhancer motifs. Genes Dev 4:10351043[Abstract]
-
Roberson MS, Schoderbek WE, Tremml G, Maurer RA 1994 Activation of the glycoprotein hormone alpha-subunit promoter by a
LIM-homeodomain transcription factor. Mol Cell Biol 14:29852993[Abstract]
-
Luo X, Ikeda Y, Schlosser DA, Parker KL 1995 Steroidogenic
factor 1 is the essential transcript of the mouse ftz-f1 gene. Mol
Endocrinol 9:12331239[Abstract]
-
Reinhart J, Mertz LM, Catt KJ 1992 Molecular cloning and
expression of cDNA encoding the murine gonadotropin-releasing hormone
receptor. J Biol Chem 267:2128121284[Abstract/Free Full Text]
-
Suzuki N, Peter W, Ciesiolka T, Gruss P, Scholer HR 1993 Mouse
oct-1 contains a composite homeodomain of human oct-1 and oct-2.
Nucleic Acids Res 21:245252[Abstract]
-
Gordon DF, Wood WM, Ridgway EC 1988 Organization and
nucleotide sequence of the mouse alpha-subunit gene of the pituitary
glycoprotein hormones. DNA 7:679690[Medline]
-
Grépin C, Dagnino L, Robitaille L, Haberstroh L, Antakly
T, Nemer M 1994 A hormone-encoding gene identifies a pathway for
cardiac but not skeletal muscle gene transcription. Mol Cell Biol 14:31153129[Abstract]
-
Philips A, Lesage S, Gingras R, Maira MH, Gauthier Y, Hugo P,
Drouin J 1997 Novel dimeric Nur77 signaling mechanisms in endocrine and
lymphoid cells. Mol Cell Biol 17:59465951[Abstract]
-
Straus W 1982 Imidazole increases the sensitivity of the
cytochemical reaction for peroxidase with diaminobenzidine at a
neutral ph. J Histochem Cytochem 30:491493[Medline]