Tissue-Specific Transcriptional Activity of a Pancreatic Islet Cell-Specific Enhancer Sequence/Pax6-Binding Site Determined in Normal Adult Tissues in Vivo Using Transgenic Mice
Stephan Beimesche,
Andrea Neubauer,
Stephan Herzig,
Rafal Grzeskowiak,
Thomas Diedrich,
Irmgard Cierny,
Doris Scholz,
Tahseen Alejel and
Willhart Knepel
Department of Molecular Pharmacology University of
Göttingen D-37075 Göttingen, Germany
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ABSTRACT
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A pancreatic islet cell-specific enhancer
sequence (PISCES) shared by the rat insulin-I, glucagon, and
somatostatin genes binds the paired domain-containing transcription
factor Pax6 and confers strong transcriptional activity in pancreatic
islet cell lines. It was found recently that Pax6 plays a major role in
islet development. In the present study, transgenic mice were used to
investigate PISCES-mediated transcription in normal adult islets
in vivo. In several independent mouse lines expressing a
PISCES-luciferase reporter transgene, the PISCES motif directed gene
expression in the adult eye, cerebellum, and discrete brain areas,
consistent with the tissue distribution of Pax6. These tissues contain
two Pax6 isoforms caused by alternative splicing, only one of which was
found to bind the PISCES motif in electrophoretic mobility shift
assays. No reporter gene expression was detected in adult pancreatic
islets or in any other peripheral organ tested. RT-PCR analysis
confirmed that Pax6 mRNA is present in adult islets. These results
demonstrate that the PISCES motif is sufficient to direct highly
tissue-specific gene expression in whole animals. The lack of
PISCES-mediated transcription in adult islets indicates that the Pax6
protein(s) expressed in adult pancreatic islets function differently
from the ones in the eye and cerebellum.
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INTRODUCTION
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The peptide hormones insulin and glucagon are critical regulators
of blood glucose concentration. Their inappropriate production and
secretion causes diabetes mellitus. These hormones are synthetized
within distinct cell types of the pancreatic islets (1). Gene
inactivation by homologous recombination in mice has shown that various
transcription factors are essential for endocrine cell differentiation
in the developing pancreas, including Pdx1 (2), Isl1 (3), and
Beta2/NeuroD (4). The lineage of the different endocrine cells may be
defined by members of the Pax family of vertebrate genes, all of which
contain a conserved sequence motif, the paired box, which encodes a
DNA-binding domain (5, 6). Inactivation of Pax4 results in the absence
of mature insulin- and somatostatin-producing cells (7), whereas Pax6
homozygous mutant mice lack glucagon-producing cells (8).
Some of these transcription factors are also expressed in mature
endocrine cells and may, thus, contribute to maintain endocrine cells
in a differentiated state. They may also directly regulate the
expression of hormone genes in terminally differentiated islets. Pdx1
is produced in both ß- and
-cells in the adult islet (9, 10, 11). It
can bind to and transactivate the insulin gene promoter (12),
particularly in combination with a heterodimer of the transcription
factors E47 and Beta2, which binds to an adjacent site on the insulin
promoter (13, 14). Isl1 is produced in all islet cells and can
transactivate the glucagon promoter (15). Also, Pax genes are expressed
in adult islets. Pax4 expression seems to be restricted largely to
ß-cells, whereas Pax6 is expressed in mature
-, ß-,
-, and
- endocrine cells (8). However, the role of all of these
transcription factors in the regulation of hormone gene transcription
in the differentiated endocrine pancreas is unclear, as evidence for
activation of islet hormone gene transcription by these factors is
based on results obtained from studies using islet tumor cell lines
(12, 13, 14, 15). Although islet cell lines have retained many characteristics
of normal islet cells and are extremely helpful for studies on islet
cell biology, some features of islet cell lines are unique or more
typical of immature islet cells (16, 17). Furthermore, genetic studies
that could potentially address the mechanism of islet homone gene
regulation in adult islets in vivo have not been
informative, because null mutations in genes that encode transcription
factors presumed to regulate islet hormone gene expression prevent
normal pancreas development (2, 3, 4, 8).
The origin of all islet endocrine cells from common progenitor cells
(18) raises the possibility that fully differentiated islet cells may
share transcriptional regulatory proteins directing insulin, glucagon,
and somatostatin gene transcription. Indeed, a DNA sequence common to
the insulin, glucagon, and somatostatin gene promoters, the pancreatic
islet cell-specific enhancer sequence (PISCES), has been detected (19).
Multiple copies of the PISCES motif are sufficient to selectively
stimulate transcriptional activity of a luciferase reporter gene in
various phenotypically distinct pancreatic islet cell lines without
affecting expression in several nonislet cell lines (20). The PISCES
motif shows high homology to the consensus recognition site of the Pax6
paired domain (19, 21), and Pax6 was found to bind and activate the
PISCES motif (22). Because mutation studies have demonstrated that the
PISCES element is important for the activity of each of the three
hormone gene promoters (19, 20, 23), the PISCES element and its binding
factor appear to be a key component of the transcription complexes
coordinately directing insulin, glucagon, and somatostatin gene
transcription in distinct islet cell types. However, these studies have
again been performed using islet tumor cell lines. The transcriptional
activity conferred by the PISCES element and its binding factor in
mature endocrine cells of adult islets in vivo remained thus
unclear. In the present study, PISCES-luciferase reporter transgenic
mice were generated and used to study the transcriptional activity of
the PISCES motif in normal mature tissues in vivo.
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RESULTS
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The PISCES Motif Directs Highly Tissue-Specific Expression in
Mice
Four copies of a 16-bp oligonucleotide containing the PISCES motif
as present in domain A of the glucagon G3 element were placed in front
of the herpes simplex virus thymidine kinase promoter truncated at -81
and linked to the luciferase reporter gene (p4xG3AT81Luc). This
construct has been shown to drive high level expression of the reporter
gene after transfection into phenotypically distinct islet cell lines
(19, 20, 23). Although homomultimeric repeats of transcriptional
elements are artificial, this organization satisfies the requirement
for multiple elements to generate a functional enhancer. This approach
has been successful for characterizing the binding sites for single
transcription factors by cell transfection and also in transgenic mice
(24, 25, 26, 27, 28). For example, an oligomerized NFAT-binding motif that directs
transcription of SV40 T-antigen in transgenic mice indicated cell
populations exhibiting constitutive or inducible NFAT activity (25),
and a minimal promoter containing three copies of a binding site for
NF
B-related transcription factors indicated tisssue-specific and
inducible transcriptional activities of distinct NF-
B/Rel proteins
(26). To determine the tissue-specific transcriptional activity of the
PISCES motif in mature tissues in vivo, transgenic mice were
generated. The PISCES-luciferase reporter gene was microinjected as a
linear fragment into pronuclei of one-cell embryos. Of six
independently derived transgenic founder mice bearing from 2 to 15
4xG3AT81Luc transgenes, five expressed the luciferase reporter gene
(Fig. 1
). The absolute level of
expression of the reporter gene was different in the independent
transgenic lines; reporter enzyme activity was, respectively, 12,700,
466,100, 2,420,000, 14,500,000, and 21,200,000 light units/mg protein
in the tissue with highest expression for each independent line (Fig. 1
). However, all transgenic lines showed a similar pattern of reporter
expression in adult animals (Figs. 1
and 2
). The PISCES motif directed gene
expression in the eye and brain with highest levels in the cerebellum
(Figs. 1
and 2
). Virtually no reporter enzyme activity was detected in
pancreas and other peripheral organs including liver, spleen, stomach,
intestine, kidney, lung, heart, and skeletal muscle (Figs. 1
and 2
).
The level for pancreatic transgene expression in the five independent
lines was, respectively, 0.0005, 0.0009, 0.003, 0.01, and 0.06% of the
tissue with highest expression. Reporter expression was also not found
in isolated pancreatic islets of Langerhans (not shown). These results
show that a short oligonucleotide containing the PISCES motif is
sufficient to direct tissue-specific gene expression in mature tissues
in vivo; the PISCES motif does not direct pancreatic
expression but directs expression in the eye and brain with highest
levels in the cerebellum.

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Figure 1. Reporter Transgene Expression in Adult Tissues
Luciferase reporter enzyme activity was measured in the indicated
tissues from five independent transgenic lines carrying the 4xG3AT81Luc
transgene. Values are light units x 10-6 per mg
protein. Mesenc., Mesencephalon; Telenc., telencephalon; Bulbus olf.,
bulbus olfactorius; Medulla obl., medulla oblongata; Dienc.,
diencephalon; Pituitary gl., pituitary gland.
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Figure 2. Relative Reporter Transgene Expression in Adult
Tissues
Luciferase reporter enzyme activity was measured in the indicated
tissues from five independent transgenic lines carrying the 4xG3AT81Luc
transgene. For each independent transgenic line, luciferase activity
measured in the tissue with highest activity is set as 100 and
luciferase activity measured in the other tissues is expressed relative
to that. Mesenc., Mesencephalon; Telenc., telencephalon; Bulbus olf.,
bulbus olfactorius; Medulla obl., medulla oblongata; Dienc.,
diencephalon; gl., gland.
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To verify that this tissue specificity is conferred by the PISCES
motif, we analyzed the expression of a transgene containing a mutated
G3A homomultimer (4xG3AmutT81Luc). This mutation functionally
inactivates the PISCES motif as shown in protein-DNA binding and cell
transfection experiments (19, 20, 23). In four independent transgenic
lines bearing from 1 to 10 transgenes, reporter enzyme expression was
very low. Figure 3
shows the expression
of the mutated transgene in several tissues of two independent
transgenic lines. The expression of the mutated transgene was low, and
its pattern of expression different from that of the wild-type
transgene (Fig. 3
, compare with Fig. 1
). These results support the
conclusion that the PISCES motif directs expression in vivo
in the mature eye and brain.

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Figure 3. Expression of a Mutated Transgene in Adult Tissues
Luciferase reporter enzyme activity was measured in the indicated
tissues from two independent transgenic lines carrying the
4xG3AmutT81Luc transgene. Values are light units x
10-6 per mg protein. The corresponding figures for
pancreatic expression of the mutated transgene are 0.000003 and
0.00001, respectively. Mesenc., Mesencephalon; Telenc., telencephalon;
Bulbus olf., bulbus olfactorius; Medulla obl., medulla oblongata;
Dienc., diencephalon; Pituitary gl., pituitary gland.
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The expression of the PISCES-luciferase transgene in the adult brain
was further analyzed in tissue sections of the brain of the second
highest expressing line at the macroscopic level using a Molecular
Light Imager (EG&G Berthold, Bad Wildbad, Germany). The sagittal
sections shown in Fig. 4
confirm that the
PISCES motif directs expression to highly restricted areas in the
brain. No reporter expression was found in the cerebral cortex,
hippocampus, or caudate putamen (Fig. 4
). The strongest luminescence
signals were detected over the cerebellum (Fig. 4
, top
panel), the optic chiasma, and the superior colliculus (Fig. 4
, bottom panel). In additional sections, some signals were
also found in the olfactory bulb, septal area, zona incerta, and
amygdala (not shown).

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Figure 4. Imaging of Transgene Expression in Sagittal
Sections of the Brain of a 4xG3AT81Luc Transgenic Mouse
Luciferase luminescence signals are superimposed in pseudocolor onto
the brightfield image. Red, High intensity;
blue, low intensity. The strongest luminescence signals
are detected over the cerebellum (top panel) and, in a
section closer to the midline, over the superior colliculus (S.C.) and
the optic chiasma/optic tract (O.C.) (lower panel).
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The expression of the PISCES-luciferase transgene was studied
also in the mouse embryo, using the line with the highest transgene
expression in the adult brain. On day 13.5 of embryonic development,
strong transgene expression was found in the brain but not in the
embryonic pancreas. Reporter enzyme activity in extracts of the head
was 1,474,165 ± 352,555 light units/mg protein (n = 6),
whereas no activity was detected in extracts from the embryonic
pancreas. Likewise, strong transgene expression in the head, but not
the pancreas, was detected on days 15.5, 16.5, 17.5, and 18.5
postcoitus (p.c.) (not shown).
PISCES-Binding Proteins (PISCES-BPs) in Cerebellum and Eye
A PISCES-BP has been characterized in nuclear extracts from islet
cell lines (19, 20, 23). This PISCES-BP has recently been identified as
the transcription factor Pax6 (22). To characterize PISCES-BP(s) in the
cerebellum and eye of adult mice, the electrophoretic mobility
shift assay was used. Labeled G3A was incubated with whole-cell
extracts from the cerebellum or eye. A protein complex that bound
specifically to labeled G3A and that was competed for by G3A, but not
by G3Amut, was detected in both extracts (Fig. 5
, compare lane 7 with 8, and lane 11
with 12, respectively). This protein complex in cerebellum and eye
extracts comigrated with PISCES-BP from an islet cell line (Fig. 5
, compare lanes 12 and 8 with lane 4) and was abolished by the addition
of a specific anti-Pax6 antiserum (not shown). Pax6 is known to be
expressed in the cerebellum and eye of adult mice (29). These results
provide no evidence that the cerebellum and eye express specific
PISCES-BPs other than Pax6. One additional, prominent complex of
slower mobility that forms on labeled G3A was detected (Fig. 5
, lanes 5
and 9); however, because this complex also binds labeled G3Amut (Fig. 5
, lanes 6 and 10), which is not active in cell transfection and
transgenic mice tests, it is not relevant to the function of the PISCES
motif. We were unable to detect PISCES-BPs in extracts from pancreas or
isolated pancreatic islets (not shown).

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Figure 5. PISCES-BPs in the Eye and Cerebellum as Revealed by
Electrophoretic Mobility Shift Assay
Nuclear extracts (lane 1) or whole-cell extracts (lanes 212) from the
eye, cerebellum (Cerebel.) or HIT islet cell line were incubated with
the probes indicated. The competitors were added at a 500-fold molar
excess. The oligonucleotide G3A contains the PISCES motif, whereas the
PISCES motif is mutated in G3Amut. F, Free probe.
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Differential PISCES Binding by Pax6 Splice Variants
The eye and brain of adult mice are known to express the Pax6 gene
as at least two isoforms (6, 30, 31). An alternatively spliced exon
(exon 5a) in vertebrate Pax6 genes is included variably in the mature
mRNA transcript, resulting in the insertion of a 14-amino acid peptide
in the paired domain (6). This protein exhibits unique DNA-binding
properties (21). To investigate the ability of these splice variants to
bind the PISCES motif, the two Pax6 paired domains (denoted Pax6 and
Pax65a) were expressed in Escherichia coli as glutathione
S-transferase fusion proteins and used in an electrophoretic
mobility shift assay. As shown in Fig. 6
, Pax6 bound very well to labeled oligonucleotides containing the
glucagon G3 enhancer-like element (G3) or domain A of the G3 element
(G3A) with the PISCES motif, whereas the extended paired domain
(Pax65a) did not. These results show that of the two alternative Pax6
proteins, only the one without the 14-amino acid insertion in the
paired domain can bind the PISCES motif.

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Figure 6. Pax6, but Not the Splice Variant Pax65a Paired
Domain, Binds the PISCES Motif as Revealed by Electrophoretic Mobility
Shift Assay
The bacterially expressed Pax6 or Pax65a paired domains were
incubated with the probes indicated. The oligonucleotides G3 and G3A
contain the PISCES motif. The oligonucleotides P6Con and 5aCon contain
high-affinity consensus binding sites for the Pax6 and Pax65a paired
domains, respectively (21 ), and served as positive controls. An
oligonucleotide (SCE) containing the cAMP response element of the rat
somatostatin gene was used as negative control. F, Free probe. Specific
complexes formed by Pax6 and Pax65a are indicated by arrows on the
left and right, respectively. Pax65a
has been shown to form several complexes when incubated with labeled
5aCon (21 ).
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Pax6 Gene Expression in Pancreatic Islets as Revealed by RT-PCR
Pax6 gene expression was studied in pancreatic islets by RT-PCR.
Two primer pairs were used, one of which flanks the alternatively
spliced exon 5a (Fig. 7
). With poly
(A)+ RNA extracted from the adult pancreas (Fig. 7
) or from
isolated pancreatic islets (not shown), the primer pair that targets
exon 2 and exon 6 detected pax6 transcripts with and without
exon 5a (499- and 457-bp fragment, respectively, Fig. 7
). The primer
pair that targets exon 6 and exon 13 (Fig. 7
) generated a single
product of the expected size (933-bp fragment, Fig. 7
). The RT-PCR
products were verified by subcloning and sequencing. These results
confirm (6, 8, 22, 32) that the Pax6 gene is expressed in adult
pancreatic islets. They indicate that the transcripts in the islets are
fully consistent with the known Pax6 cDNA, with and without the
alternatively spliced exon 5a. Similar results were obtained with poly
(A)+ RNA extracted from the embryonic pancreas on days
11.5/12.0 p.c. (Fig. 7
). With poly (A)+ RNA extracted from
the eye and cerebellum, both primer pairs generated products of the
expected size for both splice variants (not shown), consistent with
published reports of the expression of both splice variants in the eye
and brain (21, 30, 31, 33).

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Figure 7. Pax6 Gene Expression in Mouse Pancreas as Revealed
by RT-PCR
The upper part of the figure shows the relative
positions of the two primer pairs used. The Pax6 cDNA is shown with the
exons of the human gene. UTR, Untranslated region. The sizes of the
expected fragments are 499 and 457 bp with the primer pair that targets
exon 2 and exon 6 (with and without exon 5a) and 933 bp with the primer
pair that targets exon 6 and exon 13. Poly (A)+ RNA from
adult or embryonic (day 11.5/12.0 p.c.) pancreas was used. The RT-PCR
products were verified by subcloning and sequencing.
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DISCUSSION
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In the present study transgenic mice were used to investigate the
transcriptional activity of the PISCES motif, which binds the
transcription factor Pax6, in normal mature tissues in vivo.
Although the Pax6 gene is expressed in the adult eye, brain, and
pancreatic islets, this study shows that the PISCES motif directs
expression in the eye and brain but not in pancreatic islets,
suggesting that the Pax6 protein(s) in normal pancreatic islets
function differently.
This study shows that four copies of a short 16-bp oligonucleotide
containing the PISCES motif are sufficient to direct highly
cell-specific expression in transgenic mice. Reporter gene expression
was selectively activated by the PISCES motif in the eye and discrete
areas within the central nervous system of the adult mouse, with
highest levels in the cerebellum and colliculi superiores and low
activity in the olfactory bulb, septal area, zona incerta, and
amygdala. No reporter expression was found in the cerebral cortex,
hippocampus, caudate putamen, or any peripheral organ tested. The
PISCES-binding transcription factor Pax6 is known to be expressed in
mature eye tissues including the retina, lens, corneal epithelia, and
iris (34). In the cerebellar cortex of the adult brain, the granular
cell layer expresses high levels of the Pax6 gene (29). In general, the
reporter gene expression pattern observed in the present study agrees
with the known tissue distribution of Pax6 (29), suggesting that
transcriptional activity of the PISCES motif is conferred by the
transcription factor Pax6. A possible exception could be the superior
colliculus, where Pax7 rather than Pax6 is expressed (29). However, in
this region most of the fibers of the optic nerve terminate (35), and
thus the luminescence signal detected over the superior colliculus and
optic chiasma/optic tract could be generated by reporter enzyme
localized within projections of Pax6-expressing retinal ganglion cells
(30, 34, 36). Pax6 is expressed early in development, and as is
indicated by severe malformations of spontaneous mutants in
Drosophila (eyeless), mouse (Small
eye), and man (aniridia), Pax6 is essential for the
formation of the eye, nose, and central nervous system (5, 6, 29, 30, 37). Pax6 is down-regulated in most cell types as they become
postmitotic and differentiate. Expression is maintained, however,
within several cell types of the mature eye and in discrete subregions
of the adult brain (see above). The PISCES-mediated transcriptional
activity observed in the present study in the adult mouse suggests that
Pax6 protein(s) expressed in the adult eye and brain possess
independent transcriptional activity, supporting a role for Pax6 in the
differentiation and maintenance of mature eye tissues and specific
brain cell subtypes (29, 34, 38).
The gross distribution of glucagon (39, 40, 41) and somatostatin gene
products (42, 43) in the adult eye and brain partially overlap with the
expression pattern of Pax6, raising the possibility that Pax6 could
contribute to their regulation. Other genes with identified
Pax6-binding sites are the mouse
A-crystallin gene (44) and the gene
encoding the neural cell adhesion molecule (45). Even so, the Pax6
target genes in the mature eye and brain remain to be defined. It is
noteworthy that the homomultimeric PISCES minienhancer could serve to
direct the expression of proteins under study to defined regions of the
eye and brain.
The PISCES is a sequence motif of the rat glucagon, insulin-I, and
somatostatin genes (19). The finding of the present study that a
regulatory sequence of islet hormone genes is able to direct expression
in the brain exemplifies that the endocrine cells of the pancreas are
related to neurons. Although of endodermal origin, the pancreatic
islets have several characteristics in common with the brain (1, 18, 39), which may be explained at the molecular level by the fact that
pancreatic islets share several transcription factors with the brain
including Beta2/NeuroD (4), brain4 (46), as well as Pax6. Pax6 is
expressed early during pancreatic development (8, 22), and several
lines of evidence suggest that the Pax6 gene is expressed also in
mature, normal pancreatic islets. Using RT-PCR, Turque et
al. (32) detected exon 7-containing pax6 transcripts in
islets of juvenile 4- to 5-week-old mice, and in the present study exon
2 to 13-containing pax6 transcripts were found in adult
mouse islets. The mRNAs of two splice variants, with and without exon
5a, were detected, although only the paired domain without exon 5a,
when expressed in bacteria, was found to bind the PISCES motif. Sander
et al. (22) showed immunofluorescence staining of adult
mouse pancreatic islets with an antiserum directed against the paired
domain of quail Pax6. Finally, a mutated Pax6 gene of a disrupted
allele lacking the region from exon 4 to 6 was found to be
transcriptionally active in the islets of Langerhans in newborn mice
(8). Although these data indicate that the Pax6 gene is expressed in
normal pancreatic islets, they define neither the protein(s) formed nor
their functional activity. Transgenesis provides the most comprehensive
and rigorous test of cell-specific transcriptional activity for DNA
control elements. The present study shows that the PISCES motif by
itself does not possess transcriptional activity in normal pancreatic
islets of the adult or embryonic mouse, suggesting that the Pax6
protein(s) in normal islets function differently from the ones in the
eye, the brain, and pancreatic islet cell lines in which the PISCES
motif confers strong transcriptional activity (Refs. 19, 20, 23 and
the present study).
The lack of PISCES-mediated transcription in normal pancreatic islets
could be explained by unique posttranslational modifications of Pax6,
the absence in islets of essential coactivators, or the presence of
modulatory proteins, as exemplified by the homeodomain protein
Engrailed-1 in quail neuroretina, which inhibits the DNA binding of
Pax6 through direct protein-protein interaction (47). It is noteworthy
that engrailed may also be expressed in islets (48). The
lack of activity of Pax6 at the isolated PISCES motif in normal
pancreatic islets may not be unique, since Pax6 has been shown to
repress ß-crystallin gene transcription through a Pax6-binding site
of the ßB1-crystallin promoter in chicken primary lens epithelial
cells (49). Based on the expression of the Pax6 gene in normal islets
and based on the activation of G3-mediated transcription by
overexpression of a Pax6 isoform in a non-glucagon-expressing cell
line, it has been suggested that Pax6, acting through the PISCES motif,
may be required for pancreatic islet hormone gene transcription
in vivo (22). The lack of PISCES-mediated transcriptional
activity in normal islets in vivo, as determined in the
present study using transgenic mice, could indicate that islet hormone
gene transcription is maintained in normal islets independently of
Pax6. Alternatively, Pax6 may be essential for islet hormone gene
expression in pancreatic islets and act through the PISCES motif, if a
specific chromatin positioning or sequences in addition to the PISCES
element are necessary for activation of transcription by Pax6 in
pancreatic islets. Thus, Pax6 protein(s) may act through the PISCES
motif in the context of the islet hormone gene promoters. The
regulation of the POMC gene gives an example of a case in which
transgenes containing regulatory sequences were expressed in one of the
expected tissues but not in others, until additional DNA sequences were
included (50). The identification of the sequences necessary and the
proteins that bind to them will be required before conclusions can be
drawn about the role of Pax6 in hormone gene transcription in normal
pancreatic islets.
The results obtained in Pax6-/- mice clearly establish a
role of Pax6 for endocrine cell differentiation in the developing
pancreas (8). With the exception of islet hormone genes, Pax6 target
genes through which Pax6 directs pancreatic islet formation have not
yet been identified. In those genes, Pax6 or Pax65a could act through
PISCES-related or other DNA sites as well as through protein-protein
interaction. Developmental regulation of the splicing of 5'-exons of
the Pax6 gene has been shown in Drosophila, where the form
of the eyeless transcript expressed in the adult stage is
distinct from the embryonic form and also from the larval form (51). An
alternative exon
replacing the exons 03 (corresponding to the
mouse exons 14) has been found in the quail (52). However, no
evidence for development-specific formation of pax6
transcripts in mouse islets was obtained by RT-PCR in the present
study. The nature and relative roles of the Pax6 protein(s) derived
from the Pax6 and Pax65a mRNAs during pancreatic islet development
remain to be defined.
In summary, the present study shows that the PISCES motif is
sufficient to direct expression in the eye and brain, but is not
sufficient to direct expression in pancreatic islets of transgenic
mice, suggesting that the Pax6 protein(s) in normal islets function
differently from the ones in the eye, the brain, and islet cell lines.
Designed to fulfill islet-specific functions, the Pax6 protein(s) in
normal islets could thus expand the functional diversity of the Pax6
gene.
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MATERIALS AND METHODS
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Generation and Analysis of Transgenic Mice
The construction of the PISCES-luciferase reporter gene plasmids
(p4xG3AT81Luc, p4xG3AmutT81Luc) has been described previously (20).
Transgenic mice were generated according to standard procedures (53).
Reporter fusion genes free of vector sequences were obtained by
digestion with BamHI and ApaI, purified, and
microinjected into the male pronucleus of fertilized eggs (FVB/N).
Microinjected eggs were transferred to the oviducts of foster mothers
(CBA/CaJ). Genomic (tail) DNA from the founder mice and offspring was
digested with BglII, electrophoresed, and subjected to
Southern blot analysis using a 1.6-kb XbaI luciferase
fragment as probe [Megaprime DNA labeling system,
(Amersham, Arlington Heights, IL) using
[
-32P]dCTP]. Reporter gene expression was determined
by measuring reporter enzyme activity in tissue extracts or on cryostat
sections. The tissues were homogenized (200 mg wet weight per ml
ice-cold 100 mM K phosphate buffer, pH 7.8, containing 1
mM dithiothreitol, 4 mM EGTA, 4 mM
EDTA, 0.7 mM phenylmethylsulfonyl fluoride, 5 µg/ml
leupeptin, 5 µg/ml pepstatin, and 5 µg/ml aprotinin), subjected to
three cycles of freeze-thawing, and centrifuged. Luciferase activity
was measured in the supernatants as described (54, 55). Protein was
measured using a commercial kit (Bio-Rad Laboratories, Inc., Munich, Germany). Sections (40 µm) were cut with a
cryostat at -26 C and transferred onto slides (Super Frost*/Plus
microscope slides, Menzel-Gläser, Braunschweig, Germany).
Sections were covered with luciferin substrate solution (Luciferase
Assay Kit, Berthold Detection Systems, Pforzheim, Germany), and the
luminescence signal was measured with a Molecular Light Imager (EG&G
Berthold). All animal studies were conducted according to the
"Guidelines for Care and Use of Experimental Animals" and approved
by the Committee on Animal Care und Use of the local institution and
state.
Electrophoretic Mobility Shift Assays
Oligonucleotides with 5'-GATC overhangs were labeled by a
fill-in reaction using [
-32P]dCTP and Klenow enzyme
(56). The oligonucleotides used as probes or competitors have been
described previously: G3, G3A, and G3Amut (23); SCE (57); and P6Con and
5aCon (21). Nuclear extracts from the insulin-producing pancreatic
islet cell line HIT were prepared as described (57). Whole-cell
extracts from the eye and cerebellum of transgenic mice (
150 and 260
mg wet weight per 200 µl extraction buffer) as well as from HIT cells
were prepared following the protocol of Kornhauser et al.
(58). Using 15 µg of nuclear protein or 10 µl of whole-cell
extracts in 25 µl (total volume) reaction buffer containing 18
mM HEPES, pH 7.9, 50 mM NaCl, 90 mM
KCl, 2 mM MgCl2, 0.08 mM EDTA, 0.4
mM EGTA, 5 mM dithiothreitol, 0.4
mM NaF, 2 mM spermidin, 0.2 mM
phenylmethylsulfonyl fluoride, 0.2 µg/ml leupeptin, 0.28 µg/ml
pepstatin, 16 µg/ml bestatin, 0.4 µg/ml aprotinin, 25 µg of BSA,
2 µg of poly(dI-dC), 12% glycerol, and 0.1% NP-40, the assay was
performed as described (23). The Pax6 and Pax65a paired domains were
expressed in E. coli as glutathione-S-transferase
fusion proteins, purified, and used in an electrophoretic mobility
shift assay as described (21, 57).
RT-PCR
Pancreatic islets were isolated as described (59). Poly
(A)+ RNA was extracted from the adult eye, cerebellum,
pancreas, or isolated pancreatic islets using commercial kits (Micro
Fast Track or Fast Track 2.0, Invitrogen, San Diego, CA).
Poly (A)+ RNA from embryonic mouse pancreas (day 11.5/12.0
p.c.) was a generous gift from Dr. Luc St. Onge (DeveloGen,
Göttingen, Germany). RT-PCR was performed using a commercial kit
(Gene Amp Thermostable rTth Reverse Transcriptase RNA PCR Kit, Roche
Molecular Systems, Branchburg, NJ) with primers and PCR reaction
conditions as follows. Primer pair 2/6: upstream primer
5'-ACGAAAGAGAGGATGCCTC-3' (exon 2), downstream primer
5'-CCCAAGCAAAGATGGAAG-3' (exon 6); 30 sec at 95 C, 1 min at 58 C, 3 min
at 72 C, for 40 cycles; the expected products are 457 and 499 bp long
(without and with exon 5a) (30). Primer pair 6/13: upstream primer
5'-CATCTTTGCTTGGGAAATC-3' (exon 6), downstream primer
5'-AACTTGGACGGGAACTGAC-3' (exon 13); 30 sec at 95 C, 1 min at 59 C, 2
min at 72 C, for 40 cycles; the expected product is 933 bp long (30).
After agarose gel electrophoresis, the products obtained were verified
by extraction, subcloning (pCR 2.1, Invitrogen), and cycle
sequencing (Thermo Sequenase fluorescent-labeled primer cycle
sequencing kit, Amersham, Braunschweig, Germany; M13
fluorescent primer).
 |
ACKNOWLEDGMENTS
|
---|
We thank Richard L. Maas (Harvard Medical School, Boston, MA)
for pGEX-Pax6 and pGEX-Pax65a, Simon Saule (Institut Pasteur, Lille,
France) for anti-Pax6 antiserum, Klaus Becker, Ksenia Sovic, and Arnold
Hasselblatt (University of Göttingen, Göttingen, Germany)
for islet preparation, and Rainer Kembügler (EG&G
Berthold, Bad Wildbad, Germany) for his help with the Molecular
Light Imager. We thank Luc St. Onge (DeveloGen, Göttingen,
Germany) for the generous gift of poly (A)+ RNA from
embryonic pancreas and for instructing us how to dissect pancreatic
tissue from mouse embryos.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Willhart Knepel, Department of Molecular Pharmacology, University of Göttingen, Robert-Koch-Strasse 40, D-37075 Göttingen, Germany. E-mail:
wknepel{at}med.uni-goettingen.de
This work was supported by the Deutsche Forschungsgemeinschaft,
SFB402/A3.
Received for publication May 8, 1998.
Revision received February 1, 1999.
Accepted for publication February 3, 1999.
 |
REFERENCES
|
---|
-
Habener JF, Stoffers DA 1998 A newly discovered role of
transcription factors involved in pancreas development and the
pathogenesis of diabetes mellitus. Proc Assoc Am Physicians 110:1221[Medline]
-
Jonsson J, Carlsson L, Edlund T, Edlund H 1994 Insulin-promoter-factor 1 is required for pancreas development in mice.
Nature 371:606609[CrossRef][Medline]
-
Ahlgren U, Pfaff SL, Jessell TM, Edlund T, Edlund H 1997 Independent requirement for ISL1 in formation of pancreatic mesenchyme
and islet cells. Nature 385:257260[CrossRef][Medline]
-
Naya FJ, Huang HP, Qiu Y, Mutoh H, DeMayo FJ, Leiter AB, Tsai
MJ 1997 Diabetes, defective pancreatic morphogenesis, and abnormal
enteroendocrine differentiation in BETA2/NeuroD-deficient mice. Genes
Dev 11:23232334[Abstract/Free Full Text]
-
Mansouri A, Hallonet M, Gruss P 1996 Pax genes and
their roles in cell differentiation and development. Curr Opin Cell
Biol 8:851857[CrossRef][Medline]
-
Callaerts P, Halder G, Gehring WJ 1997 Pax-6 in
development and evolution. Annu Rev Neurosci 20:483532[CrossRef][Medline]
-
Sosa-Pineda B, Chowdhury K, Torres M, Oliver G, Gruss P 1997 The Pax4 gene is essential for differentiation of
insulin-producing ß cells in the mammalian pancreas. Nature 386:399402[CrossRef][Medline]
-
St-Onge L, Sosa-Pineda B, Chowdhury K, Mansouri A, Gruss P 1997 Pax6 is required for differentiation of
glucagon-producing
-cells in mouse pancreas. Nature 387:406409[CrossRef][Medline]
-
Leonard J, Peers B, Johnson T, Ferreri K, Lee S, Montminy MR 1993 Characterization of somatostatin transactivating factor-1, a novel
homeobox factor that stimulates somatostatin expression in pancreatic
islet cells. Mol Endocrinol 7:12751283[Abstract]
-
Miller CP, McGehee Jr RE, Habener JF 1994 IDX-1: a new
homeodomain transcription factor expressed in rat pancreatic islets and
duodenum that transactivates the somatostatin gene. EMBO J 13:11451156[Abstract]
-
Guz Y, Montminy MR, Stein R, Leonard J, Gamer LW, Wright CVE,
Teitelman G 1995 Expression of Stf-1, a putative insulin gene
transcription factor, in b-cells of pancreas, duodenal epithelium and
pancreatic exocrine and endocrine progenitors during ontogeny.
Development 121:1118[Abstract/Free Full Text]
-
Ohlsson H, Karlsson K, Edlund T 1993 IPF1, a
homeodomain-containing transactivator of the insulin gene. EMBO J 12:42514259[Abstract]
-
Peers B, Leonard J, Sharma S, Teitelman G, Montminy MR 1994 Insulin expression in pancreatic islet cells relies on cooperative
interactions between the helix loop helix factor E47 and the homeobox
factor STF-1. Mol Endocrinol 8:17981806[Abstract]
-
Naya FJ, Stellrecht CMM, Tsai MJ 1995 Tissue-specific
regulation of the insulin gene by a novel basic helix-loop-helix
transcription factor. Genes Dev 9:10091019[Abstract]
-
Wang M, Drucker DJ 1995 The LIM domain homeobox gene
isl-1 is a positive regulator of islet cell-specific
proglucagon gene transcription. J Biol Chem 270:1264612652[Abstract/Free Full Text]
-
Philippe J, Chick WL, Habener JF 1987 Multipotential
phenotypic expression of genes encoding peptide hormones in rat
insulinoma cell lines. J Clin Invest 79:351358[Medline]
-
Clark SA, Burnham BL, Chick WL 1990 Modulation of
glucose-induced insulin secretion from a rat clonal ß-cell line.
Endocrinology 127:27792788[Abstract]
-
Alpert S, Hanahan D, Teitelman G 1988 Hybrid insulin genes
reveal a developmental lineage for pancreatic endocrine cells and imply
a relationship with neurons. Cell 53:295308[Medline]
-
Knepel W, Vallejo M, Chafitz JA, Habener JF 1991 The
pancreatic islet-specific glucagon G3 transcription factors recognize
control elements in the rat somatostatin and insulin-I genes. Mol
Endocrinol 5:14571466[Abstract]
-
Wrege A, Diedrich T, Hochhuth C, Knepel W 1995 Transcriptional
activity of domain A of the rat glucagon G3 element conferred by an
islet-specific nuclear protein that also binds to similar pancreatic
islet cell-specific enhancer sequences (PISCES). Gene Expr 4:205216[Medline]
-
Epstein JA, Glaser T, Cai J, Jepeal L, Walton DS, Maas RL 1994 Two independent and interactive DNA-binding subdomains of the Pax6
paired domain are regulated by alternative splicing. Genes Dev 8:20222034[Abstract]
-
Sander M, Neubüser A, Kalamaras J, Ee HC, Martin GR,
German MS 1997 Genetic analysis reveals that Pax6 is required for
normal transcription of pancreatic hormone genes and islet development.
Genes Dev 11:16621673[Abstract]
-
Knepel W, Jepeal L, Habener JF 1990 A pancreatic islet
cell-specific enhancer-like element in the glucagon gene contains two
domains binding distinct cellular proteins. J Biol Chem 265:87258735[Abstract/Free Full Text]
-
Kruse F, Rose SD, Swift GH, Hammer RE, MacDonald RJ 1993 An
endocrine-specific element is an integral component of an
exocrine-specific pancreatic enhancer. Genes Dev 7:774786[Abstract]
-
Verweij CL, Guidos C, Crabtree GR 1990 Cell type specificity
and activation requirements for NFAT-1 (nuclear factor of activated
T-cells) transcriptional activity determined by a new method using
transgenic mice to assay transcriptional activity of an individual
nuclear factor. J Biol Chem 265:1578815795[Abstract/Free Full Text]
-
Lernbecher T, Müller U, Wirth T 1993 Distinct
NF-
B/Rel transcription factors are responsible for tissue-specific
and inducible gene activation. Nature 365:767770[CrossRef][Medline]
-
Rincón M, Flavell RA 1996 Regulation of AP-1 and NFAT
transcription factors during thymic selection of T cells. Mol Cell Biol 16:10741084[Abstract]
-
Rincón M, Flavell RA 1997 Transcription mediated by NFAT
is highly inducible in effector CD4+ T helper 2 (Th2) cells
but not in Th1 cells. Mol Cell Biol 17:15221534[Abstract]
-
Stoykova A, Gruss P 1994 Roles of pax-genes in
developing and adult brain as suggested by expression patterns. J
Neurosci 14:13951412[Abstract]
-
Walther C, Gruss P 1991 Pax-6, a murine paired box
gene, is expressed in the developing CNS. Development 113:14351449[Abstract]
-
Davis JA, Reed RR 1996 Role of Olf-1 and Pax-6 transcription
factors in neurodevelopment. J Neurosci 16:50825094[Abstract/Free Full Text]
-
Turque N, Plaza S, Radvanyi F, Carriere C, Saule S 1994 Pax-QNR/Pax6, a paired box- and homeobox-containing gene
expressed in neurons, is also expressed in pancreatic endocrine cells.
Mol Endocrinol 8:929938[Abstract]
-
Ton CCT, Hirvonen H, Miwa H, Weil MM, Monaghan P, Jordan T,
van Heyningen V, Hastie ND, Meijers-Heijboer H, Drechsler M,
Royer-Pokora B, Collins F, Swaroop A, Strong LC, Saunders GF 1991 Positional cloning and characterization of a paired box- and
homeobox-containing gene from the aniridia region. Cell 67:10591074[Medline]
-
Macdonald R, Wilson SW 1996 Pax proteins and eye development.
Curr Opin Neurobiol 6:4956[CrossRef][Medline]
-
Zeman W, Innes JRM 1963 Craigies Neuroanatomy of the Rat.
Academic Press Inc, New York
-
Hitchcock PF, Macdonald RE, VanDeRyt JT, Wilson SW 1996 Antibodies against Pax6 immunostain amacrine and ganglion cells and
neuronal progenitors, but not rod precursors, in the normal and
regenerating retina of the goldfish. J Neurobiol 29:399413[CrossRef][Medline]
-
Glaser T, Jepeal L, Edwards JG, Young SR, Favor J, Maas RL 1994 Pax6 gene dosage effect in a family with congenital
cataracts, aniridia, anophthalmia and central nervous system defects.
Nat Genet 7:463471[Medline]
-
Hanson I, Van Heyningen V 1995 Pax6: more than meets the eye.
Trends Genet 11:268272[CrossRef][Medline]
-
Efrat S, Teitelman G, Anwar M, Ruggiero D, Hanahan D 1988 Glucagon gene regulatory region directs oncoprotein expression to
neurons and pancreatic
cells. Neuron 1:605613[Medline]
-
Lee YC, Campos RV, Drucker DJ 1993 Region- and age-specific
differences in proglucagon gene expression in the central nervous
system of wild-type and glucagon-simian virus-40 T-antigen transgenic
mice. Endocrinology 133:171177[Abstract]
-
Drucker DJ, Asa S 1988 Glucagon gene expression in vertebrate
brain. J Biol Chem 263:1347513478[Abstract/Free Full Text]
-
Kiyama H, Emson PC 1990 Distribution of somatostatin mRNA in
the rat nervous system as visualized by a novel non-radioactive
in situ hybridization histochemistry procedure. Neuroscience 38:223244[CrossRef][Medline]
-
Larsen JNB 1995 Somatostatin in the retina. Acta Ophthalmol
Scand Suppl 218:124[Medline]
-
Cvekl A, Kashanchi F, Sax CM, Brady JN, Piatigorsky J 1995 Transcriptional regulation of the mouse
A-crystallin gene:
activation dependent on a cyclic AMP-responsive element (DE1/CRE) and a
Pax-6-binding site. Mol Cell Biol 15:653660[Abstract]
-
Holst BD, Wang Y, Jones FS, Edelman GM 1997 A binding site for
Pax proteins regulates expression of the gene for the neural cell
adhesion molecule in the embryonic spinal cord. Proc Natl Acad Sci USA 94:14651470[Abstract/Free Full Text]
-
Hussain MA, Lee J, Miller CP, Habener JF 1997 POU domain
transcription factor brain 4 confers pancreatic
-cell-specific
expression of the proglucagon gene through interaction with a novel
proximal promoter G1 element. Mol Cell Biol 17:71867194[Abstract]
-
Plaza S, Langlois MC, Turque N, LeCornet S, Bailly M,
Bègue A, Quatannens B, Dozier C, Saule S 1997 The
homeobox-containing engrailed (En-1) product downregulates the
expression of Pax-6 through a DNA binding-independent mechanism. Cell
Growth Differ 8:11151125[Abstract]
-
Rudnick A, Ling TY, Odagiri H, Rutter WJ, German MS 1994 Pancreatic beta cells express a diverse set of homeobox genes. Proc
Natl Acad Sci USA 91:1220312207[Abstract/Free Full Text]
-
Duncan MK, Haynes JIII, Cvekl A, Piatigorsky J 1998 Dual roles
for Pax6: a transcriptional repressor of lens fiber cell-specific
ß-crystallin genes. Mol Cell Biol 18:55795586[Abstract/Free Full Text]
-
Young JI, Otero V, Cerdán MG, Falzone TL, Chan EC, Low
MJ, Rubinstein M 1998 Authentic cell-specific and developmentally
regulated expression of pro-opiomelanocortin genomic fragments in
hypothalamic and hindbrain neurons of transgenic mice. J Neurosci 18:66316640[Abstract/Free Full Text]
-
Sheng G, Thouvenot E, Schmucker D, Wilson DS, Desplan C 1997 Direct regulation of rhodopsin 1 by Pax-6/eyeless
in Drosophila: evidence for a conserved function in
photoreceptors. Genes Dev 11:11221131[Abstract]
-
Dozier C, Carrière C, Grévin D, Martin P,
Quatannens B, Stéhelin D, Saule S 1993 Structure and DNA-binding
properties of Pax-QNR, a paired box- and homeobox-containing
gene. Cell Growth Differ 4:281289[Abstract]
-
Hogan B, Beddington R, Costantini F, Lacy E 1994 Manipulating
the Mouse Embryo, ed 2. Cold Spring Harbor Laboratory, Cold Spring
Harbor, NY
-
Schwaninger M, Lux G, Blume R, Oetjen E, Hidaka H, Knepel W 1993 Membrane depolarization and calcium influx induce glucagon gene
transcription in pancreatic islet cells through the cyclic
AMP-responsive element. J Biol Chem 268:51685177[Abstract/Free Full Text]
-
Schwaninger M, Blume R, Krüger M, Lux G, Oetjen E,
Knepel W 1995 Involvement of the Ca2+-dependent phosphatase
calcineurin in gene transcription that is stimulated by cAMP through
cAMP response elements. J Biol Chem 270:88608866[Abstract/Free Full Text]
-
Fürstenau U, Schwaninger M, Blume R, Kennerknecht I,
Knepel W 1997 Characterization of a novel protein kinase C response
element in the glucagon gene. Mol Cell Biol 17:18051816[Abstract]
-
Oetjen E, Diedrich T, Eggers A, Eckert B, Knepel W 1994 Distinct properties of the cAMP-responsive element of the rat insulin I
gene. J Biol Chem 269:2703627044[Abstract/Free Full Text]
-
Kornhauser JM, Nelson DE, Mayo KE, Takahashi JS 1992 Regulation of jun-B messenger RNA and AP-1 activity by light
and a circadian clock. Science 255:15811584[Medline]
-
Schulz A, Hasselblatt A 1988 Phentolamine, a deceptive tool to
investigate sympathetic nervous control of insulin release. Naunyn
Schmiedebergs Arch Pharmacol 337:637643[Medline]