Essential Requirement for Pax6 in Control of Enteroendocrine Proglucagon Gene Transcription
Mary E. Hill,
Sylvia L. Asa and
Daniel J. Drucker
Department of Medicine (M.F.H., D.J.D.) The Banting and Best
Diabetes Centre Toronto General Hospital Toronto, Ontario M5G
2C4, Canada
Department of Pathology (S.L.A.) The
Mount Sinai Hospital University of Toronto Toronto, Ontario M5G
2C4, Canada
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ABSTRACT
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The primary function of islet A cells is the
synthesis and secretion of glucagon, an essential hormonal regulator of
glucose homeostasis. The proglucagon gene is also expressed in
enteroendocrine L cells of the intestinal epithelium, which produce
glucagon-like peptide 1 (GLP-1) and glucagon-like peptide
2 (GLP-2), regulators of insulin secretion and intestinal growth,
respectively. We show here that Pax6, a critical
determinant of islet cell development and proglucagon gene expression
in islet A cells, is also essential for glucagon gene transcription in
the small and large intestine. Pax6 is expressed in
enteroendocrine cells, binds to the G1 and G3 elements in the
proglucagon promoter, and activates proglucagon gene transcription. The
dominant negative Pax6 allele,
SEYNeu, represses proglucagon gene
transcription in enteroendocrine cells. Mice homozygous for the
SEYNeu mutation exhibit markedly reduced
levels of proglucagon mRNA transcripts in the small and large
intestine, and GLP-1 or GLP-2-immunopositive
enteroendocrine cells were not detected in the intestinal mucosa. These
findings implicate an essential role for Pax6 in the
development and function of glucagon-producing cells in both pancreatic
and intestinal endodermal lineages.
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INTRODUCTION
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The islets of Langerhans contain distinct populations of
specialized endocrine cells that synthesize and secrete insulin,
glucagon, and somatostatin, hormones that regulate metabolic processes
and, ultimately, glucose homeostasis. Characterization of the molecular
factors important for islet development and islet hormone gene
expression is of significant interest, in that inadequate development,
dysfunction, or destruction of islet ß-cells produces diabetes
mellitus in affected individuals. Isolation and analysis of insulin
gene transcription factors have provided considerable information about
the control of both insulin gene expression and islet cell development
and differentiation, as many transcription factors play dual roles in
regulating both insulin gene transcription and formation and
organization of differentiated islet cell types in the developing
pancreas (1, 2, 3, 4).
Given the central importance of the ß-cell and its product, insulin,
for glucoregulation, the majority of studies of islet transcription
factors have focused on the insulin gene. One of the first ß-cell
transcription factors to be isolated, isl-1, does not appear
to be essential for control of insulin gene transcription (5); however
isl-1 does activate proglucagon gene transcription, and mice
homozygous for a null isl-1 mutation exhibit defective islet
development and A cell formation (6, 7). In contrast, the
Pdx-1 transcription factor is required for insulin gene
transcription in the adult ß-cell (4) and, remarkably,
Pdx-1-/- mice fail to develop a pancreas (8). Furthermore,
human subjects with heterozygous loss of function mutations in
Pdx-1 develop maturity-onset diabetes and, similarly,
heterozygous Pdx-1+/- mice develop glucose intolerance,
providing important evidence for the central role of homeobox
transcription factors for ß-cell function in vivo (4, 9).
Pdx-1 is a comparatively weak transactivator of insulin gene
transcription and appears to function synergistically with
BETA2/NeuroD, a helix-loop-helix protein that together with E47,
binds to an adjacent site in the insulin gene promoter (3). Remarkably,
BETA2/NeuroD is also essential for both insulin gene transcription
and islet development. Mice with a null mutation in the BETA2/NeuroD
gene exhibit severe abnormalities in islet cell formation and a marked
reduction in the number of ß cells and rapidly develop diabetes and
die in the neonatal period (10). Although isl-1,
Pdx-1, and BETA2/NeuroD were originally isolated as insulin
gene transcription factors, analysis of genetic mutations in
transcription factors not previously implicated in the control of
insulin gene expression has revealed an essential and unexpected role
for specific genes in islet development.
Targeted inactivation of the gene encoding the Nkx2.2 homeodomain
protein results in mice with arrested ß-cell development, leading to
diabetes in the postnatal period (11). Mice with disruption of both
Pax4 alleles fail to develop mature ß- and
-cells and
die within 72 h of birth due to growth retardation and
dehydration. Intriguingly, pax4-/- mice have increased
numbers of islet A cells, consistent with a possible block in islet
cell differentiation (12). In contrast, islets from mice with a
targeted inactivation in the Pax6 gene contain ß- and
-cells but no A cells, suggesting that Pax6 is essential
for the formation of the pancreatic A cell lineage (13). Furthermore,
mice with the small eye SEYNeu Pax6
mutation also exhibit markedly reduced numbers of islet A cells and
decreased levels of pancreatic glucagon (2). Taken together, these
observations emphasize the essential importance of islet transcription
factor genes for development and maturation of mature islet cells
in vivo.
In contrast to the restricted expression of the insulin gene in the
pancreatic ß-cell, the proglucagon gene is expressed in both
pancreatic islets and enteroendocrine cells of the intestine. Although
abnormalities in islet A cell development and proglucagon gene
expression are evident in studies of mice with null mutations in islet
transcription factors (2, 7, 13), considerably less information is
available about the molecular determinants of enteroendocrine cell
development and intestinal proglucagon gene expression in
vivo. The finding that mice with targeted or naturally occurring
Pax6 mutations develop striking abnormalities in the numbers
of islet A cells and decreased pancreatic proglucagon gene expression
prompted us to determine whether Pax6 might also be
important for proglucagon gene expression in the intestine. We report
here that Pax6 is an essential transcriptional regulator of
proglucagon gene expression in enteroendocrine cells of the small and
large bowel.
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RESULTS
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As studies of Pax6 mutant and wild-type mice localized
Pax6 expression to islet cells, including glucagon-producing
A cells (2, 13, 14), we assessed whether Pax-6 is also
expressed in proglucagon-producing intestinal enteroendocrine cells.
Northern blot analysis using total and polyA+ RNA (Fig. 1
) detected Pax6 mRNA
transcripts in RNA from the intestinal GLUTag intestinal endocrine cell
line and, with a longer exposure, InR1-G9 islet cells. After recent
studies demonstrating that Pax6 activates islet proglucagon
gene transcription through the proglucagon gene G3 enhancer element
(2), we initially assessed the Pax6-dependent activation of
the proglucagon gene promoter in BHK fibroblasts. A reporter plasmid
that contains the G1-G3 proximal rat proglucagon promoter-enhancer
elements in their natural genomic orientation (Fig. 2C
), [-476]GLU-Luc, was significantly
activated by cotransfection with wild-type Pax6, but not by
the dominant negative Pax6 mutant allele,
SEYNeu, in BHK fibroblasts (Fig. 2A
).
Remarkably, deletion of both the G3 and G2 enhancer sequences did not
abrogate the Pax6 activation of proglucagon promoter
activity, as both [220]GLU-Luc (lacking G3) and [-168]GLU-Luc
(lacking G3 and G2) were activated by Pax6 (Fig. 2A
).
Furthermore, deletion of the G3, G2, and G4 enhancer elements did not
eliminate Pax6-dependent activation of proglucagon promoter
activity, as a plasmid containing only G1 sequences, [-93]GLU-Luc,
was still activated by Pax6 (Fig. 2A
). These observations,
taken together with the presence of a putative Pax6 binding site at
approximately -90 to -60 of the proximal rat proglucagon gene
promoter (15), suggested that the G1 element alone might support
Pax6-dependent transcriptional activation. These results
suggest that the G3 element may not be essential for
pax6-dependent activation of the proglucagon promoter in
fibroblasts.

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Figure 1. Northern Blot Analysis of Pax-6, Proglucagon, and
18s mRNA Transcripts in Total and polyA+ RNA from the Enteroendocrine
GLUTag and Islet InR1-G9 Cell Lines
The times of exposure were 48 and 12 h for Pax-6
and glucagon blots, respectively. With a longer exposure (not shown),
Pax-6 mRNA transcripts were detected in the InR1-G9 cell
RNA. The relative migration positions of the 28s and 18s RNAs are
shown.
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Figure 2. Relative Transcriptional Activity of Proglucagon
Promoter Luciferase Plasmids in BHK Fibroblasts (A) InR1-G9 Islet Cells
(B), and GLUTag Enteroendocrine Cells (C)
The structure of the proximal rat proglucagon promoter and the G1-G4
elements is shown in panel c. GLU-Luc plasmids contained 476, 220, 168,
or 93 bp of rat proglucagon gene 5'-flanking sequences (15 ) fused to
the luciferase coding region, or four copies of the G1 proglucagon gene
enhancer region ligated upstream of a minimal proglucagon promoter
[4G1]GLU-Luc. Relative luciferase activity (RLA) was normalized to
the values obtained after transfection of the promoterless expression
vector, SK-Luc, in the same experiments. Luciferase reporter plasmids
were transfected alone with pBS (Bluescript, Stratagene,
La Jolla, CA), with the promoterless expression vector pBAT14
alone, or with either the wild-type Pax-6 or the
dominant negative Pax-6
SEYNeu cDNA. All transfections were done in
quadruplicate, and the data shown represent the mean ±
SEM from three different experiments. *, P
<.05; **, P <.01; ***, P < 0.001.
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As studies of islet A cells and pancreatic glucagon biosynthesis in
Pax6 mutant mice infer a major role for Pax6 in
the regulation of pancreatic proglucagon gene expression via the G3
element in islet A cells (2), we assessed the activity of proglucagon
promoter-luciferase plasmids, with or without the G3 element, in
InR1-G9 islet A cells. In keeping with the data obtained in BHK
fibroblasts, Pax6 activated the proglucagon promoter in
InR1-G9 cells, in a G3-independent manner (Fig. 2B
). The greatest
degree of Pax6-dependent proglucagon promoter activation was
observed with [-168]GLU-Luc, a reporter that contains the G1 and G4,
but not the G2 or G3 enhancer elements. Furthermore, Pax6
significantly increased the activity of [4G1]GLU-Luc in InR1-G9
cells, and the dominant negative SEYNeu cDNA
markedly repressed the basal activity of [-93]GLU-Luc in islet cells
(Fig. 2B
). In contrast, the transcriptional activity of a minimal PRL
promoter was not inhibited after cotransfection with the
SEYNeu cDNA. These findings clearly demonstrate
that Pax6 activates the proglucagon promoter, not only in
transfected BHK cells but also in islet cells, in a G3-independent
manner.
To determine whether Pax6 also regulates proglucagon gene
transcription in intestinal endocrine cells, we carried out
transfection experiments using the GLUTag enteroendocrine cell line.
Pax6 significantly activated the transcriptional activity of
proglucagon promoter plasmids in GLUTag cells (Fig. 2C
), demonstrating
that Pax6 is transcriptionally active in intestinal
enteroendocrine cells, and not only in islet cell types. Furthermore,
the Pax6-dependent activation of -[220]GLU-Luc (Fig. 2C
)
demonstrates that the G3 element is not absolutely required for
Pax6 activation in enteroendocrine cells. Moreover,
transfection of the dominant negative SEYNeu
allele significantly inhibited the transcriptional activity of both
-[93]GLU-Luc and [4G1]GLU-Luc, consistent with a role for the G1
element in Pax6-dependent transcriptional activity in GLUTag
enteroendocrine cells.
The finding that Pax6 activated
proglucagon gene transcription via both the G3 (2) and G1 elements
prompted us to assess whether Pax6 was capable of binding
both these promoter elements in electrophoretic mobility shift assay
(EMSA) studies in vitro. After transfection of
Pax6 into BHK cells, a unique DNA-protein complex was
detected using the G1 element probe, which was not detected in
wild-type BHK cells or cells transfected with the expression vector
alone (Fig. 3A
, complex G1B).
Furthermore, an identically migrating prominent G1B complex was
observed in GLUTag nuclear extracts, and a similar, although less
abundant complex, was detected using InR1-G9 extract (not shown). Using
a G3 element probe containing a previously identified Pax6
binding site (2), a major DNA-protein complex (G3C) was detected in
nuclear extracts prepared from Pax6-transfected BHK cells. A
similar G3C complex was detected in GLUTag nuclear extracts (Fig. 3A
).

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Figure 3. EMSA Experiments with the Proglucagon Gene G1 and
G3 Elements
A, Nuclear extracts from wild-type BHK fibroblasts (with or
without transfected Pax-6) and GLUTag enteroendocrine
cells were incubated with either G1 or G3 probes and analyzed as
described in Materials and Methods. The slightly
different migration positions of the G3C complexes are attributable to
specific electrophoresis conditions, as evidenced by comparable
migration abnormalities of the free probe in the various lanes.
Specific complexes are designated by arrows. FP, Free
probe. B, Competition for G1 and G3 complex formation in GLUTag cells
using increasing (10- to 1000-fold) molar amounts of excess unlabeled
homologous competitor oligonucleotide. C, EMSA experiments with
Pax-6 or cdx-2/3 antisera in GLUTag cell
extracts. G1 or G3 probes were incubated with no extract (lanes 1 and
5) nuclear extract only (-) or extract plus either
Pax-6 (lanes 3 and 7) or cdx-2/3 (lanes 4
and 8) antisera. D and E, EMSA experiments using GLUTag extracts and
either the G3 (panel D) or G1 (panel E) elements as probes, and the G3,
rat proglucagon gene promoter Gluc sequence (-110/-95), G1, insulin
(Ins), or somatostatin (SMS) PISCES elements as competitor
oligonucleotide sequences. FP, Free probe.
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All 3 GLUTag complexes, G1AC, were diminished after competition for
G1 binding with increasing amounts of unlabeled G1 competitor
oligonucleotides (Fig. 3B
), but no competition was observed with
heterologous nonspecific competitor DNA (not shown). Similarly, the
three major complexes observed using GLUTag extracts and the G3 probe,
G3A-C, were markedly reduced after competition with increasing amounts
of unlabeled G3 sequences (Fig. 3B
). Taken together, these experiments
strongly suggest that Pax6 forms a complex with both the G1
and G3 elements in GLUTag enteroendocrine cells.
To provide additional evidence for the presence of Pax6 in
GLUTag DNA-protein complexes detected with either the G1 or G3 probes,
EMSA experiments were carried out in the presence or absence of
Pax6 antisera (14). The G1B complex was markedly diminished
after preincubation of GLUTag extracts with Pax6 antisera
(Fig. 3C
), strongly suggesting that endogenous Pax6 binds to
the proglucagon promoter via the G1 element in enteroendocrine cells.
Similarly, the GLUTag G3C complex was almost completely eliminated
after preincubation with Pax6 antisera (Fig. 3C
). In
contrast, neither the GLUTag G1B nor G3C complexes were diminished
after preincubation with antisera against cdx-2/3 or after
incubation with nonimmune sera (not shown).
The finding that Pax6 binds to both the G3 and G1
elements suggests that Pax6 recognizes DNA sequences common to both G3
and G1. Furthermore, a core DNA sequence designated the PISCES
(pancreatic islet cell-specific enhancer sequence) element has been
identified in the 5'-flanking region of the insulin, somatostatin, and
glucagon genes (16, 17). To ascertain whether DNA sequences
corresponding to putative PISCES elements could compete for the binding
to the G3 and G1 elements, a series of EMSA competition experiments
were performed (Fig. 3
, D and E). An oligonucleotide from the rat
glucagon gene (-110/-95) 5'-flanking region just 5'- to the boundary
of G1 did not compete for complex formation with either the G3 or G1
probes. In contrast, the G1 element modestly diminished the formation
of the G3C complex, whereas the insulin gene PISCES element almost
completely prevented the formation of the G3C complex (Fig. 3D
).
Similarly, both the insulin gene PISCES element and the G3 sequence
competed for the formation of the G1B complex, whereas the somatostatin
element was a comparably ineffective competitor for Pax6
binding to G3 (Fig. 3D
) or G1 (not shown). Taken together, these
findings illustrate that the core element common to the G3, G1, and
insulin gene PISCES element is capable of competing for Pax6
binding in GLUTag extracts in vitro.
The results of the RNA, transfection, and EMSA experiments demonstrated
that Pax6 is expressed in intestinal GLUTag cells and
activates enteroendocrine proglucagon gene transcription via binding to
and activation of the G3 and G1 enhancer elements. To explore the
relevance of Pax6 for intestinal proglucagon gene expression
in vivo, we analyzed enteroendocrine cell populations in
mice with a dominant negative Pax6
SEYNeu mutation. These mice have previously been
shown to exhibit disordered islet formation, markedly reduced numbers
of islet A cells, and decreased levels of pancreatic glucagon (2). As
SEYNeu homozygous mice die shortly after birth,
live SEYNeu -/- mice and both +/- and +/+
littermates were obtained on neonatal day 1, generally within 1 h
after birth, euthanized, and genotyped (Fig. 4A
) using PCR and restriction enzyme
analysis as described previously (18). We first analyzed proglucagon
mRNA transcripts in the small and large bowel of wild-type and mutant
mice by RT-PCR. Although proglucagon mRNA transcripts were comparable
in RNA from +/+ and +/- mice, the levels were markedly reduced in both
small and large bowel RNA from SEYNeu -/- mice
(Fig. 4B
).

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Figure 4. Glucagon Gene Expression in
SEYNeu Mice
A, PCR analysis of genotyping at the Pax-6 locus in +/+ wild-type, +/-
heterozygous, and -/- SEYNeu mice. The
SEYNeu allele contains a unique
HindII site that generates 140- and 80-bp fragments
after enzymatic digestion. B, RT-PCR analysis of intestinal gene
expression in +/+ and SEYNeu mice.
Intestinal RNA from neonatal day 1 age- and sex-matched +/+, +/-, and
-/- SEYNeu littermates. RNA from small and
large bowel was reverse transcribed and analyzed by RT-PCR using
primers for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and
proglucagon, over a range of cycle numbers from 1925, as described in
Materials and Methods.
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We next analyzed the distribution and morphology of endocrine cells in
the islets and gut of mice with the homozygousSEYNeu mutation. Consistent with previously reported
findings (2), islets from SEYNeu mice were small
and poorly developed with abnormal distribution of hormone-containing
cells (Fig. 5
). Cells containing
glucagon, although reduced in number, were readily identified (Fig. 5
)
but did not show the usual pattern of distribution at the periphery of
islets as seen in control wild-type animals. Insulin immunoreactivity
was reduced and was found most prominently in single cells and small
clusters adjacent to ducts rather than in well formed islets (Fig. 5
).
Somatostatin-containing cells were reduced in number in
SEYNeu mice compared with controls. Staining for
pancreatic polypeptide was not markedly different in
SEYNeu and control mice. Cells containing
peptide YY (PYY) were numerous in the pancreases of
SEYNeu mice but were not distributed in normal
islets as they were in control animals (Fig. 5
).

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Figure 5. Pancreatic Islet Morphology in +/+ Control and
SEYNeu Mice
Glucagon immunoreactivity (G) is present in the islets of neonatal day
1 SEYNeu mice, but the distribution of the
immunoreactive cells is distorted compared with the normal peripheral
location of glucagon in control (+/+) animals. Insulin positivity (I)
is reduced in SEYNeu mice, and it is
localized predominantly in scattered individual cells, whereas
insulin-containing cells are found in well formed islets in +/+
controls (d). Somatostatin-containing (S) cells are fewer in the
pancreas of SEYNeu mice than in controls.
Staining for pancreatic polypeptide (PP) is not different in
SEYNeu and control mice. Cells containing
PYY are numerous in both SEYNeu and control
animals, but the distribution within well formed islets is not seen in
SEYNeu mice.
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The intestinal morphology of the small and large intestine in neonatal
day 1 SEYNeu mice appeared normal, as assessed
by conventional light microscopy. However, immunolocalization studies
of intestinal endocrine cells showed total absence of glucagon-like
peptide 1 (GLP-1) and GLP-2 immunoreactivity in
both the small and large bowel (Fig. 6
)
whereas enteroendocrine cells containing these peptides were readily
identified in control +/+ mice (Fig. 6
). In contrast to the absence of
intestinal endocrine cells expressing GLP-1 or GLP-2,
endocrine cells containing immunopositivity for PYY, cholecystokinin,
serotonin, and secretin, were found in the usual distribution in the
small and large bowel of SEYNeu mice (Fig. 7
). As GLP-2 stimulates growth of the
mucosal epithelium in mice (19), we analyzed intestinal morphology of
the small bowel in -/- mice. No significant differences in small
bowel crypt plus villus height were observed after assessment of
multiple histological sections of small bowel from +/+ andSEYNeu mice (data not shown).

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Figure 6. Absence of GLP-1 and GLP-2
Immunoreactivity in the Gut of SEYNeu Mice
GLP-1 immunostaining yields entirely negative results in
the bowel of a neonatal day 1 SEYNeu mouse,
whereas a comparable area in the gut of a +/+ control mouse has
numerous positive cells. GLP-2 immunostaining is also completely
negative in the bowel of a SEYNeu mouse but
several GLP-2-immunoreactive cells are present in the bowel of a +/+
control mouse. No GLP-1- or GLP-2-immunopositive cells
were seen in several dozen histological sections from the small and
large bowel of three different SEYNe mice.
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Figure 7. Localization of Enteroendocrine Cells in the Gut of
SEYNeu Mice
In the bowel of neonatal day 1 SEYNeu mice,
enteroendocrine cells are detected that exhibit immunoreactivity for
PYY, cholecystokinin (CCK), serotonin (SER), and secretin (S) in a
distribution that is unchanged from control +/+ mice (not shown).
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DISCUSSION
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Enteroendocrine cells of the small and large bowel exhibit a
regional distribution along the small and large bowel that is unique to
specific hormonal subtypes (20). As enteroendocrine cells and
pancreatic islets are both believed to arise from endodermal origin, it
is not surprising that several endocrine genes, including secretin,
gastrin, and PYY, are transiently expressed in the fetal endocrine
pancreas, after which expression in adult islets is extinguished but is
maintained in gastrointestinal endocrine cells throughout adult life
(21, 22). Although enteroendocrine cells appear to be derived from a
common pluripotential crypt precursor capable of giving rise to
multiple differentiated intestinal cell lineages (23), little is known
about the molecular determinants of intestinal endocrine cell
formation. Furthermore, the hierarchy and complexity of transcription
factors that regulate endocrine cell lineage in the gut remain poorly
understood.
Analysis of mice with a targeted disruption of the helix-loop-helix
transcription factor BETA2 has provided important insights into the
genetic control of endocrine cell lineages in the gut (10). The
secretin gene, a transcriptional target for BETA2, is transiently
expressed in fetal islet ß-cells, after which expression in the adult
is restricted to enteroendocrine cells (24). BETA2-/- mice exhibit
striking abnormalities in islet morphogenesis with a severe reduction
in mature islet ß-cells, and a complete absence of secretin- and
cholecystokinin-producing intestinal enteroendocrine cells; in
contrast, the numbers of enteroendocrine cells expressing the
proglucagon and somatostatin genes were normal despite the lack of
BETA2 expression (10). The findings reported here extend our
understanding of enteroendocrine cell biology by demonstrating that
Pax6 is essential for expression of the proglucagon gene in
the intestinal epithelium. Whether the absence of Pax6
primarily perturbs endocrine cell formation or, specifically, hormone
gene transcription is difficult to determine, as cell lineage markers
for the proglucagon enteroendocrine cell population have not been
identified. Nevertheless, proglucagon mRNA transcripts were not
completely absent from the intestine of -/- mice, suggesting that
SEYNeu mice contain at least a few putative
enteroendocrine cells that support proglucagon gene transcription
in vivo.
The finding that Pax6 plays an essential role in expression
of both pancreatic and intestinal proglucagon gene expression further
supports the existence of shared mechanisms for regulating hormone gene
expression in both islet and intestinal endocrine cells. DNA-binding
protein(s) from islet cell lines were originally identified that bound
to highly similar promoter sequences, designated the PISCES motif,
present in the 5'-flanking regions of the insulin, somatostatin, and
proglucagon gene promoters (Refs. 16, 25 and Fig. 8
). Multiple DNA-protein complexes have
been detected using the glucagon gene G3 element as a probe in EMSA
experiments, as shown here and in previous studies (16). Furthermore,
the somatostatin and insulin PISCES elements compete for binding with
multiple DNA complexes formed by G3 (16). The demonstration that
Pax6 binds to the PISCES element in the insulin,
proglucagon, and somatostatin promoters, taken together with the
competition for DNA-protein complex formation on G3 using PISCES
elements from islet hormone promoters, is consistent with the
hypothesis that these elements recognize one or more highly similar, if
not identical, transcription factor complexes (16). The major reduction
in islet hormone gene expression in Pax6
SEYNeu mice strongly implicates Pax6
as a PISCES-binding protein that is functionally essential for
transcriptional regulation in the endocrine pancreas (2). Our results
extend the functional concept of the PISCES motif to include
regulation, via Pax6, of both islet and enteroendocrine gene
expression.

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Figure 8. GLP-1-Immunopositive Cells in the
Islets and Intestine of a Newborn Wild-Type (+/+) and
SEYNeu (-/-) Mouse
The organization and number of GLP-1-immunopositive islet
cells is abnormal, and GLP-1- or GLP-2-immunopositive
intestinal endocrine cells are not detected in the
SEYNeu mouse.
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Pax-6 immunoreactive cells have been detected in the
mouse fore/midgut endoderm as early as embryonic day 9 (E9.0)
(2), and by day E9.5, a few glucagon-positive cells are detected at the
time of the appearance of the pancreatic bud that also appear to
coexpress Pax-6. At E15.5, numerous cells in the endocrine
pancreas express Pax-6 and either glucagon, insulin,
somatostatin, or pancreatic polypeptide. The ontogeny of
Pax6 expression in the developing gut has not been reported.
The murine intestinal epithelium undergoes a complex pattern of
cytodifferentiation beginning at approximately E15, and distinct
enteroendocrine cell populations are detectable in the small intestine
by E17 (26). Similarly, endocrine cells are first detected in the mouse
colon by E15.5, with the earliest cell type detected expressing PYY,
followed by the appearance of additional hormonal phenotypes between
E17.5 and E18.5 (27). Glucagon-immunopositive cells are infrequent
before E19 in the developing small bowel, but detectable by E16.5 in
the murine colon (27). Whether intestinal Pax6 expression is
exclusively localized to the glucagon-immunopositive enteroendocrine
cell lineage in the developing and adult gut remains to be
determined.
As the levels of proglucagon mRNA transcripts were markedly
reduced in SEYNeu mice, these mutant mice
provide an opportunity to assess the importance of the
proglucagon-derived peptides for fetal growth and development.
Although intestinal-derived GLP-2 has been recently identified as a
potent intestinal growth factor in adult mice (19), the role, if any,
of GLP-2 (or GLP-1) in the development of the fetal gut is
not known. The observation that the small and large bowel appear
histologically normal in SEYNeu mice despite the
marked reduction in intestinal proglucagon gene expression (and hence
GLP-2 biosynthesis) suggests that normal levels of GLP-2 are not
essential for growth and differentiation of the intestinal mucosal
epithelium during fetal development in vivo. In summary, the
results presented here demonstrate that a single transcription factor,
Pax6, is essential for both islet cell formation and islet
and enteroendocrine gene transcription, via interaction with a DNA
element common to the promoters of genes expressed in the endocrine
cells of the pancreas and gut.
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MATERIALS AND METHODS
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Plasmids
The plasmids pBat14, pBat14.mPax6, and pBat14.mPax6
SEYNeu were kindly provided by M. S. German
(University of California, San Francisco, CA). These plasmids are
eukarykotic expression vectors under the control of the cytomegalovirus
(CMV) promoter (2). The 5'-deleted rat proglucagon gene promoter
sequences, subcloned in the promoterless plasmid SK-Luc immediately
adjacent to the coding sequence of the firefly luciferase reporter
gene, have been previously described (28, 29). Synthetic
oligonucleotides corresponding to specific rat proglucagon gene G1
sequences (15) were annealed, separated by agarose gel electrophoresis,
gel purified, and ligated into the [-82]GLU-Luc plasmid (28). This
new plasmid was designated [4G1]GLU-Luc and contained four copies of
the G1 sequence in a 5'-3' orientation adjacent to the -82 to +58
fragment of the proximal rat proglucagon promoter. The plasmid
[-36]PRL-Luc was a kind gift from H. P. Elsholtz (University of
Toronto, Toronto, Ontario, Canada). The CMV promoter plasmid, CMV-Luc,
and the promoterless plasmid, SK-Luc, were used as positive and
negative controls, respectively, in each transfection experiment.
Cell Culture and Transfections
All cell culture reagents used were obtained from Life Technologies/Gibco BRL (Toronto, Ontario, Canada).
Cell lines were maintained in DMEM (4.5 g glucose/liter). Baby hamster
kidney (BHK) fibroblasts and InR1-G9 islet cells (30) were grown in
DMEM supplemented with 5% calf serum; mouse enteroendocrine GLUTag
cells (31) were grown in DMEM supplemented with 10% FCS. BHK cells and
InR1-G9 cells were transfected by the calcium phosphate precipitation
method with 5 µg reporter plasmid DNA + 5 µg expression plasmid DNA
per 60-mm dish. GLUTag cells were electroporated with 10 µg reporter
plasmid + 10 µg expression plasmid. All cells were harvested 1620 h
after transfection for analysis of luciferase activity as described
previously (28, 32), and values were normalized relative to the
background luciferase activity obtained after transfection of SK-Luc in
the same experiment. Statistical analysis was performed using
Students t test.
RNA Analysis
RNA was prepared from cells and tissue by the acid ethanol
precipitation method previously described (33). Poly A+ RNA was
isolated using a Qiagen Poly A Kit (Mississauga, Ontario,
Canada). Electrophoresis of RNA, transfer to nylon membrane,
hybridization, and washing were carried out as previously described
(34). For RT-PCR, first-strand cDNA synthesis was generated from total
RNA using the SuperScript Preamplification System from Life Technologies. Target cDNA was then amplified using specific
oligonucleotide primer pairs by the PCR method. Primers for mouse
proglucagon were 5'-TGAAGACCATTTACTTTGTGGCT-3' and
5'-CTGGTGGCAAGATTGTCCAGAAT-3'; primers for glyceraldehyde-3-phosphate
dehydrogenase were 5'TCCACCACCCTGTTGCTGTAG-3' and
5'-GACCACAGTCCATGACATCACT-3'. Reactions were denatured at 94 C for 1
min and then annealed at 66 C for 45 sec, followed by extension at 72 C
for 1 min for 1925 cycles.
Mice and Genotyping
Small eye SEYNeu +/- mice were kindly
provided by Brigid Hogan (Vanderbilt University, Nashville, TN) and
maintained in the animal facility of the Toronto Hospital.
SEYNeu +/- were mated to generate homozygous,
heterozygous, and wild-type offspring, which were distinguished by PCR
and restriction enzyme analysis of the amplification product as
previously described (18).
EMSAs
Nuclear proteins from GLUTag, InR1-G9, and BHK cells were
prepared as previously described (28, 32). Synthetic oligonucleotides
corresponding to specific proglucagon gene G1 and G3 sequences (15)
were annealed, labeled with [32P]ATP using Klenow enzyme,
and purified in a G50 spin column. EMSAs were performed as described
previously (28, 32). For supershift experiments, nuclear extracts were
preincubated with 1:10 diluted anti-Pax6 antiserum (a
generous gift from Dr. S. Saule, Lille, France) or nonimmune antisera
at 4 C for 30 min before addition of 32P-labeled DNA probe
and subsequent incubation at 30 C for 30 min. All reaction mixes were
loaded onto a 5% nondenaturing polyacrylamide gel, and after
electrophoresis, the gel was exposed to x-ray film for 2448 h.
Oligonucleotide competitor sequences from the rat insulin and glucagon
gene PISCES elements were synthesized as per Fig. 4A
in Ref. 16 .
Histology and Immunocytochemistry
Tissues were processed for immunohistochemistry and/or
intestinal morphometry as previously described (35, 36).
Formalin-fixed, paraffin-embedded tissue was sectioned at 4 µm for
imunohistochemistry using the streptavidin-biotin-peroxidase complex
technique. Primary antisera and antibodies were directed against the
following antigens and were used at the specified dilutions: insulin
(monoclonal antibody from BioGenex Laboratories, Inc., San
Ramon, CA) 1:40 for 30 min; glucagon (polyclonal antiserum from
Immunon, Pittsburg, PA) 1:200 for 30 min; GLP-1
(polyclonal antiserum prepared by D. J. Drucker) 1:1500 for 30
min; GLP-2 (polyclonal antiserum prepared by D. J. Drucker) 1:2500
for 30 min; PYY (polyclonal antiserum from Peninsula Laboratories Inc., Belmont CA) 1:2000 for 30 min; somatostatin (polyclonal
antiserum from DAKO Corp., Carpinteria, CA) prediluted
preparation further diluted 1:40 overnight; pancreatic polypeptide
(polyclonal antiserum from DAKO Corp.) 1:6000 for 30 min;
cholecystokinin (polyclonal antiserum from Serotec,
Oxford, U.K.) 1:1000 for 30 min; serotonin (monoclonal antibody from
DAKO Corp.) 1:50 for 60 min; secretin (polyclonal
antiserum from BioGenex Laboratories, Inc., San Ramon, CA)
prediluted preparation further diluted 1:5 overnight; gastrin
(polyclonal antiserum from Immunostain, Los Angeles, CA) prediluted
preparation further diluted 1:20 overnight; vasoactive intestinal
peptide (polyclonal antiserum from Zymed Laboratories, Inc., San Francisco, CA) prediluted preparation further diluted
1:10 overnight. The reactions were visualized using
3,3'-diaminobenzidine and hydrogen peroxide. Appropriate positive and
negative controls were performed in each case. The gastrointestinal
tract from newborn +/+, +/- and SEYNe -/-
mice was removed and fixed in 10% buffered formalin. Intestinal
micrometry was performed with the use of a Leitz (Westar,
Germany) microscope with a video camera connected to a computer
monitor. The microscope was calibrated at x10 magnification, and crypt
plus villus height in the small bowel was measured by examining at
least 10 longitudinally oriented villi from each animal and is
expressed in micrometers (mean ± SEM).
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. D. Drucker, The Toronto Hospital, 200 Elizabeth Street CCRW3838, Toronto, Ontario M5G 2C4, Canada.
This work was supported by an operating grant to D.J.D. from the
Medical Research Council (MRC) of Canada. D.J.D. is a Senior Scientist
of the MRC.
Received for publication February 3, 1999.
Revision received May 5, 1999.
Accepted for publication May 25, 1999.
 |
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