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Molecular and Cellular Biology, June 2000, p. 4445-4454, Vol. 20, No. 12
Hormone and Metabolic Research Unit,
Université catholique de Louvain and Christian de Duve Institute
of Cellular Pathology,1 and Department
of Pathology, Université catholique de
Louvain,3 1200 Brussels, Center for
Transgene Technology and Gene Therapy, Flanders Interuniversity
Institute for Biotechnology, 3000 Leuven,5
Belgium; Department of Developmental Biology, Hagedorn Research
Institute, 2820 Gentofte, Denmark2; and
Institut de Génétique et de Biologie
Moléculaire et Cellulaire, CNRS/INSERM, Université Louis
Pasteur, 67404 Illkirch Cedex, C. U. Strasbourg,
France4
Received 4 October 1999/Returned for modification 28 October
1999/Accepted 7 March 2000
Hepatocyte nuclear factor 6 (HNF-6) is the prototype of a new class
of cut homeodomain transcription factors. During mouse development,
HNF-6 is expressed in the epithelial cells that are precursors of the
exocrine and endocrine pancreatic cells. We have investigated the role
of HNF-6 in pancreas differentiation by inactivating its gene in the
mouse. In hnf6 The pancreas contains exocrine cells
that produce digestive enzymes, ducts through which these enzymes
transit to the gut, and endocrine cells, organized in islets of
Langerhans, that produce insulin, glucagon, somatostatin, and
pancreatic polypeptide (PP). The pancreas arises from the primitive
gut epithelium as a dorsal bud and a ventral bud, which later fuse to
form a single organ (reviewed in reference 29). The
pancreatic epithelium, surrounded by mesenchyme, then proliferates and
branches into multiple evaginations. During the initial stage of
pancreas development (embryonic day 9.5 [e9.5] to approximately e14.5
in the mouse), the pancreatic epithelium consists mainly of cells that
are the precursors of the acinar, ductal, and endocrine cells
(29). Starting at e9.5, early glucagon-expressing cells are
found in the epithelium. Around e12, glucagon cells are found both
within the epithelium and in small clusters that are distinct from the
epithelium. The fate of these clusters is unknown. Starting around
e14.5 to e15, a wave of differentiation is associated with an increase
in the synthesis of digestive enzymes and hormones, ultimately
resulting in the formation of the acini, ducts, and islets of
Langerhans around e18 to e19. The insulin-expressing cells ( A number of transcription factors are involved in endocrine pancreas
development (reviewed in references 8 and
25). They belong to the class of basic
helix-loop-helix factors (Ngn-3, NeuroD/Beta2, and Hes-1) or are
homeoproteins of the LIM (Isl-1), paired-box (Pax-4 and Pax-6),
Antennapedia (Hb9 and Pdx-1; also known as IPF-1, IDX-1, STF-1, IUF-1,
and GSF), or NK-2 (Nkx2.2) class. Targeted disruption of the genes
coding for these transcription factors indicated that they are required
for pancreatic morphogenesis and/or for the differentiation of one or
several pancreatic endocrine cell types (1, 2, 10, 13, 17, 18, 23,
24, 26, 31, 33-36). The temporal and cellular expression
profiles of these factors and the results of gene disruption
experiments led to the proposal that pancreatic endocrine cell
differentiation relies on the activation of a cascade of transcription
factors (8, 16). Moreover, it was proposed recently that
pancreatic endocrine cell differentiation is controlled by a lateral
specification mechanism involving the Notch signaling pathway, in which
Ngn-3 is the earliest marker of endocrine cell precursors (3, 10, 17).
We isolated previously a cDNA coding for the transcription factor
hepatocyte nuclear factor 6 (HNF-6) (22). HNF-6 belongs to
the new ONECUT class of cut homeodomain proteins, whose members contain
a single cut domain and a divergent homeodomain (21, 22). In
the mouse embryo, hnf6 is expressed in several tissues, including the epithelial cells of the pancreas, starting at the onset
of its development (20, 30). During formation of the acini,
ducts, and islets, the expression of hnf6 becomes restricted to the acini and ductal cells (20, 30). Transient
transfection experiments have identified target genes for HNF-6. These
include hnf3 Construction of targeting vector and generation of knockout
mice.
The targeting construct was made by subcloning
hnf6 gene fragments from a strain 129 mouse genomic library
in the pPNT vector. R1 embryonic stem (ES) cells (a gift from A. Nagy)
derived from blastocysts of mouse strain 129 were electroporated with
the linearized construct and were selected with G418 and ganciclovir.
Independent ES clones containing an inactivated hnf6 allele
were aggregated with Swiss strain morula-stage embryos as described
previously (6), and the embryos were transferred into
pseudopregnant Swiss foster mothers. Two chimeric males from
independent clones were test bred with Swiss mice for germ line
transmission. Heterozygous offspring was intercrossed to generate
hnf6 Immunohistochemistry.
Dissected pancreas, fixed in Bouin's
solution or in 4% paraformaldehyde in phosphate-buffered saline, was
embedded in paraffin, sectioned at 5 µm, and immunostained. Primary
antibodies were mouse antiinsulin (Novo BioLabs clone HUI-018), mouse
antiglucagon (Novo BioLabs clone GLU-001), rabbit anti-islet amyloid
polypeptide, rabbit anti-glucose transporter 2 (Glut-2) (Chemicon
AB1342 or gift from B. Thorens), rabbit anti-Pdx-1, mouse
antisomatostatin (Novo BioLabs clone SOM-018), rabbit anti-PP (Lilly),
rabbit anti-Pax-6, mouse anti-Isl-1, and rabbit anti-Nkx6.1. Primary
antibodies were detected by immunoperoxidase using biotinylated sheep
anti-rabbit or anti-mouse immunoglobulin G (Boehringer Mannheim/Roche),
a streptavidin-peroxidase conjugate (Boehringer Mannheim/Roche), and
DAB+ (Dako). For immunofluorescence, primary antibodies were detected
with secondary antibodies coupled to Texas red or Cy-2 (Jackson Immunochemicals).
In situ hybridization.
Nonradioactive in situ
cohybridization with RNA antisense probes labeled with digoxigenin-UTP
or fluorescein-UTP was performed as described in reference
9. The Ngn-3 probe is 0.75 kb long and encompasses
the entire Ngn-3 coding sequence (7). The HNF-6 probe spans
nucleotides 1214 to 1607 of the mouse HNF-6 cDNA (nucleotide numbering
as in GenBank accession no. U95945).
Multiplex RT-PCR analysis.
Multiplex reverse
transcription-PCR (RT-PCR), which allows coamplification of several
cDNAs from total RNA, was performed as described elsewhere
(15). Amplification products were quantified using a
PhosphorImager, and relative mRNA concentrations were calculated as
ratios to the coamplified internal standard. Primer sequences were
5'-TGGCGCCTCATCCCTTGGATG-3' and
5'-CAGTCACCCACTTCTGCTTCG-3' (Ngn-3),
5'-CTGGTTCCCTGAGGGTTTCAA-3' and
5'-GGAACTTCTTGGTCTCCAGGT-3' (Notch-1),
5'-CAACATGGGCCGCTGTCCTC-3' and
5'-CACATCTGCTTGGCAGTTGATC-3' (Notch-2),
5'-GCAGCTGTGAACAACGTGGAG-3' and
5'-AACCGCACAATGTCCTGGTGC-3' (Notch-3),
5'-TCAACACGACACCGGACAAACC-3' and
5'-GGTACTTCCCCAACACGCTCG-3' (Hes-1),
5'-ACCCTTCACCAATGACTCCTATG-3' and
5'-ATGATGACTGCAGCAAATCGC-3' (TATA binding protein [TBP]),
and 5'-GACCTGCAGAGCTCCAATCAAC-3' and
5'-CACGACCCTCAGTACCAAAGGG-3' (glucose-6-phosphate
dehydrogenase [G6PDH]), 5'-CAGCACCTCACGCCCACCTC-3' and
5'-CTTCCCATGTTCTTGCTCTTTCC-3' (HNF-6).
Cloning of the mouse ngn3 gene promoter.
A
7.0-kb-long genomic XbaI-XhoI fragment was cloned
in pBluescript (10), and a 5.1-kb region from 4.95 kb
upstream to 0.15 kb downstream of the ATG initiator codon was sequenced
using Li-Cor 4000L and Beckman CEQ 2000 automated DNA sequencers.
Transfections.
Rat-1 fibroblasts were grown in Dulbecco's
modified Eagle medium (DMEM) supplemented with 10% fetal calf serum.
Cells (3 × 105) grown in serum-free DMEM on
60-mm-diameter dishes were cotransfected by lipofection using DOTAP and
3 µg of a reporter vector containing the ngn3 gene
promoter cloned upstream of the firefly luciferase gene, 1.5 µg of
the pECE-HNF6 Electrophoretic mobility shift assays (EMSA).
Recombinant
HNF-6 was in vitro transcribed and translated using a wheat germ
extract (TNT expression system; Promega). Five microliters of the
reaction mix was incubated on ice for 20 min in a final volume of 20 µl containing 10 mM HEPES (pH 7.6), 1 mM dithiothreitol, 1 mM
MgCl2, 0.5 mM EGTA, 50 mM KCl, 10% (vol/vol) glycerol,
3 µg of poly(dI-dC), and the 32P-labeled probe
(30,000 cpm). The samples were loaded on a 6% acrylamide gel
(acrylamide/bisacrylamide ratio of 29:1) in 0.25× Tris-borate-EDTA
buffer and electrophoresed at 200 V. The sense-strand sequences of the
double-stranded oligonucleotide probes, derived from the mouse
ngn3 gene, were
5'-CTTCCCGATAGCATCCATAGTGGGGCGGGG-3' (proximal site) and
5'-GCTCAGTGCCAAATCCATGTGTCAGCTTCT-3'
(distal site) (the HNF-6 binding consensus is underlined).
Glucose, insulin, and glucagon measurements.
Blood glucose
(tail vein) was measured using an Elite glucometer (Bayer). Insulin and
glucagon levels were measured by radioimmunoassay on 25 µl of plasma
using the sensitive rat insulin radioimmunoassay and glucagon
radioimmunoassay pancreas-specific kits (Linco).
Targeted disruption of the hnf6 gene and generation of
HNF-6 knockout mice.
We inactivated the hnf6 gene in
the mouse by homologous recombination (Fig.
1). A neomycin resistance gene
(neo) cassette was used to replace the proximal promoter
region and the first exon. This exon codes for a region of the protein
that is essential for DNA binding and transcriptional activity
(21). The strategy for disrupting the hnf6 gene
is shown in Fig. 1A. The recombinant ES clones were identified by
Southern blotting using probes located 5' and 3' from the recombination
site (Fig. 1B). The resulting transgenic mice were genotyped by PCR as
in Fig. 1C. Lack of hnf6 expression in
hnf6
0270-7306/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.
Transcription Factor Hepatocyte Nuclear Factor 6 Regulates Pancreatic Endocrine Cell Differentiation and Controls
Expression of the Proendocrine Gene ngn3
ABSTRACT
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ embryos, the exocrine
pancreas appeared to be normal but endocrine cell differentiation was
impaired. The expression of neurogenin 3 (Ngn-3), a transcription
factor that is essential for determination of endocrine cell
precursors, was almost abolished. Consistent with this, we demonstrated
that HNF-6 binds to and stimulates the ngn3 gene promoter.
At birth, only a few endocrine cells were found and the islets of
Langerhans were missing. Later, the number of endocrine cells increased
and islets appeared. However, the architecture of the islets was
perturbed, and their 
cells were deficient in glucose transporter 2 expression. Adult hnf6/ mice were diabetic.
Taken together, our data demonstrate that HNF-6 controls pancreatic
endocrine differentiation at the precursor stage and identify HNF-6 as
the first positive regulator of the proendocrine gene ngn3
in the pancreas. They also suggest that HNF-6 is a
candidate gene for diabetes mellitus in humans.
INTRODUCTION
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
cells)
are then in the core of the islets, and the cells expressing glucagon (
cells), somatostatin (
cells), and PP are located at their periphery (reviewed in references 29 and
32).
, which is coexpressed with hnf6
in the developing pancreatic epithelium (20, 30). Since the
expression of Pdx-1, a factor whose absence leads to pancreatic
agenesis (18), is controlled by HNF-3 (36) and
since HNF-3 expression is stimulated by HNF-6, it has been proposed
that HNF-6 is a key component of the pancreatic transcription factor
cascade (11). To investigate the role of HNF-6 in pancreatic
cell differentiation, we inactivated its gene by homologous
recombination in the mouse.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ progeny. The progeny derived from each
of these two males displayed the same phenotype. The phenotype of the
hnf6+/ mice was normal. These mice were
therefore used as controls together with wild-type littermates.
expression vector, and 500 ng of pRL138 plasmid coding
for Renilla luciferase as internal control. After 5 h,
the medium was replaced by DMEM plus 10% fetal calf serum and further
incubated for 40 h before measuring luciferase activities with a
Dual-Luciferase kit (Promega). Luciferase activities were measured with
a TD-20/20 Luminometer (Promega) and expressed as the ratio of reporter
activity (firefly luciferase) to internal control activity
(Renilla luciferase). pECE-HNF6 and pRL138 have been
described elsewhere (21, 22). The ngn3 promoter
reporter vector p3957ngn3-luc contains a 3,957-bp-long
BstuI-BstuI genomic fragment (from 
4573 to
617 relative to the ATG initiator codon) cloned in the
SmaI site of pGL3 basic (Promega).
RESULTS
Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
/ pancreas was confirmed by RT-PCR (Fig.
1D). hnf6+/ mice were phenotypically normal.
Cross-breeding of these mice produced hnf6/
offspring at the frequency of 22% (83/376). The
hnf6/ mice were growth retarded at birth and
showed a reduced growth rate (Fig. 1E). Seventy-five percent of them
died between postnatal day 1 (P1) and P10, probably as a result of
liver failure. The surviving mice reached adulthood.
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FIG. 1.
Targeted disruption of the hnf6 gene and
generation of hnf6 / mice. (A) Scheme of the
hnf6 gene, targeting construct, and product of homologous
recombination. The three exons are shown as black boxes. Cut and homeo
refer to the cut domain and homeodomain; neo and tk refer to the
neomycin resistance and thymidine kinase genes. The NotI
site derived from the polylinker of a genomic phage clone. (B) Southern
blot analysis of DNA isolated from six ES cell clones resistant to G418
and ganciclovir. Correct homologous recombination at the 5' end was
confirmed by hybridization of EcoRI-digested DNA with the 5'
probe. This probe detected an 8.5-kb wild-type and a 6.5-kb recombinant
fragment. Correct homologous recombination at the 3' end was confirmed
by hybridization of ScaI-digested DNA with the 3' probe,
which detected a 12.5-kb wild-type and an 8.5-kb recombinant fragment.
(C) PCR genotyping of tail DNA. Primer pairs amplifying sequences
neo (5'-CTGTGCTCGACGTTGTCACTG-3' and
5'-GATCCCCTCAGAAGAACTCGT-3') and of exon 1 of the
hnf6 gene (5'-CAGCACCTCACGCCCACCTC-3' and
5'-CAGCCACTTCCACATCCTCCG-3') were added simultaneously in
the PCR. (D) Multiplex RT-PCR analysis of total RNA from e14.5 pancreas
with primers located in exons 1 and 3 of the hnf6 gene and
with sequences of TBP as internal control. hnf6 mRNA was
undetectable in hnf6/ pancreas, and the
ratio of HNF-6 to TBP amplification products in
hnf6+/ mice was 52% of that in
hnf6+/+ mice. (E) Weight gain curve of a
representative hnf6/ mouse shows a reduced
growth rate compared to a wild-type littermate.
Defective endocrine cell differentiation in HNF-6 knockout
mice.
To analyze the pancreas of hnf6/
mice, we first performed histological and immunohistochemical
examinations on embryos at various stages of development. Exocrine
tissue was histologically normal. In keeping with this, the expression
of the exocrine marker p48 (19) was normal at e14.5 (not
shown). In contrast, endocrine development was severely inhibited in
all embryos. Indeed, the number of glucagon-expressing cells was
reduced by 85% in hnf6/ embryos compared to
control embryos at e12.5 and e15.5 (Fig. 2C to F). This reduction involved the
glucagon-expressing cells found interspersed in the epithelium.
However, glucagon expression was normal at e10.75 (Fig. 2A and B), and
the number of clusters of early glucagon cells was normal at e12.5 and
e15.5 (arrows in Fig. 2C to F). No insulin was found at e15.5 in
hnf6/ embryos, in contrast to control
animals, in which insulin-expressing cells were found scattered
throughout the epithelium (Fig. 2I and J). At P4,
hnf6/ animals showed a markedly reduced
number of cells (Fig. 2G and H), cells (Fig. 2K and L), cells (Fig. 2M and N), and PP cells (Fig. 2O and P). These four cell
types were scattered within, and in the vicinity of, pancreatic ducts,
instead of being clustered in typical islets as in control littermates.
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/ mice, the endocrine cells became
organized in islets only 2 to 3 weeks after birth. The architecture of
the islets was abnormal, as they showed no mantle of cells around
the core of cells at 5 weeks (Fig. 3A
and B) and at 10 weeks many cells were found throughout the islets,
instead of being located at their periphery (not shown). The
differentiation state of islet cells in 5-week-old
hnf6/ mice was investigated by monitoring
the coexpression of hormonal and metabolic markers. Glut-2, which in
wild-type mice is expressed in cells, was undetectable by
immunofluorescence in most of the hnf6/
mice. In the hnf6/ mice in which some Glut-2
could be visualized, its expression was markedly reduced (Fig. 3C and
D). Unlike in control mice, IAPP (amylin) was absent from several
insulin-positive cells (Fig. 3E and F).
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/ mice, (i)
endocrine pancreas development is severely inhibited; (ii) the
appearance of the islets of Langerhans is delayed; (iii) when the
islets form, they display a perturbed architecture and contain cells
that have not reached full maturity.
Analysis of differentiation markers in
hnf6/ mice.
To explore how endocrine
pancreas development is perturbed in hnf6/
embryos, we analyzed by immunohistochemistry the expression of pancreatic transcription factors at various stages of development. Pdx-1 and Nkx6.1 are markers of the early pancreatic epithelium. Their
expression indicates that endodermal cells have been specified to a
pancreatic fate (27). Isl-1 and Pax-6 are expressed in postmitotic cells that have started to differentiate into either one of
the four endocrine cell types (1, 8, 31, 34).
/ embryos
than in control embryos (Fig. 4M to P and S to V). In line with the
normal expression of glucagon in the early glucagon cell clusters (Fig.
2D and F), the expression of isl1 and pax6 was
detected at normal levels in these cell clusters (arrows in Fig. 4M, N,
S, T, and V).
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/
embryos showed cystic structures. These were delineated by cells that
had characteristics of the early pancreatic epithelium, as they all
expressed pdx1 and nkx6.1 (Fig. 4F and J). Within
the epithelium lining the cysts, a few cells expressed the endocrine
markers isl1 and pax6 (Fig. 4P and V). After
birth, the cysts developed as enlarged ducts or as large cavities (up
to 2 cm). Their epithelium expressed pdx1 (Fig. 3H),
contrary to normal ductal cells (Fig. 3G). Insulin- or
glucagon-expressing cells were then detected within, or in close
association with, the epithelium lining the cysts (Fig. 3B, D, and F)
or the pancreatic ducts (Fig. 3I and J). Insulin-negative, Glut-2-positive cells were also seen in the epithelium lining the cysts
at 5 weeks (data not shown), which is interesting since glut2 is expressed not only in mature cells but also in
the undifferentiated pancreatic epithelium (28).
We conclude from these experiments that in the absence of HNF-6, the
pancreatic epithelium is specified. However, this epithelium fails to
give rise, between e10.75 and e12.5, to the expected pool of
isl1- and pax6-expressing cells. Instead, this
epithelium generates cystic structures delineated by pdx1-
and nkx6.1-expressing cells.
Reduced expression of the proendocrine gene ngn3 in
hnf6/ embryos.
A lateral specification
mechanism is involved in pancreatic endocrine cell differentiation
(3). In this mechanism, the proendocrine gene
ngn3, which is the earliest marker of endocrine cell
precursors, plays a dual role. First, Ngn-3 is essential for endocrine
differentiation (3, 10). Second, it is proposed that Ngn-3
stimulates the synthesis of Notch ligands, which activate the Notch
pathway in neighboring Notch-expressing cells. This increases the
expression of hes1, which decreases that of ngn3 and consequently inhibits endocrine differentiation.
/ embryos. In contrast, the mRNA coded
by ngn3 was nearly undetectable in
hnf6/ embryos. Consistent with this, in situ
hybridization experiments showed that the number of
ngn3-positive cells was strongly reduced in e14.5
hnf6/ embryos compared to wild-type embryos
(Fig. 5B). In the few ngn3-positive cells, labeling was
weaker than in wild-type embryos. These observations suggested that
HNF-6 controls directly or indirectly the expression of the
ngn3 gene. We therefore determined by in situ
cohybridization if ngn3 and hnf6 are coexpressed
in the pancreatic epithelium of normal embryos during development. The
results showed that hnf6 is expressed throughout the
pancreatic epithelium and that the ngn3-positive cells
coexpress hnf6 (Fig. 5C and data not shown). To investigate
whether HNF-6 can directly control transcription of the ngn3
gene, we searched for HNF-6 binding sites in the ngn3 promoter. As no information on this promoter was available, we cloned a
fragment of the mouse ngn3 gene (10) and
sequenced 4.95 kb upstream of the coding region. We identified a TATA
box consensus and found two nucleotide sequences that match
(21) the HNF-6 DNA-binding consensus
5'-(A/T/G)(A/T)(A/G)TC(A/C)ATN(A/T/G)-3'. These are located
453 bp (5'-GCATCCATAG-3') and 3,187 bp
(5'-AAATCCATGT-3') upstream of the TATA box. In EMSA, each
of these sites bound in vitro-synthesized HNF-6 (Fig. 5D). The
difference in the intensity of the retarded complexes generated with
the two probes indicated that the distal site binds HNF-6 with higher
affinity than the proximal one. To test if HNF-6 can stimulate
transcription from the ngn3 gene promoter, transient
transfection experiments were then performed with a 3,957-bp-long
ngn3 gene fragment containing the two HNF-6 sites cloned
upstream of the luciferase gene. The results showed that this
ngn3 gene fragment displays promoter activity and that
cotransfection of an HNF-6 expression vector stimulates this activity
fourfold (Fig. 5E).
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Perturbed glucose homeostasis in hnf6/
mice.
The reduction in the number of endocrine cells and the
absence of islets of Langerhans at birth prompted us to determine how glycemia is controlled in the postnatal period. Blood glucose, glucagon, and insulin levels were measured in 4-day-old mice. The mice
were starved for 4 h to standardize for the feeding status. Figure
6A shows that blood glucose levels were
significantly lower in hnf6/ mice than in
their wild-type littermates. In hnf6/ mice
the insulin levels were below the threshold sensitivity of the assay
(<0.05 ng/ml; n = 4), in contrast to the wild-type mice, for which a value of 0.46 ± 0.04 ng/ml (n = 3) was found. Glucagon levels were reduced by approximately 20%
in the hnf6/ mice compared to wild-type mice
(Fig. 6B). We interpreted the data as follows. Low insulinemia in
hnf6/ mice is consistent with the fasting
state of the animals and with the low number of cells in the
pancreas (Fig. 2K and L). Low glucagonemia is consistent with the low
number of pancreatic cells (Fig. 2G and H). The apparent
discrepancy in hnf6/ mice between a strong
reduction in pancreatic cell number and a mildly reduced blood
glucagon level can be explained by the fasting state, which stimulates
glucagon secretion. In these mice, however, glucagon secretion is not
sufficient to ensure normal glucagonemia and normoglycemia. We conclude
that glucose homeostasis is perturbed in newborn
hnf6/ mice, at least in part as a
consequence of inappropriate insulinemia and glucagonemia.
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/ mice had islets
of Langerhans, but the morphology of the islets was abnormal (Fig. 3).
To further investigate glucose homeostasis in adult
hnf6/ mice, we performed glucose tolerance
tests on 10-week-old animals. hnf6+/ mice
showed normal fasting glycemia and normal blood glucose response curves
(data not shown). In contrast, hnf6/ mice
exhibited elevated fasting glycemia and were glucose intolerant (Fig.
6C). The perturbed glucose response curve was, at least in part, a
consequence of insufficient glucose-induced insulin response, as shown
by the low levels of insulin 1 h after glucose injection (Fig.
6D). In addition, glucagonemia, measured after an overnight fasting
period, was slightly lower than in the wild-type mice. We conclude that
lack of HNF-6 results in diabetes.
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DISCUSSION |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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In the present work we addressed the question of the role of the
ONECUT transcription factor HNF-6 in pancreatic cell differentiation. The early, specified, pancreatic epithelium contains precursors of the
exocrine and endocrine cells (29), and it expresses
pdx1, nkx6.1, and hnf6. Our results
show that HNF-6 is dispensable for reaching this specified state.
Indeed, expression of pdx1 and nkx6.1 was normal
at the initiation of pancreas development (e10.75) in the
hnf6/ embryos. Further differentiation of
the pancreatic epithelium into exocrine pancreatic cells was unaffected
in hnf6/ embryos. In contrast, the number of
the four endocrine cell types was reduced and the islets of Langerhans
were absent at birth, indicating that HNF-6 is involved in endocrine
cell differentiation.
Ngn-3 was recently characterized as a proendocrine factor, since its
expression is required for endocrine determination (3) and
its absence in ngn3/ mice results in lack of
pancreatic endocrine cells (10). In wild-type embryos,
ngn3 expression starts around e9.5, peaks around e13.5 to
e15.5, and disappears postnatally. It is proposed (3, 17)
that expression of ngn3 induces both endocrine
differentiation and the synthesis of Notch ligands. Binding of Notch
ligands to receptors of neighboring cells would stimulate in these
cells the synthesis of Hes-1, a factor that represses ngn3
expression. In hnf6/ embryos, the
concentration of Ngn-3 mRNA was very low at e14.5 to e17.5. This was
not a consequence of increased hes1 gene expression, since
the levels of Hes-1 mRNA were normal. Instead, our observations suggested that HNF-6 stimulates ngn3 expression and that
absence of HNF-6 results in reduced ngn3 gene activation.
Indeed, we showed that HNF-6 can bind to and activate the
ngn3 promoter. Given the known requirement of Ngn-3 for
endocrine differentiation, it is not surprising that the
ngn3 deficiency seen in hnf6/
embryos is associated with impaired endocrine differentiation. To
explain the pancreatic endocrine phenotype of the
hnf6/ embryos, we propose that the absence
of HNF-6 leads, in most cells, to a reduction of ngn3 gene
expression below the threshold required to induce endocrine
differentiation. In a few cells, ngn3 expression would be
above the threshold, consistent with the known cell-to-cell variation
in transcriptional response of a particular gene (5). In
these cells, endocrine differentiation would be initiated and allowed
to proceed along the next steps characterized by expression of
differentiation markers such as Pax-6 and Isl-1. The low number of
cells that express these markers at e12.5 and e15.5 would therefore
reflect the reduction in the number of precursor cells that have
entered the endocrine differentiation pathway.
From our in situ hybridization experiments, we conclude that all cells of the pancreatic epithelium at e14.5 express hnf6 but only a fraction express ngn3. This raises the question as to how ngn3 gene activation by HNF-6 is restricted to a subpopulation of HNF-6-expressing cells. The uniform distribution of hnf6 expression throughout the epithelium may suggest that HNF-6 acts as an accessory protein for a cell-type-restricted transcription factor with which it would cooperate to activate the ngn3 gene promoter. Alternatively, the activity of HNF-6 could be modulated by cell-type-specific mechanisms.
According to the lateral specification model (see above),
hes1 is activated by Notch and represses endocrine
differentiation (3, 17). However, the broad expression of
hes1 in the pancreatic epithelium, including in cells that
are not in contact with differentiating endocrine cells, suggested that
the role of hes1 is not restricted to lateral specification
(17). Our data on hnf6/ mice
further support this interpretation. Indeed, according to the lateral
specification model, one would expect reduced expression of
ngn3 to be associated with reduced expression of
hes1. This was not the case in the
hnf6/ mice. Our data therefore clearly point
to the existence of Ngn-3-independent mechanisms of hes1
gene activation.
After birth, the number of endocrine cells increased in the pancreas of
hnf6/ mice. These cells were observed near
enlarged ducts and near cysts. They were most likely at the origin of
the islets formed postnatally in the hnf6/
mice. Endocrine cells located near ducts have been observed in rats
during pancreas regeneration after partial pancreatectomy (4). The presence of endocrine cells within the cystic
epithelium of hnf6/ mice suggests that it
retained some differentiation potential. However, most of the cells
lining the cysts expressed pdx1 and glut2, but
not insulin, suggesting that these cells had failed to complete
differentiation. No expression of ngn3 was detected by
RT-PCR or by in situ hybridization in the pancreas of
hnf6/ mice after birth (data not shown).
Other factors of the ONECUT class have been identified in humans
(14), and their expression in the pancreas should be
analyzed to determine if they may participate in the control of
pancreatic development.
HNF-6 stimulates hnf3 (20, 30) and HNF-3
stimulates pdx1 in the pancreas (36). This
suggested that HNF-6 could control indirectly pdx1 gene
expression. Our data would dismiss this hypothesis since expression of
pdx1, as shown here, and of hnf3 (data not shown) was unaffected in hnf6/ embryos.
However, we cannot exclude that a HNF-6
HNF-3Pdx-1 cascade
functions in wild-type embryos and that, in the absence of HNF-6,
activation of the hnf3 gene is compensated for by other factors.
Glucose homeostasis was perturbed in hnf6/
mice. At birth, their insulinemia was very low, consistent with the low
number of pancreatic cells. Contrary to what has been observed in
knockout mice that have few or no cells (10, 18, 24, 31,
33), the newborn hnf6/ mice were
hypoglycemic both in the fasted (Fig. 6) and in the fed (data not
shown) state. Blood glucose levels depend on the balance between
intestinal glucose transport, glucose consumption, and hepatic
gluconeogenesis. Understanding the mechanisms by which newborn
hnf6/ mice control glucose homeostasis
therefore requires characterization of these parameters. The fact that
fasted newborn hnf6/ mice did survive rules
out hypoglycemia as a cause of the high mortality rate (75%) seen
between P1 and P10. It is more likely that these mice die from liver
failure. Indeed, work in progress in our laboratory suggests that
hnf6/ mice have a variable liver phenotype
characterized by abnormal biliary differentiation and liver necrosis.
The pancreatic phenotype in newborn animals is transient. Five- to
ten-week-old mice had islets of Langerhans, but they became diabetic.
They had normal levels of pancreatic glucokinase mRNA (data not shown).
However, full differentiation was not reached since islet morphology
was abnormal and since IAPP was not expressed in several cells.
Moreover, the glucose transporter Glut-2 was barely detectable in the
cells of hnf6/ mice. This may, at least
in part, explain their diabetic phenotype. Indeed, Glut-2-deficient
mice have diabetes because of a decreased glucose-induced insulin
response (12), as seen here in the
hnf6/ mice. We conclude from our work that
HNF6 is a candidate gene for diabetes mellitus in humans.
| |
ACKNOWLEDGMENTS |
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We thank L. Hue, J. C. Henquin, and J. Rahier for discussions and C. Bouzin, S. Fierens, V. Lannoy, L. Maisin, Y. Peignois, E. Gils, T. Vancoetsem, and K. Wijnens for help. Anti-Pax-6 and anti-Glut-2 antibodies were from S. Saule and B. Thorens, respectively. The monoclonal antibodies against Isl-1 and Nkx2.2, developed by T. Jessel, were obtained from the DSHB developed under the auspices of NICHM and maintained at the University of Iowa.
This work was supported by grants from the Belgian State Program on Interuniversity Poles of Attraction, the D.G. Higher Education and Scientific Research of the French Community of Belgium, the Fund for Scientific Medical Research, and the National Fund for Scientific Research (Belgium). O.D.M. and J.J. were supported by grants from the National Institutes of Health and the Juvenile Diabetes Foundation. G.G. and F.G. were supported by a grant from the Association pour la Recherche sur le Cancer, by funds from the Institut National de la Recherche Scientifique, from the Centre National de la Recherche Scientifique, and from the Hôpital Universitaire de Strasbourg. F.P.L. is Senior Research Associate of the National Fund for Scientific Research (Belgium).
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FOOTNOTES |
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* Corresponding author. Mailing address: Box 7529, 75 Ave. Hippocrate, B-1200 Brussels, Belgium. Phone: +32 2 764 7583. Fax: +32 2 764 7507. E-mail: lemaigre{at}horm.ucl.ac.be.
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REFERENCES |
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Top
Abstract
Introduction
Materials and Methods
Results
Discussion
References
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Molecular and Cellular Biology, June 2000, p. 4445-4454, Vol. 20, No. 12
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